AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-γ, alone and in combination with lamivudine. METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support prod...AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-γ, alone and in combination with lamivudine. METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization. RESULTS: IFN-γat 0.1 to 5μg/L efficiently down regulated HBsAg expression in transduced HepG2 cells. At 5μg/L, IFN-γalso suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-γshowed no additive effect, sequential treatment first with lamivudine and then IFN-γwas found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2μmol/L lamivudine for two days, followed by 1μg/L IFN-γfor another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2μmol/L lamivudine for two days, followed by 5μg/L IFN-γfor six days showed a 72% reduction in HBV cccDNA pool. CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-γand lamivudine, especially in IFN-αnon-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.展开更多
Background Astragafi Radix, the root of Astragalus membranceus （Fish） Bunge Var. mongholicus （Bge）, is a crude drug considered as one of the effective traditional Chinese anti-ageing material. The two isomers of 4...Background Astragafi Radix, the root of Astragalus membranceus （Fish） Bunge Var. mongholicus （Bge）, is a crude drug considered as one of the effective traditional Chinese anti-ageing material. The two isomers of 4-hydroxy-5-hydroxymethyl-[1,3]dioxolan-2,6＇-spirane-5＇,6＇,7＇,8＇-tetrahydro-indolizine-3＇-carbaldehyde （HDTIC）, HDTIC-1 and HDTIC-2, were first extracted from the herb in 2002. We demonstrated previously that 0.1 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2 strongly delay replicaUve senescence of human fetal lung diploid fibroblasts （2BS）. In this study, we chose them to investigate their effects on the expression of senescence-associated genes to explore the mechanism of how HDTIC delays replicative senescence. Methods The effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 were observed in vitro by RT-PCR and Western blot. The anti-oxidative activities of the compounds were also observed by phenotype alteration after treatment with antioxidants. Results There was an obvious expression of p16 in the control senescent cells. However, in the 2BS cells, after 56 population doublings （PDs） grown from PD28 in 0.1 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2, there was a weak mRNA expression of p16 and no protein expression of pl 6 was observed. The expression level of p21 increased with cell ageing Moreover, there was no difference between the expression level of p21 in the control cells and that in the same PD cells cultured with HDTIC compounds. The results also showed that 2BS cells exposed to 100 μmol/L H202 for 5 minutes retum to their non-senescent phenotype and continue to be confluent after incubating the damaged cells with HDTIC-1 （1.0 μmol/L ） or HDTIC-2 （10 μmol/L ） for I hour. Conclusions Expression of p16 by 2BS cells was strongly inhibited by HDTIC compounds, which could contribute to their delayed replicative senescence by the way of p16^INK4a/Rb/MAPK. The anti-oxidative activities of HDTIC-1 and HDTIC-2, described in this study for the first time, mig展开更多
AIM:To analyze the antiviral mechanism of Epigallocatechin gallate(EGCG)against hepatitis B virus(HBV) replication.METHODS:In this research,the HBV-replicating cell line HepG2.117 was used to investigate the antiviral...AIM:To analyze the antiviral mechanism of Epigallocatechin gallate(EGCG)against hepatitis B virus(HBV) replication.METHODS:In this research,the HBV-replicating cell line HepG2.117 was used to investigate the antiviral mechanism of EGCG.Cytotoxicity of EGCG was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Hepatitis B virus e antigen(HBeAg)and hepatitis B virus surface antigen(HBsAg)in the supernatant were detected by enzyme-linked immunosorbent assay.Precore mRNA and pregenomic RNA(pgRNA) levels were determined by semi-quantitative reverse transcription polymerase chain reaction(PCR)assay.The effect of EGCG on HBV core promoter activity was measured by dual luciferase reporter assay.HBV covalently closed circular DNA and replicative intermediates of DNA were quantified by real-time PCR assay.RESULTS:When HepG2.117 cells were grown in the presence of EGCG,the expression of HBeAg was suppressed,however,the expression of HBsAg was not affected.HBV precore mRNA level was also downregulated by EGCG,while the transcription of precore mRNA was not impaired.The synthesis of both HBV covalently closed circular DNA and replicative intermediates of DNA were reduced by EGCG treatment to a similar extent,however,HBV pgRNA transcripted from chromosome-integrated HBV genome was not affected by EGCG treatment,indicating that EGCG targets only replicative intermediates of DNA synthesis.CONCLUSION:In HepG2.117 cells,EGCG inhibits HBV replication by impairing HBV replicative intermediates of DNA synthesis and such inhibition results in reduced production of HBV covalently closed circular DNA.展开更多
AIM： To develop a conditionally replicative gene-viral vector system called CNHK500-p53, which contains dual promoters within the E1 region, and combines the advantages of oncolytic virus and gene therapies for hepat...AIM： To develop a conditionally replicative gene-viral vector system called CNHK500-p53, which contains dual promoters within the E1 region, and combines the advantages of oncolytic virus and gene therapies for hepatocellular carcinoma （HCC）. METHODS： CNHK500-p53 was constructed by using human telomerase reverse transcriptase （hTERT） promoter to drive adenovirus E1a gene and hypoxia response element （HRE） promoter to drive adenovirus E1b gene. p53 gene expressing cassette was inserted into the genome of replicative virus. Viral replication experiments, cytopathic effect （CPE） and methyl thiazolyl tetrazolium （MTT） assay were performed to test the selective replication and oncolytic efficacy of CNHK500-p53. RESULTS： Immunohistochemistry verified that infection with CNHK500-p53 was associated with selective replication of adenovirus and production of p53 protein in telomerase-positive and hypoxia-inducible factordependent HCC cells, p53 protein secreted from HepG2, infected with CNHK500-p53 was significantly higher than that infected with nonreplicative adenovirus Ad-p53 in vitro （388 ± 34.6 μg/L vs 76.3 ± 13.17 μg/L）. Viral replication experiments showed that replication of CNHK500-p53 and CNHK500 or WtAd5, was much stronger than that of Ad-p53 in tested HCC cell lines. CPE and H1-F assay indicated that CNHK500-p53 selectively replicated in and killed HCC cells while leaving normal cells unaffected. CONCLUSION： A more efficient gene-viral system is developed by combining selective oncolysis with exogenous expression of p53 against HCC cells.展开更多
