Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fer...Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization （IVF） technique. Methods： Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction （PCR） was used to detect HB S and pre-Core/Core （pre-C/C） coding genes both in one- and two-cell embryos. Reverse transcription-PCR （RT-PCR） analysis was used to study the expression of the two genes. Fluorescence in situ hybridization （FISH） analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Results： Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Conclusion： Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.展开更多
AIM: To demonstrate the relationship between H-ras oncogene and hepatocellular carcinoma (HCC) metastasis. METHODS: Activated H-ras oncogene was transfected into SMMC 7721, a cell line derived from human HCC, by calci...AIM: To demonstrate the relationship between H-ras oncogene and hepatocellular carcinoma (HCC) metastasis. METHODS: Activated H-ras oncogene was transfected into SMMC 7721, a cell line derived from human HCC, by calcium phosphate transfection method. Some metastasis-related parameters were detected in vitro, including adhesion assay, migration assay, expression of collagenase IV(c IV ase) and epidermal growth factor receptor (EGFR). RESULTS: The abilities of H-ras-transfected cell clones in adhesion to laminin (LN) or fibronectin (FN), migration, c IV ase secretion increased markedly, and the expression of EGFR elevated moderately. More importantly, these alterations were consistent positively with the expression of p21, the protein product of H-ras oncogene. CONCLUSION: H-ras oncogene could induce the metastatic phenotype of HCC cell in vitro to raise its metastatic potential.展开更多
Background: Corn and soybean meal(SBM) are two of the most common feed ingredients used in pig feeds.However, a variety of antinutritional factors(ANFs) present in corn and SBM can interfere with the bioavailability o...Background: Corn and soybean meal(SBM) are two of the most common feed ingredients used in pig feeds.However, a variety of antinutritional factors(ANFs) present in corn and SBM can interfere with the bioavailability of nutrients and have negative health effects on the pigs. In the present study, two-stage fermentation using Bacillus subtilis followed by Enterococcus faecium was carried out to degrade ANFs and improve the nutritional quality of corn and SBM mixed feed. Furthermore, the microbial composition and in vitro nutrient digestibility of inoculated mixed feed were determined and compared those of the uninoculated controls.Results: During the fermentation process, B. subtilis and lactic acid bacteria(LAB) were the main dominant bacteria in the solid-state fermented inoculated feed, and fermentation produced a large amount of lactic acid(170 mmo L/kg),which resulted in a lower pH(5.0 vs. 6.4) than the fermented uninoculated feed. The amounts of soybean antigenic proteins(β-conglycinin and glycinin) in mixed feed were significantly decreased after first-stage fermentation with B. subtilis. Inoculated mixed feed following two-stage fermentation contained greater concentratioin of crude protein(CP), ash and total phosphorus(P) compared to uninoculated feed, whereas the concentrations of neutral detergent fiber(NDF), hemicellulose and phytate P in fermendted inoculated feed declined(P < 0.05) by 38%, 53%, and 46%,respectively. Notably, the content of trichloroacetic acid soluble protein(TCA-SP), particularly that of small peptides and free amino acids(AA), increased 6.5 fold following two-stage fermentation. There was no difference in the total AA content between fermented inoculated and uninoculated feed. However, aromatic AAs(Phe and Tyr) and Lys in inoculated feed increased, and some polar AAs, including Arg, Asp, and Glu, decreased compared with the uninoculated feed. In vitro dry matter and CP digestibility of inoculated feed improved(P < 0.05) compared with the uninoculated feed.Conclusions: Our results s展开更多
CRISPR/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRN...CRISPR/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.展开更多