The present study presents cytogenetics/cytology of haploidization in the origin of a new, fast growing diploid, small cell-type (F-dPCs). The sequence of events was haploid groupings of the chromosomes in normal, hum...The present study presents cytogenetics/cytology of haploidization in the origin of a new, fast growing diploid, small cell-type (F-dPCs). The sequence of events was haploid groupings of the chromosomes in normal, human metaphase cells, followed by genomic doubling to homozygousdiploidy. These events were responses to DNA replication stress fromamino acid glutamine deprivation. Importantly, these homozygous cells outgrew normal fibroblasts in 2 - 3 passages—they had gained proliferative advantage (GPA), presumably from loss (LOH) of tumor suppressor genes. They were morphologically changed cells with rounded nuclei that grew in a “streaming” growth pattern and with changed form and size of mitosis, similar to some hyperplasias. The grouping of the chromosomes in metaphase cells was asymmetric with a narrow range around the median (23) (no micro-nuclei), suggesting genetic control. The root-origin of haploidization was evidenced by maternal and paternal genomes occupying separate territories in metaphase cells, which assumedly permitted independent segregations of bichromatid chromosomes. In near-haploid ALL-L1 leukemia the loss of virtually, whole chromosomal complements was judged by SNP array analyses, as a primary event before genomic doubling to hyperdiploidy with LOH. From the present data such specific, non-random loss of chromosomes strongly suggested, a haploidization process capable of genomic doubling, as observed for the “birth” of the small, F-dPCs. This suggestion was supported by this type of leukemia being the L1-type, where L1 signifies small cells. The possibility now exists that a tumorigenic process can be initiated directly from diploid cells through haploid (near-haploid) distributed chromosomes in normal metaphase cells. This event followed by monosomic doublings to UPDs would lead to massive LOH and a return to para-diploidy, a frequent occurrence in many types of tumors. The present simple, cultural derivations of the extraordinary F-dPCs allow GPA-identification and experimental展开更多
Background The accumulation of free radicals and advanced glycation end products （AGEs） in cell plays a very important role in replicative senescence. Aminoguanidine （AG） has potential antioxidant effects and decr...Background The accumulation of free radicals and advanced glycation end products （AGEs） in cell plays a very important role in replicative senescence. Aminoguanidine （AG） has potential antioxidant effects and decreases AGE levels. This study aimed to investigate its effect on replicative senescence in vitro. Methods The effects of aminoguanidine on morphology, replicative lifespan, cell growth and proliferation, AGEs, DNA damage, DNA repair ability and telomere length were observed in human fetal lung diploid fibroblasts （2BS）. Results Aminoguanidine maintained the non-senescent phenotype of 2BS cells even at late population doubling （PD） and increased cumulative population doublings by at least 17-21 PDs. Aminoguanidine also improved the potentials of growth and proliferation of 2BS cells as detected by the MTT assay. The AGE levels of late PD cells grown from early PD in DMEM containing aminiguanidine decreased significantly compared with those of late PD control cells and were similar to those of young control cells. In addition, the cells pretreated with aminoguanidine had a significant reduction in DNA strand breaks when they were exposed to 200 μmol/L H2O2 for 5 minutes which indicated that the compound had a strong potential to protect genomic DNA against oxidative stress. And most of the cells exposed to 100 μmol/L H2O2 had much shorter comet tails and smaller tail areas after incubation with aminoguanidine-supplemented DMEM, which indicated that the compound strongly improved the DNA repair abilities of 2BS cells. Moreover, PD55 cells grown from PD28 in 2 mmol/L or 4 mmol/L aminoguanidine retain telomere lengths of 7.94 kb or 8.12 kb, which was 0.83 kb or 1.11 kb longer than that of the control cells. Conclusion Aminoguanidine delays replicative senescence of 2BS cells and the senescence-delaying effect of aminoguanidine appear to be due to its many biological properties including its potential for proliferation improvement, its inhibitory effect of AGE formation, antioxidant effect, im展开更多
DNA damage may compromise genome integrity and lead to cell death. Cells have evolved a variety of processes to respond to DNA damage including damage repair and tolerance mechanisms, as well as damage checkpoints. Th...DNA damage may compromise genome integrity and lead to cell death. Cells have evolved a variety of processes to respond to DNA damage including damage repair and tolerance mechanisms, as well as damage checkpoints. The DNA damage tolerance(DDT) pathway promotes the bypass of single-stranded DNA lesions encountered by DNA polymerases during DNA replication. This prevents the stalling of DNA replication. Two mechanistically distinct DDT branches have been characterized. One is translesion synthesis(TLS) in which a replicative DNA polymerase is temporarily replaced by a specialized TLS polymerase that has the ability to replicate across DNA lesions. TLS is mechanistically simple and straightforward, but it is intrinsically error-prone. The other is the error-free template switching(TS) mechanism in which the stalled nascent strand switches from the damaged template to the undamaged newly synthesized sister strand for extension past the lesion. Error-free TS is a complex but preferable process for bypassing DNA lesions. However, our current understanding of this pathway is sketchy. An increasing number of factors are being found to participate or regulate this important mechanism, which is the focus of this editorial.展开更多
In order to determine the replication sites of hepatitis C virus, the in situ hybridization and immunohistochemical technique using digoxin-labeled 531bp plus-strand and minus-strand HCVRNA probes were employed to det...In order to determine the replication sites of hepatitis C virus, the in situ hybridization and immunohistochemical technique using digoxin-labeled 531bp plus-strand and minus-strand HCVRNA probes were employed to detect HCVRNA in the liver tissues, bone marrow mononuclear cells and peripheral blood mononuclear cells (PBMCs) from the patients with chronic hepatitis C, and in HCV transfected COS cells. The results showed that both plus-strand and minusstrand HCVRNA were detected in 80% of liver tissues (4/5). Plus-strand HCVRNA could be detected in 90% of PBMCs and bone marrow mononuclear cells (18/20), minus-strand HCVRNA in 25% of PBMCs. In HCV transfected COS cells, plus-strand HCVRNA distributed evenly in 20% cellular nuclei and cytoplasms. No minus-strand HCVRNA was detected in the bone marrow mononuclear cells and HCV transfected COS cells. The positive signal appeared in more cells when the liver tissues, PBMCs and marrow mononuclear cells were hybridized by plus-strand probes than when hybridized by minus-strand probes. Our results suggested that the hepatocytic cytoplasms and PBMC cytoplasms were the replication sites of HCV, but the marrow mononuclear cells were not the replication sites of HCV although they were infected by HCV. HCV infection might be accounted for the pathogenesis of chronic hepatitis and relapse of hepatitis C after liver transplantation.展开更多
Objective： Oncolytic adenovirus, also called conditionally replicating adenovirus （CRAD）, has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Her...Objective： Oncolytic adenovirus, also called conditionally replicating adenovirus （CRAD）, has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage. Methods： A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase （hTERT）, the coding sequence of E1A gene, an internal ribosome entry site sequence （IRES） and the coding sequence of herpes simplex virus-thymidine kinase （HSV-TK） was constructed. Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK. The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured. The infection efficiency was determined by flow cytometry. Results： Ad-ETK deliverys E1A and HSV-TK gene, which selectively replicates in hTERT-positive tumor cells, and the progeny virus can reach up to 150 IU/cell. Our in vitro study showed that Ad-ETK plus ganciclovir （GCV） induced an obvious cell death. Conclusion： An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy.展开更多
Two sublines of microalgae Dunaliella viridis Teodor. were obtained: CuS—a subline sensitive to toxic concentration of copper ions and CuR—a subline resistant to toxic concentration of copper ions. The chronological...Two sublines of microalgae Dunaliella viridis Teodor. were obtained: CuS—a subline sensitive to toxic concentration of copper ions and CuR—a subline resistant to toxic concentration of copper ions. The chronological aging of cultures was revealed in increase of DNA (polyploidization) and triacylglycerols (TAG) content in microalgae cells. The adaptation of D. viridis to toxic concentrations of copper ions resulted in formation of an adaptive epygenotype characterized by increased content of carbonylated proteins and decreased content of proline compared to the CuS-subline. The adaptation of D. viridis to toxic concentrations of copper ions resulted in the increase of the rate of chronological aging compared to the CuS-subline. Four subcultures with different rates of transplanting were obtained from each subline. The rate of replicative aging was shown to be dependent on the rate of chronological aging of subcultures (passage rate). The pre-adaptation of D. viridis to toxic concentrations of copper ions was accompanied by increase of accumulation rate of TAG and DNA in cells, which was interpreted as a sign of cellular aging during the progressive passages of the algae culture.展开更多
This study examined the replicative senescence of primary-culture human vocal fold fibroblasts, in terms of changes in gross chromosomal structure with sub- culturing, and population doubling time (PDT). The mRNA expr...This study examined the replicative senescence of primary-culture human vocal fold fibroblasts, in terms of changes in gross chromosomal structure with sub- culturing, and population doubling time (PDT). The mRNA expressions of 14 target genes were also examined. The objectives were to identify the onset of senescence for establishing the acceptable limit of sub-culturing, and to better understand the effect of cellular aging on matrix protein regulation in the vocal fold. Gross chromosomal changes in vocal fold fibroblasts from a 58-year-old woman were karyotyped with Giemsa stain. Proliferation of the fibroblasts was determined with cell recovery and PDT calculation. Transcript levels of the target genes were found by RT-PCR. Onset of significant chromosomal anomalies was seen with passage 5. For mRNA expressions, significant increases with passaging were observed in collagenase, macrophage elastase, lysyl oxidase and fibromodulin, whereas significant downregulation was detected in decorin, procollagen I, hyaluronic acid-synthase 2 and collagen III. This modulation pattern suggested that fibroblasts underwent in vitro aging, consistent with the significant increase in PDT. The inception of senescence did not occur until passage 5. These findings may facilitate the development of representative in vitro models for testing tissue engineering approaches involving primary-culture fibroblasts.展开更多
Objective: To evaluate the therapeutic efficacy of replicative adenovirus CNHK500 in the treatment of hepatocellular carcinoma. Methods: Virus proliferation assay, cell viability assay and Western blot were performed ...Objective: To evaluate the therapeutic efficacy of replicative adenovirus CNHK500 in the treatment of hepatocellular carcinoma. Methods: Virus proliferation assay, cell viability assay and Western blot were performed to assess the selective replication and cytolysis of CNHK500 in telomerase positive liver cancer cells Hep3B, HepGII, SMMC7721 and in normal cells. Results: The replicative multiples of CNHK500 in HepGII, Hep3B and SMMC7221 after 96 h of virus proliferation were 52 000, 396 984.9 and 632 911.3 fold respectively, similar to those of wtAd5. However, CNHK500 demonstrated more significant attenuated replicative ability in normal cell lines than wtAd5. CNHK500 replicated only 3.1-100 fold at 96 h, while the wtAd5 still reached 3160-17 357 fold. CNHK500 could cause half of HepGII cells death within 7 days at MOI 2, in Hep3B cell lines the IC50 was as low as MOI 0.01, whereas the IC50 in BJ cell was as high as MOI 1000. CNHK500 E1A protein could only be detected in hepatocellular cancer cells but not in normal cells under normoxia. E1B protein could only be detected under hypoxia condition at a MOI of 1. Conclusion: CNHK500 can efficiently replicate in and kill liver cancer cells as well as wtAd5 do while it is severely attenuated in proliferation and cytolysis among normal cells. It would be a prominsing strategy for liver cancer tratment.展开更多
AIM： To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted in telomerase-positive hepatoceUular carcinoma.METHODS： CNHK300, ONYX-015 （55 kDa protein deleted adenovirus） and wtAd5 （wild...AIM： To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted in telomerase-positive hepatoceUular carcinoma.METHODS： CNHK300, ONYX-015 （55 kDa protein deleted adenovirus） and wtAd5 （wild type adenovirus 5） were compared, and virus proliferation assay, cell viability assay, Western blot and fluorescence microscopy were used to evaluate the proliferation and cytolysis selectivity of CNHK300.RESULTS： The replicative multiples in Hep3B and HepG Ⅰ after 48 Ⅱ of CNHK300 proliferation were 40625 and 65326 fold, respectively, similar to that of wtAd5.. However, CNHK300 exhibited attenuated replicative ability in normal fibroblast cell line BJ. CNHK300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection （MOI）, but could not affect growth of normal cells even at a high MOI.CONCLUSION： CNHK300 is a cancer-selective replication-competent adenovirus which can cause oncolysis of liver cancer cells as well as wtAd5 （wild type adenovirus 5）, but had severely attenuated replicative and cytolytic ability in normal cells. This novel strategy of cancer treatment offers a promising treatment platform.展开更多
Cartilage defects are a challenge to treat clinically due to the avascular nature of cartilage.Low immunogenicity and extensive proliferation and multidifferentiation potential make fetal stem cells a promising source...Cartilage defects are a challenge to treat clinically due to the avascular nature of cartilage.Low immunogenicity and extensive proliferation and multidifferentiation potential make fetal stem cells a promising source for regenerative medicine.In this study,we aimed to determine whether fetal synovium-derived stem cells(FSDSCs)exhibited replicative senescence and whether expansion on decellularized extracellular matrix(dECM)deposited by adult SDSCs(AECM)promoted FSDSCs’chondrogenic potential.FSDSCs from passage 2 and 9 were compared for chondrogenic potential,using Alcian blue staining for sulfated glycosaminoglycans(GAGs),biochemical analysis for DNA and GAG amounts,and real-time PCR for chondrogenic genes including ACAN and COL2A1.Passage 3 FSDSCs were expanded for one passage on plastic flasks(PL),AECM,or dECM deposited by fetal SDSCs(FECM).During expansion,cell proliferation was evaluated using flow cytometry for proliferation index,stem cell surface markers,and resistance to hydrogen peroxide.During chondrogenic induction,expanded FSDSCs were evaluated for tri-lineage differentiation capacity.We found that cell expansion enhanced FSDSCs’chondrogenic potential at least up to passage 9.Expansion on dECMs promoted FSDSCs’proliferative and survival capacity and adipogenic differentiation but not osteogenic capacity.AECM-primed FSDSCs exhibited an enhanced chondrogenic potential.展开更多
Malondialdehyde(MDA)is a well known inducer of carbonyl stress in a variety of human cells,however,its effects on human bone marrow mesenchymal stem cells(hMSCs)have not been documented.In this study,the effects of MD...Malondialdehyde(MDA)is a well known inducer of carbonyl stress in a variety of human cells,however,its effects on human bone marrow mesenchymal stem cells(hMSCs)have not been documented.In this study,the effects of MDA concentration on the growth rate and proliferation of hMSCs in vitro were assessed.Under high concentrations of MDA,the cell count was decreased and the population doubling time(PDT)was lengthened.Flow cytometry(FCM)demonstrated that MDA triggered cells to undergo apoptosis,in parallel with the findings in MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]assay which showed that it can also impair cellular viability.Surprisingly,FCM also determined that the percentage of hMSCs in G2/M-and S-phases also increased in a dose-dependent manner with respect to MDA concentration.These results strongly suggest that even though hMSCs were severely impaired by high concentrations of MDA,they were still able to send signals that resulted in accelerated cellular proliferation process.This study provided important insights on how carbonyl stress affects cell cycle and proliferation of hMSCs.展开更多