OBJECTIVE To study the inhibitory effect of oridonin on the growth of cancer cells. METHODS Fifteen human cancer cell lines were subjected to various concentrations of oridonin in culture medium. The inhibitory rate o...OBJECTIVE To study the inhibitory effect of oridonin on the growth of cancer cells. METHODS Fifteen human cancer cell lines were subjected to various concentrations of oridonin in culture medium. The inhibitory rate of cell growth was measured by the MTT assay, and compared with a negative control and 5-Fu-positive control. RESULTS The 50% inhibiting concentration （IC50） and maximal inhibition （Imax） of oridonin shown by studying the growth of the cancer cell lines were as follows： leukemias （HL60 cells： 3.9 μg/ml and 73.8%, K562 cells： 4.3 μg/ml and 76.2%）; esophageal cancers（SHEEC cells： 15.4 μg/ml and 99.2%, Eca109 cells： 15.1 μg/ml and 84.6%, TEl cells： 4.0 μg/ml and 70.2%）; gastric cancers （BGC823 cells： 7.6 μg/ml and 98.7%, SGC7901 cells： 12.3 μg/ml and 85.7%）; colon cancers （HT29 cells： 13.6 μg/ml and 97.2%, HCT cells： 14.5 μg/ml and 96.5%）; liver cancers （Bel7402 cells： 15.2 μg/ml and 89.2%, HepG2 cells： 7.1 μg/ml and 88.3%）; pancreatic cancer （PC3 cells： 11.3 μg/ml and 68.4%）; lung cancer （A549 cells： 18.6 μg/ml and 98.0% ）; breast cancer （MCF7 cells： 18.4 μg/ml and 84.7%）; uterine cervix cancer （Hela cells： 13.7 μg/ml and 98.5%）. CONCLUSION Oridonin had a relatively wide anti-tumor spectrum, and a relatively strong inhibitory effect on the growth of the 15 human cancer cells. Inhibitory effects were concentration dependent.展开更多
The aim of the current study was to investigate the effects of a lactic acid bacteria inoculant（Lactobacillus plantarum, LP）, fibrolytic enzyme（EN）, combination of LP and EN（LP＋EN） on fermentation quality, nutr...The aim of the current study was to investigate the effects of a lactic acid bacteria inoculant（Lactobacillus plantarum, LP）, fibrolytic enzyme（EN）, combination of LP and EN（LP＋EN） on fermentation quality, nutritive characteristics and in vitro digestibility of total mixed ration（TMR） silages containing 0, 7.5 and 15.0%（on dry matter basis） of rape（Brassica campestris L.） straw（RS）（denoted as CTMR, LTMR and MTMR, respectively）. After ensiling for 60 days, TMR silages without additives were well preserved, but MTMR had higher p H than CTMR and LTMR. There were no differences in other parameters of fermentation quality, microbial composition, nutrition and in vitro digestibility between CTMR and LTMR except for yeast and mold number and crude protein（CP） content. CTMR and LTMR silage had higher CP content, in vitro neutral detergent fiber digestibility（IVNDFD） and in vitro acid detergent fiber digestibility（IVADFD）, lower acid detergent fiber（ADF） content than MTMR silage. LP and EN decreased p H and increased dry matter（DM） recovery of TMR silages. LP＋EN improved the fermentation quality, nutritive characteristics and in vitro digestibility of TMR silages, showed by lower p H, ADF content, higher lactic acid content, in vitro dry matter digestibility（IVDMD） and IVNDFD. Therefore, It was suggested that TMR silage contained 7.5% RS on a DM basis and treated with LP＋EN can be as a useful feed for ruminant.展开更多
Infertility in humans is surprisingly common occurring in approximately 15% of the population wishing to start a family. Despite this, the molecular and genetic factors underlying the cause of infertility remain large...Infertility in humans is surprisingly common occurring in approximately 15% of the population wishing to start a family. Despite this, the molecular and genetic factors underlying the cause of infertility remain largely undiscovered. Nevertheless, more and more genetic factors associated with infertility are being identified. This review will focus on our current understanding of the chromosomal basis of male infertility specifically： chromosomal aneuploidy, structural and numerical karyotype abnormalities and Y chromosomal microdeletions. Chromosomal aneuploidy is the leading cause of pregnancy loss and developmental disabilities in humans. Aneuploidy is predominantly maternal in origin, but concerns have been raised regarding the safety of intracytoplasmic sperm injection as infertile men have significantly higher levels of sperm aneuploidy compared to their fertile counterparts. Males with numerical or structural karyotype abnormalities are also at an increased risk of producing aneuploid sperm. Our current understanding of how sperm aneuploidy translates to embryo aneuploidy will be reviewed, as well as the application of preimplantation genetic diagnosis （PGD） in such cases. Clinical recommendations where possible will be made, as well as discussion of the use of emerging array technology in PGD and its potential applications in male infertility.展开更多
AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patie...AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.展开更多