All non-immortalized mesenchymal stem cells have a limited proliferative potential,that is,replicative senescence(RS)is an integral characteristic of the life of all mesenchymal stem cells(MSCs).It is known that one o...All non-immortalized mesenchymal stem cells have a limited proliferative potential,that is,replicative senescence(RS)is an integral characteristic of the life of all mesenchymal stem cells(MSCs).It is known that one of the important signs of RS is a decrease of cell motility,and that violations of migration processes contribute to the deterioration of tissue regeneration.Therefore,the characterization of the properties of the cell line associated with RS is a prerequisite for the effective use of MSCs in restorative medicine.One of the key proteins regulating cell motility is the small GTPase RhoA.The main purpose of this work was to study the nuclear-cytoplasmic redistribution of the RhoA protein during RS in MSC lines recently obtained and characterized in our laboratory.The study found that a comparative analysis of the intracellular localization of RhoA in three cell lines(MSCWJ-1,FetMSC,DF2)showed a decrease in the nuclear localization of RhoA during RS.展开更多
Perinatal transmission of Human immunodeficiency virus(HIV),also called mother-to-child transmission(MTCT),accounts for 90% of infections in infants worldwide and occurs in 30%-45% of children born to untreated HIV-1 ...Perinatal transmission of Human immunodeficiency virus(HIV),also called mother-to-child transmission(MTCT),accounts for 90% of infections in infants worldwide and occurs in 30%-45% of children born to untreated HIV-1 infected mothers.Among HIV-1 infected mothers,some viruses are transmitted from mothers to their infants while others are not.The relationship between virologic properties and the pathogenesis caused by HIV-1 remains unclear.Previous studies have demonstrated that one obvious source of selective pressure in the perinatal transmission of HIV-1 is maternal neutralizing antibodies.Recent studies have shown that viruses which are successfully transmitted to the child have growth advantages over those not transmitted,when those two viruses are grown together.Furthermore,the higher fitness is determined by the gp120 protein of the virus envelope.This suggests that the selective transmission of viruses with higher fitness occurred exclusively,regardless of transmission routes.There are many factors contributing to the selective transmission and HIV replicative fitness is an important one that should not be neglected.This review summarizes current knowledge of the role of HIV replicative fitness in HIV MTCT transmission and the determinants of viral fitness upon MTCT.展开更多
Objective： To evaluate the tumor selectivity and therapeutic efficiency of replication-competent adenovirus CNHK300 on human breast cancer cells. Methods： RT-PCR was used to detect the hTERT mRNA activity in various...Objective： To evaluate the tumor selectivity and therapeutic efficiency of replication-competent adenovirus CNHK300 on human breast cancer cells. Methods： RT-PCR was used to detect the hTERT mRNA activity in various breast cancer and normal fibroblast cell lines. Virus proliferation assay, cell viability assay and Western blot were applied to evaluate the proliferation and cytolysis selectivity of CNHK300. Results： The telomerase activity of MCF-7, BT-549 and SK-BR-3 was positive, while telomerase in MRC-5 and BJ was negative. The progeny virus titers in MCF-7, BT-549 and SK-BR-3 after 48 h of CNHK300 exposure was 40 625, 1 265 and 20 000 fold higher than those of 0 h, even slightly higher than those of wtAd5 （except in SK-BR-3）. ONYX-015 virus proliferation ability was weaker than that of CNHK300 in cancer cells. However, CNHK300 exhibited attenuated replicative ability as compared with wtAd5 in MRC-5 and BJ. The CNHK300 replicatative multiple was 63 and 192 fold at 48 h respectively, while the wtAd5 still multiplied 3 160-4 846 fold. CNHK300 could cause about half of breast cancer cells to die within 7 days at MOI 10 pfu/cell and below, whereas the IC50 in BJ and MRC-5 was as high as MOI 100 pfu/cell. CNHK300 E1A protein could be detected in breast cancer cells and 293 cells but not in normal fibroblast cells. Conclusion： hTERT promoter can successfully modulate the CNHK300 to be selectively replicated in breast cancer cells positive for telomerase, which may be a potential treatment strategy in breast cancer.展开更多
OBJECTIVE: To investigate the dynamic alternations of HBV markers of active HBV replicationrecipients receiving lamivudine prophylaxis after liver transplantation.METHODS: Serial liver biopsy samples and sera were obt...OBJECTIVE: To investigate the dynamic alternations of HBV markers of active HBV replicationrecipients receiving lamivudine prophylaxis after liver transplantation.METHODS: Serial liver biopsy samples and sera were obtained from 15 recipients and examined withenzyme-linked radioinmmunoassay for HBsAg, HBeAg, HBsAb, HBcAb and HBeAb, and fluorescentquantitative assay for quantitation of HBV DNA in serum. Immunohistochemical staining of HBsAg,HBcAg and HBV DNA hybridization in situ were used to detect HBV markers in liver biopsy samples.RESULTS: 100 mg lamivudine taken orally every, day for 2 weeks before transplantation enabled 12(80%) of 15 active viral replication recipients (HBV DNA positive) to converse to HBV DNA negative.HBsAb, HBcAb and HBeAb in serum emerged in 1-2 weeks after liver transplantation, and disappearedgradually within 6 months; HBV DNA fluorescent quantitative assay showed constant negativity in serum.Immunohistochemical staining of HBsAg, HBcAg and HBV DNA hybridization in situ in liver biopsysamples showed negative results synchorously. Eight of the 15 HBV active replication recipients lostHBV markers thoroughly both in serology and tissue staining as well as HBV DNA hybridization in situ ofserial liver biopsy samples from 12 to 44 weeks after liver transplantation. Should any of HBsAg, HBeAgin serology and HBsAg, HBcAg in immunohistochemical staining was positive, or HBV DNA detectablein serum, or HBV DNA hybridization in situ in liver tissue positive, allograft HBV reinfection or De novoliver allograft infection could be diagnosed. Furthermore, if associated with elevation of ALT andbilirubin, the diagnosis of HBV hepatitis recurrence could be established.CONCLUSION: Allograft HBV reinfection or De nuvo liver allograft infection in active viral replicationrecipients could be prevented with lamivudine regimen, and further clearance of HBV may be possible ifproper measures are taken.展开更多
基金Supported by a grant from the Dabur Research Foundation, India and a Senior Research Fellowship of the CSIR, Gov. of India (to MKP)
文摘AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-γ, alone and in combination with lamivudine. METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization. RESULTS: IFN-γat 0.1 to 5μg/L efficiently down regulated HBsAg expression in transduced HepG2 cells. At 5μg/L, IFN-γalso suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-γshowed no additive effect, sequential treatment first with lamivudine and then IFN-γwas found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2μmol/L lamivudine for two days, followed by 1μg/L IFN-γfor another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2μmol/L lamivudine for two days, followed by 5μg/L IFN-γfor six days showed a 72% reduction in HBV cccDNA pool. CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-γand lamivudine, especially in IFN-αnon-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.
基金This work was-supported by the grants from the National Nature Science Foundation of China （No. 30672469） and Beijing Nature Science Foundation （No. 7062030）.
文摘Background Astragafi Radix, the root of Astragalus membranceus （Fish） Bunge Var. mongholicus （Bge）, is a crude drug considered as one of the effective traditional Chinese anti-ageing material. The two isomers of 4-hydroxy-5-hydroxymethyl-[1,3]dioxolan-2,6＇-spirane-5＇,6＇,7＇,8＇-tetrahydro-indolizine-3＇-carbaldehyde （HDTIC）, HDTIC-1 and HDTIC-2, were first extracted from the herb in 2002. We demonstrated previously that 0.1 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2 strongly delay replicaUve senescence of human fetal lung diploid fibroblasts （2BS）. In this study, we chose them to investigate their effects on the expression of senescence-associated genes to explore the mechanism of how HDTIC delays replicative senescence. Methods The effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 were observed in vitro by RT-PCR and Western blot. The anti-oxidative activities of the compounds were also observed by phenotype alteration after treatment with antioxidants. Results There was an obvious expression of p16 in the control senescent cells. However, in the 2BS cells, after 56 population doublings （PDs） grown from PD28 in 0.1 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2, there was a weak mRNA expression of p16 and no protein expression of pl 6 was observed. The expression level of p21 increased with cell ageing Moreover, there was no difference between the expression level of p21 in the control cells and that in the same PD cells cultured with HDTIC compounds. The results also showed that 2BS cells exposed to 100 μmol/L H202 for 5 minutes retum to their non-senescent phenotype and continue to be confluent after incubating the damaged cells with HDTIC-1 （1.0 μmol/L ） or HDTIC-2 （10 μmol/L ） for I hour. Conclusions Expression of p16 by 2BS cells was strongly inhibited by HDTIC compounds, which could contribute to their delayed replicative senescence by the way of p16^INK4a/Rb/MAPK. The anti-oxidative activities of HDTIC-1 and HDTIC-2, described in this study for the first time, mig
基金Supported by National Technology and Science Key Project (2008ZX10002-010)the Important National Science and Technology Specific Projects(2009ZX09301-014)
文摘AIM:To analyze the antiviral mechanism of Epigallocatechin gallate(EGCG)against hepatitis B virus(HBV) replication.METHODS:In this research,the HBV-replicating cell line HepG2.117 was used to investigate the antiviral mechanism of EGCG.Cytotoxicity of EGCG was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Hepatitis B virus e antigen(HBeAg)and hepatitis B virus surface antigen(HBsAg)in the supernatant were detected by enzyme-linked immunosorbent assay.Precore mRNA and pregenomic RNA(pgRNA) levels were determined by semi-quantitative reverse transcription polymerase chain reaction(PCR)assay.The effect of EGCG on HBV core promoter activity was measured by dual luciferase reporter assay.HBV covalently closed circular DNA and replicative intermediates of DNA were quantified by real-time PCR assay.RESULTS:When HepG2.117 cells were grown in the presence of EGCG,the expression of HBeAg was suppressed,however,the expression of HBsAg was not affected.HBV precore mRNA level was also downregulated by EGCG,while the transcription of precore mRNA was not impaired.The synthesis of both HBV covalently closed circular DNA and replicative intermediates of DNA were reduced by EGCG treatment to a similar extent,however,HBV pgRNA transcripted from chromosome-integrated HBV genome was not affected by EGCG treatment,indicating that EGCG targets only replicative intermediates of DNA synthesis.CONCLUSION:In HepG2.117 cells,EGCG inhibits HBV replication by impairing HBV replicative intermediates of DNA synthesis and such inhibition results in reduced production of HBV covalently closed circular DNA.
基金Supported by the Major State Basic Research Development Program (973 Program) of China, No. 2003CB515507
文摘AIM： To develop a conditionally replicative gene-viral vector system called CNHK500-p53, which contains dual promoters within the E1 region, and combines the advantages of oncolytic virus and gene therapies for hepatocellular carcinoma （HCC）. METHODS： CNHK500-p53 was constructed by using human telomerase reverse transcriptase （hTERT） promoter to drive adenovirus E1a gene and hypoxia response element （HRE） promoter to drive adenovirus E1b gene. p53 gene expressing cassette was inserted into the genome of replicative virus. Viral replication experiments, cytopathic effect （CPE） and methyl thiazolyl tetrazolium （MTT） assay were performed to test the selective replication and oncolytic efficacy of CNHK500-p53. RESULTS： Immunohistochemistry verified that infection with CNHK500-p53 was associated with selective replication of adenovirus and production of p53 protein in telomerase-positive and hypoxia-inducible factordependent HCC cells, p53 protein secreted from HepG2, infected with CNHK500-p53 was significantly higher than that infected with nonreplicative adenovirus Ad-p53 in vitro （388 ± 34.6 μg/L vs 76.3 ± 13.17 μg/L）. Viral replication experiments showed that replication of CNHK500-p53 and CNHK500 or WtAd5, was much stronger than that of Ad-p53 in tested HCC cell lines. CPE and H1-F assay indicated that CNHK500-p53 selectively replicated in and killed HCC cells while leaving normal cells unaffected. CONCLUSION： A more efficient gene-viral system is developed by combining selective oncolysis with exogenous expression of p53 against HCC cells.