Background Endometriosis affects natural fertility through various approaches, and in vitro fertilization （IVF） is a good treatment. But the IVF result of endometriosis patients is still under debate. We investigate...Background Endometriosis affects natural fertility through various approaches, and in vitro fertilization （IVF） is a good treatment. But the IVF result of endometriosis patients is still under debate. We investigated the effect of endometriosis on IVF by analyzing the data from a single reproductive center. Methods A retrospective, database-searched cohort study was performed. Relevant information was collected from the electronic records of women who underwent IVF/intracytoplasmic sperm injection between January 2006 and December 2010 in the Assisted Reproductive Unit of Sir Run Run Shaw Hospital. Patients with endometriosis were enrolled the study group. The rest of the women formed the control group. The main outcome was the clinical pregnancy rate. Secondary outcomes were oocytes retrieved number, fertilization rate, high-quality embryo rate, number of high-quality embryo for embryo transplantation, and implantation embryo/high-quality embryo ratio （IE/HQE ratio）. Comparisons were performed by the z2-test and independent t-test. Results The endometriosis group （n=177） had a markedly lower oocytes retrieved number, fertilization rate, implantation rate, and clinical pregnancy rate （7.6_＋5.1, 63.6%, 27.7%, and 45.2%, respectively） compared with the non-endometriosis group （n=4267; 11.8＋7.3, 68.4%, 36.2%, and 55.2%, respectively）. Stratified analysis showed that this difference was found in the subgroup younger than 35-years old, while only fertilization rate and implantation rate were different in the elder subgroup. The ratio of high-quality embryos transferred is lower in endometriosis group （53.7% vs. 71.8%, P 〈0.05）, but there is no difference in IE/HQE ratio between two groups. There is no significant difference in fertilization rate, implantation rate, and clinical pregnancy rate between mild and severe endometriosis patients. Conclusions Endometriosis patients suffer a decreasing IVF pregnancy rates mainly caused by reducing oocytes number and fertilization rate, regardless 展开更多
Background:With the development of assisted reproductive technology(ART)and its increasing success rate in the mainland of China,more attention has been paid to the safety of ART.In this study,we explored the associat...Background:With the development of assisted reproductive technology(ART)and its increasing success rate in the mainland of China,more attention has been paid to the safety of ART.In this study,we explored the associations between conception by ART and pregnancy/perinatal complications,and neonatal outcomes compared with similar outcomes following spontaneous conception.Methods:This retrospective cohort study of pregnancies over a 3-year period(2013-2015)was performed at Beijing Obstetrics and Gynecology Hospital,Beijing,China.Subjects were divided into two groups:conception by ART(n=2256)or spontaneous conception(n=6768).According to different fertilization modes,the ART group was divided into in vitro fertilization(IVF,n=1873)and intracytoplasmic sperm injection(ICSI,n=383)subgroups.The ART group was also divided into two different embryo transfer methods;fresh embryo transfer(ET,n=1583)and frozen embryo transfer(FET,n=673)subgroups.Pregnancy complications,perinatal complications,and neonatal outcomes of the enrolled subjects were investigated and analyzed by univariate analysis and multivariate logistic regression.Results:After adjustment for maternal age,gravidity,parity,maternal education,smoking,alcohol consumption,and body mass index(BMI),pregnancies conceived by ART were associated with a significantly increased incidence of gestational diabetes mellitus(GDM;OR 1.88,95%CI 1.56-2.27),gestational hypertension(OR 2.18,95%CI 1.83-2.60),and intrahepatic cholestasis of pregnancy(ICP)(OR 2.79,95%CI 2.15-3.64),compared with spontaneous conception.These associations were similar for the singleton group.In the twin group,only the incidence of ICP was significantly higher than in controls.We found that pregnancies conceived by ART were associated with perinatal complications,including placental abruption(OR 2.14,95%CI 1.33-3.45),premature rupture of membranes(PROM;OR 1.24,95%CI 1.06-1.45),postpartum hemorrhage(OR 2.89,95%CI 2.33-3.59)and polyhydramnios(OR 2.01,95%CI 1.29-3.16).The singleton group had a similar result展开更多