文摘The present study presents cytogenetics/cytology of haploidization in the origin of a new, fast growing diploid, small cell-type (F-dPCs). The sequence of events was haploid groupings of the chromosomes in normal, human metaphase cells, followed by genomic doubling to homozygousdiploidy. These events were responses to DNA replication stress fromamino acid glutamine deprivation. Importantly, these homozygous cells outgrew normal fibroblasts in 2 - 3 passages—they had gained proliferative advantage (GPA), presumably from loss (LOH) of tumor suppressor genes. They were morphologically changed cells with rounded nuclei that grew in a “streaming” growth pattern and with changed form and size of mitosis, similar to some hyperplasias. The grouping of the chromosomes in metaphase cells was asymmetric with a narrow range around the median (23) (no micro-nuclei), suggesting genetic control. The root-origin of haploidization was evidenced by maternal and paternal genomes occupying separate territories in metaphase cells, which assumedly permitted independent segregations of bichromatid chromosomes. In near-haploid ALL-L1 leukemia the loss of virtually, whole chromosomal complements was judged by SNP array analyses, as a primary event before genomic doubling to hyperdiploidy with LOH. From the present data such specific, non-random loss of chromosomes strongly suggested, a haploidization process capable of genomic doubling, as observed for the “birth” of the small, F-dPCs. This suggestion was supported by this type of leukemia being the L1-type, where L1 signifies small cells. The possibility now exists that a tumorigenic process can be initiated directly from diploid cells through haploid (near-haploid) distributed chromosomes in normal metaphase cells. This event followed by monosomic doublings to UPDs would lead to massive LOH and a return to para-diploidy, a frequent occurrence in many types of tumors. The present simple, cultural derivations of the extraordinary F-dPCs allow GPA-identification and experimental
基金This work was supported by the grants from the National Natural Science Foundation of China(No.30672469)the Beijing Natural Science Foundation(No.7062030)
文摘Background The accumulation of free radicals and advanced glycation end products （AGEs） in cell plays a very important role in replicative senescence. Aminoguanidine （AG） has potential antioxidant effects and decreases AGE levels. This study aimed to investigate its effect on replicative senescence in vitro. Methods The effects of aminoguanidine on morphology, replicative lifespan, cell growth and proliferation, AGEs, DNA damage, DNA repair ability and telomere length were observed in human fetal lung diploid fibroblasts （2BS）. Results Aminoguanidine maintained the non-senescent phenotype of 2BS cells even at late population doubling （PD） and increased cumulative population doublings by at least 17-21 PDs. Aminoguanidine also improved the potentials of growth and proliferation of 2BS cells as detected by the MTT assay. The AGE levels of late PD cells grown from early PD in DMEM containing aminiguanidine decreased significantly compared with those of late PD control cells and were similar to those of young control cells. In addition, the cells pretreated with aminoguanidine had a significant reduction in DNA strand breaks when they were exposed to 200 μmol/L H2O2 for 5 minutes which indicated that the compound had a strong potential to protect genomic DNA against oxidative stress. And most of the cells exposed to 100 μmol/L H2O2 had much shorter comet tails and smaller tail areas after incubation with aminoguanidine-supplemented DMEM, which indicated that the compound strongly improved the DNA repair abilities of 2BS cells. Moreover, PD55 cells grown from PD28 in 2 mmol/L or 4 mmol/L aminoguanidine retain telomere lengths of 7.94 kb or 8.12 kb, which was 0.83 kb or 1.11 kb longer than that of the control cells. Conclusion Aminoguanidine delays replicative senescence of 2BS cells and the senescence-delaying effect of aminoguanidine appear to be due to its many biological properties including its potential for proliferation improvement, its inhibitory effect of AGE formation, antioxidant effect, im
基金Supported by United States National Science Foundation,No.MCB-1158008
文摘DNA damage may compromise genome integrity and lead to cell death. Cells have evolved a variety of processes to respond to DNA damage including damage repair and tolerance mechanisms, as well as damage checkpoints. The DNA damage tolerance(DDT) pathway promotes the bypass of single-stranded DNA lesions encountered by DNA polymerases during DNA replication. This prevents the stalling of DNA replication. Two mechanistically distinct DDT branches have been characterized. One is translesion synthesis(TLS) in which a replicative DNA polymerase is temporarily replaced by a specialized TLS polymerase that has the ability to replicate across DNA lesions. TLS is mechanistically simple and straightforward, but it is intrinsically error-prone. The other is the error-free template switching(TS) mechanism in which the stalled nascent strand switches from the damaged template to the undamaged newly synthesized sister strand for extension past the lesion. Error-free TS is a complex but preferable process for bypassing DNA lesions. However, our current understanding of this pathway is sketchy. An increasing number of factors are being found to participate or regulate this important mechanism, which is the focus of this editorial.
文摘In order to determine the replication sites of hepatitis C virus, the in situ hybridization and immunohistochemical technique using digoxin-labeled 531bp plus-strand and minus-strand HCVRNA probes were employed to detect HCVRNA in the liver tissues, bone marrow mononuclear cells and peripheral blood mononuclear cells (PBMCs) from the patients with chronic hepatitis C, and in HCV transfected COS cells. The results showed that both plus-strand and minusstrand HCVRNA were detected in 80% of liver tissues (4/5). Plus-strand HCVRNA could be detected in 90% of PBMCs and bone marrow mononuclear cells (18/20), minus-strand HCVRNA in 25% of PBMCs. In HCV transfected COS cells, plus-strand HCVRNA distributed evenly in 20% cellular nuclei and cytoplasms. No minus-strand HCVRNA was detected in the bone marrow mononuclear cells and HCV transfected COS cells. The positive signal appeared in more cells when the liver tissues, PBMCs and marrow mononuclear cells were hybridized by plus-strand probes than when hybridized by minus-strand probes. Our results suggested that the hepatocytic cytoplasms and PBMC cytoplasms were the replication sites of HCV, but the marrow mononuclear cells were not the replication sites of HCV although they were infected by HCV. HCV infection might be accounted for the pathogenesis of chronic hepatitis and relapse of hepatitis C after liver transplantation.
文摘Objective： Oncolytic adenovirus, also called conditionally replicating adenovirus （CRAD）, has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage. Methods： A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase （hTERT）, the coding sequence of E1A gene, an internal ribosome entry site sequence （IRES） and the coding sequence of herpes simplex virus-thymidine kinase （HSV-TK） was constructed. Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK. The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured. The infection efficiency was determined by flow cytometry. Results： Ad-ETK deliverys E1A and HSV-TK gene, which selectively replicates in hTERT-positive tumor cells, and the progeny virus can reach up to 150 IU/cell. Our in vitro study showed that Ad-ETK plus ganciclovir （GCV） induced an obvious cell death. Conclusion： An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy.
文摘Two sublines of microalgae Dunaliella viridis Teodor. were obtained: CuS—a subline sensitive to toxic concentration of copper ions and CuR—a subline resistant to toxic concentration of copper ions. The chronological aging of cultures was revealed in increase of DNA (polyploidization) and triacylglycerols (TAG) content in microalgae cells. The adaptation of D. viridis to toxic concentrations of copper ions resulted in formation of an adaptive epygenotype characterized by increased content of carbonylated proteins and decreased content of proline compared to the CuS-subline. The adaptation of D. viridis to toxic concentrations of copper ions resulted in the increase of the rate of chronological aging compared to the CuS-subline. Four subcultures with different rates of transplanting were obtained from each subline. The rate of replicative aging was shown to be dependent on the rate of chronological aging of subcultures (passage rate). The pre-adaptation of D. viridis to toxic concentrations of copper ions was accompanied by increase of accumulation rate of TAG and DNA in cells, which was interpreted as a sign of cellular aging during the progressive passages of the algae culture.
文摘This study examined the replicative senescence of primary-culture human vocal fold fibroblasts, in terms of changes in gross chromosomal structure with sub- culturing, and population doubling time (PDT). The mRNA expressions of 14 target genes were also examined. The objectives were to identify the onset of senescence for establishing the acceptable limit of sub-culturing, and to better understand the effect of cellular aging on matrix protein regulation in the vocal fold. Gross chromosomal changes in vocal fold fibroblasts from a 58-year-old woman were karyotyped with Giemsa stain. Proliferation of the fibroblasts was determined with cell recovery and PDT calculation. Transcript levels of the target genes were found by RT-PCR. Onset of significant chromosomal anomalies was seen with passage 5. For mRNA expressions, significant increases with passaging were observed in collagenase, macrophage elastase, lysyl oxidase and fibromodulin, whereas significant downregulation was detected in decorin, procollagen I, hyaluronic acid-synthase 2 and collagen III. This modulation pattern suggested that fibroblasts underwent in vitro aging, consistent with the significant increase in PDT. The inception of senescence did not occur until passage 5. These findings may facilitate the development of representative in vitro models for testing tissue engineering approaches involving primary-culture fibroblasts.
基金This work is supported by International Cooperation Important Project of National Natural Science Foundation of China(No.30120160824)the State 863 High Technology R&D Project of China(No.2001AA217031).
文摘Objective: To evaluate the therapeutic efficacy of replicative adenovirus CNHK500 in the treatment of hepatocellular carcinoma. Methods: Virus proliferation assay, cell viability assay and Western blot were performed to assess the selective replication and cytolysis of CNHK500 in telomerase positive liver cancer cells Hep3B, HepGII, SMMC7721 and in normal cells. Results: The replicative multiples of CNHK500 in HepGII, Hep3B and SMMC7221 after 96 h of virus proliferation were 52 000, 396 984.9 and 632 911.3 fold respectively, similar to those of wtAd5. However, CNHK500 demonstrated more significant attenuated replicative ability in normal cell lines than wtAd5. CNHK500 replicated only 3.1-100 fold at 96 h, while the wtAd5 still reached 3160-17 357 fold. CNHK500 could cause half of HepGII cells death within 7 days at MOI 2, in Hep3B cell lines the IC50 was as low as MOI 0.01, whereas the IC50 in BJ cell was as high as MOI 1000. CNHK500 E1A protein could only be detected in hepatocellular cancer cells but not in normal cells under normoxia. E1B protein could only be detected under hypoxia condition at a MOI of 1. Conclusion: CNHK500 can efficiently replicate in and kill liver cancer cells as well as wtAd5 do while it is severely attenuated in proliferation and cytolysis among normal cells. It would be a prominsing strategy for liver cancer tratment.
基金International Cooperative Key Project of the National Natural Science Foundation of China, No. 30120160824the State 863 High Technology R&D Project of China, No. 2001AA217031
文摘AIM： To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted in telomerase-positive hepatoceUular carcinoma.METHODS： CNHK300, ONYX-015 （55 kDa protein deleted adenovirus） and wtAd5 （wild type adenovirus 5） were compared, and virus proliferation assay, cell viability assay, Western blot and fluorescence microscopy were used to evaluate the proliferation and cytolysis selectivity of CNHK300.RESULTS： The replicative multiples in Hep3B and HepG Ⅰ after 48 Ⅱ of CNHK300 proliferation were 40625 and 65326 fold, respectively, similar to that of wtAd5.. However, CNHK300 exhibited attenuated replicative ability in normal fibroblast cell line BJ. CNHK300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection （MOI）, but could not affect growth of normal cells even at a high MOI.CONCLUSION： CNHK300 is a cancer-selective replication-competent adenovirus which can cause oncolysis of liver cancer cells as well as wtAd5 （wild type adenovirus 5）, but had severely attenuated replicative and cytolytic ability in normal cells. This novel strategy of cancer treatment offers a promising treatment platform.
基金This project was partially supported by Research Grants from the AO Foundation(S-12-19P)National Institutes of Health(NIH)(no.1 R03 AR062763-01A1).
文摘Cartilage defects are a challenge to treat clinically due to the avascular nature of cartilage.Low immunogenicity and extensive proliferation and multidifferentiation potential make fetal stem cells a promising source for regenerative medicine.In this study,we aimed to determine whether fetal synovium-derived stem cells(FSDSCs)exhibited replicative senescence and whether expansion on decellularized extracellular matrix(dECM)deposited by adult SDSCs(AECM)promoted FSDSCs’chondrogenic potential.FSDSCs from passage 2 and 9 were compared for chondrogenic potential,using Alcian blue staining for sulfated glycosaminoglycans(GAGs),biochemical analysis for DNA and GAG amounts,and real-time PCR for chondrogenic genes including ACAN and COL2A1.Passage 3 FSDSCs were expanded for one passage on plastic flasks(PL),AECM,or dECM deposited by fetal SDSCs(FECM).During expansion,cell proliferation was evaluated using flow cytometry for proliferation index,stem cell surface markers,and resistance to hydrogen peroxide.During chondrogenic induction,expanded FSDSCs were evaluated for tri-lineage differentiation capacity.We found that cell expansion enhanced FSDSCs’chondrogenic potential at least up to passage 9.Expansion on dECMs promoted FSDSCs’proliferative and survival capacity and adipogenic differentiation but not osteogenic capacity.AECM-primed FSDSCs exhibited an enhanced chondrogenic potential.
基金supported by the National Natural Sciences Foundation of China (No.30470637).
文摘Malondialdehyde(MDA)is a well known inducer of carbonyl stress in a variety of human cells,however,its effects on human bone marrow mesenchymal stem cells(hMSCs)have not been documented.In this study,the effects of MDA concentration on the growth rate and proliferation of hMSCs in vitro were assessed.Under high concentrations of MDA,the cell count was decreased and the population doubling time(PDT)was lengthened.Flow cytometry(FCM)demonstrated that MDA triggered cells to undergo apoptosis,in parallel with the findings in MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]assay which showed that it can also impair cellular viability.Surprisingly,FCM also determined that the percentage of hMSCs in G2/M-and S-phases also increased in a dose-dependent manner with respect to MDA concentration.These results strongly suggest that even though hMSCs were severely impaired by high concentrations of MDA,they were still able to send signals that resulted in accelerated cellular proliferation process.This study provided important insights on how carbonyl stress affects cell cycle and proliferation of hMSCs.
基金This work was supported by following grants:Grant from the Director’s Fund of the Institute of Cytology,Russian Academy of SciencesState Assignment No.АААА-А19-119020190093Grant-Subsidy No.075-15-2021-1063(15BRC.21.0011).
文摘All non-immortalized mesenchymal stem cells have a limited proliferative potential,that is,replicative senescence(RS)is an integral characteristic of the life of all mesenchymal stem cells(MSCs).It is known that one of the important signs of RS is a decrease of cell motility,and that violations of migration processes contribute to the deterioration of tissue regeneration.Therefore,the characterization of the properties of the cell line associated with RS is a prerequisite for the effective use of MSCs in restorative medicine.One of the key proteins regulating cell motility is the small GTPase RhoA.The main purpose of this work was to study the nuclear-cytoplasmic redistribution of the RhoA protein during RS in MSC lines recently obtained and characterized in our laboratory.The study found that a comparative analysis of the intracellular localization of RhoA in three cell lines(MSCWJ-1,FetMSC,DF2)showed a decrease in the nuclear localization of RhoA during RS.
基金The grants of National Science Found-ation of China(30970162)Program of International Collaboration of Tianjin Municipal Science and Technology Commission(08ZCGHHZ01800)
文摘Perinatal transmission of Human immunodeficiency virus(HIV),also called mother-to-child transmission(MTCT),accounts for 90% of infections in infants worldwide and occurs in 30%-45% of children born to untreated HIV-1 infected mothers.Among HIV-1 infected mothers,some viruses are transmitted from mothers to their infants while others are not.The relationship between virologic properties and the pathogenesis caused by HIV-1 remains unclear.Previous studies have demonstrated that one obvious source of selective pressure in the perinatal transmission of HIV-1 is maternal neutralizing antibodies.Recent studies have shown that viruses which are successfully transmitted to the child have growth advantages over those not transmitted,when those two viruses are grown together.Furthermore,the higher fitness is determined by the gp120 protein of the virus envelope.This suggests that the selective transmission of viruses with higher fitness occurred exclusively,regardless of transmission routes.There are many factors contributing to the selective transmission and HIV replicative fitness is an important one that should not be neglected.This review summarizes current knowledge of the role of HIV replicative fitness in HIV MTCT transmission and the determinants of viral fitness upon MTCT.
基金This work was supported by International Cooperation Important Project of National Natural Sciences Foundation of China(No. 30120160824) and the State 863 High Technology R&D Project of China (No. 2001AA217031)
文摘Objective： To evaluate the tumor selectivity and therapeutic efficiency of replication-competent adenovirus CNHK300 on human breast cancer cells. Methods： RT-PCR was used to detect the hTERT mRNA activity in various breast cancer and normal fibroblast cell lines. Virus proliferation assay, cell viability assay and Western blot were applied to evaluate the proliferation and cytolysis selectivity of CNHK300. Results： The telomerase activity of MCF-7, BT-549 and SK-BR-3 was positive, while telomerase in MRC-5 and BJ was negative. The progeny virus titers in MCF-7, BT-549 and SK-BR-3 after 48 h of CNHK300 exposure was 40 625, 1 265 and 20 000 fold higher than those of 0 h, even slightly higher than those of wtAd5 （except in SK-BR-3）. ONYX-015 virus proliferation ability was weaker than that of CNHK300 in cancer cells. However, CNHK300 exhibited attenuated replicative ability as compared with wtAd5 in MRC-5 and BJ. The CNHK300 replicatative multiple was 63 and 192 fold at 48 h respectively, while the wtAd5 still multiplied 3 160-4 846 fold. CNHK300 could cause about half of breast cancer cells to die within 7 days at MOI 10 pfu/cell and below, whereas the IC50 in BJ and MRC-5 was as high as MOI 100 pfu/cell. CNHK300 E1A protein could be detected in breast cancer cells and 293 cells but not in normal fibroblast cells. Conclusion： hTERT promoter can successfully modulate the CNHK300 to be selectively replicated in breast cancer cells positive for telomerase, which may be a potential treatment strategy in breast cancer.
文摘OBJECTIVE: To investigate the dynamic alternations of HBV markers of active HBV replicationrecipients receiving lamivudine prophylaxis after liver transplantation.METHODS: Serial liver biopsy samples and sera were obtained from 15 recipients and examined withenzyme-linked radioinmmunoassay for HBsAg, HBeAg, HBsAb, HBcAb and HBeAb, and fluorescentquantitative assay for quantitation of HBV DNA in serum. Immunohistochemical staining of HBsAg,HBcAg and HBV DNA hybridization in situ were used to detect HBV markers in liver biopsy samples.RESULTS: 100 mg lamivudine taken orally every, day for 2 weeks before transplantation enabled 12(80%) of 15 active viral replication recipients (HBV DNA positive) to converse to HBV DNA negative.HBsAb, HBcAb and HBeAb in serum emerged in 1-2 weeks after liver transplantation, and disappearedgradually within 6 months; HBV DNA fluorescent quantitative assay showed constant negativity in serum.Immunohistochemical staining of HBsAg, HBcAg and HBV DNA hybridization in situ in liver biopsysamples showed negative results synchorously. Eight of the 15 HBV active replication recipients lostHBV markers thoroughly both in serology and tissue staining as well as HBV DNA hybridization in situ ofserial liver biopsy samples from 12 to 44 weeks after liver transplantation. Should any of HBsAg, HBeAgin serology and HBsAg, HBcAg in immunohistochemical staining was positive, or HBV DNA detectablein serum, or HBV DNA hybridization in situ in liver tissue positive, allograft HBV reinfection or De novoliver allograft infection could be diagnosed. Furthermore, if associated with elevation of ALT andbilirubin, the diagnosis of HBV hepatitis recurrence could be established.CONCLUSION: Allograft HBV reinfection or De nuvo liver allograft infection in active viral replicationrecipients could be prevented with lamivudine regimen, and further clearance of HBV may be possible ifproper measures are taken.