Objective： To compare the clinical effect of Chinese medicine （CM） and Western medicine （WM） for controlling the recurrence of pelvic endometriosis after a conservative operation. Methods： The study was a multi-...Objective： To compare the clinical effect of Chinese medicine （CM） and Western medicine （WM） for controlling the recurrence of pelvic endometriosis after a conservative operation. Methods： The study was a multi-center, randomized, parallel controlled and prospective clinical trial. Patients were randomly divided into two groups： CM group （106 cases） and WM group （102 cases）. Drugs were given to patients during 1--5 days of the first menstruation after a conservative operation in both groups. Patients with stages I and I1 （revised American Fertility Society） were treated for 3 months, while the patients with stages Ill and IV were treated for 6 months. The patients in the CM group were treated using three types of Chinese herbal medicine based on syndrome differentiation. Patients in the WM group were treated using gonadotropin releasing hormone agonist （GnRH-a） or gestrinone. Patients treated with GnRH-a received add-back therapy of Tibolone Tablets once a day after 4 months of treatment. Any cases of dysmenorrheal chronic pelvic pain, menstruation and any adverse reactions of patients were recorded once a month during the preoperative and postoperative periods and once every 3 months during the follow-up period. During the preoperative, postoperative and the follow-up periods, patients underwent type B ultrasonography of the pelvis and measurements of serum CA125 levels, gynecologic examination, routine evaluations of blood, urine, hepatic function （glutamate pyruvate transaminase）, renal function （blood urea nitrogen） and electrocardiograms. Dudng the follow-up period they underwent type B pelvic ultrasonography, measurement of serum CA125 levels and further gynecologic examinations. The two treatments were compared for clinical recurrence rates, pregnancy rates and the incidence of adverse reactions. Results： The incidence and timing of recurrence of endometriosis were not significantly different between the two groups. The first pregnancy achieved by the patient in the 展开更多
This is a retrospective,observational study to evaluate the effect of maternal age on the outcomes of in vitro fertilization and embryo transfer(IVF-ET).11830 IVF-ET cycles from 10268 women were included.Four groups o...This is a retrospective,observational study to evaluate the effect of maternal age on the outcomes of in vitro fertilization and embryo transfer(IVF-ET).11830 IVF-ET cycles from 10268 women were included.Four groups of different maternal age periods were compared.The groups were 21 30 years old group(4549 cycles),31-35 years old group(4424 cycles),36-40 years old group(2429 cycles),and over 40 years old group(428 cycles).The mean starting dose of Gn and mean total dose of Gn in each cycle were significantly higher(P<0.01),while the mean retrieved oocyte number was significantly lower(P<0.01) in groups of higher maternal age period than those in each of the lower groups.The biochemical pregnancy rate and the clinical pregnancy rate were significantly lower(P<0.01),while the miscarriage rate was significantly higher(P<0.01) in groups of higher maternal age period than those in the lower groups.No difference was found in two-pronuclear zygotes(2PN) rate and good quality embryo rate among different groups.Birth defect rate was also comparable in the born babies in different groups.In the group with patients' age over 40 years old,the pregnancy rate was 26.87%,the clinical pregnancy rate was 19.39%,while the miscarriage rate after clinical pregnancy was 36.14%.To draw the conclusion,patients with higher maternal age had worse IVF outcomes.In women of fertile age,patients between 20 and 30 years old have the best IVF outcomes.Patients over 40 years old have poor IVF outcome and high miscarriage rate,which suggested the necessity of preimplantation genetic screening(PGS).展开更多
AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by i...AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.展开更多
OBJECTIVE： To determine the cardioprotective ef- fect of magnesium lithospermate B （MLB） on myo- cardial ischemia/reperfusion （MI/R） injury and to in- vestigate the antioxidant potential in vivo and in vitro. MET...OBJECTIVE： To determine the cardioprotective ef- fect of magnesium lithospermate B （MLB） on myo- cardial ischemia/reperfusion （MI/R） injury and to in- vestigate the antioxidant potential in vivo and in vitro. METHODS： MI/R injury was induced by the occlu- sion of left anterior descending coronary artery for 30 min followed by reperfusion for 3 h in rats. After reperfusion, hearts were harvested to assess infarct size, histopathological damages, the levels of superoxide dismutase （SOD）, catalase （CAT）, glutathione peroxidase （GPx）, reduced glutathione （GSH） and malondialdehyde （MDA）. Blood samples were collected to determine serum levels of creatine kinase-MB （CK-MB）, cardiac troponin （cTnl） and lactate dehydrogenase （LDH）. Furthermore, simulatedischemia/reperfusion （SI/R） injury in vitro was established by oxygen and glucose deprivation （OGD） for 2 h followed by 24-hour recovery period in cardiomyocytes. The activity of LDH in the cultured su- pernatant and the levels of intracellular reactive oxygen species （ROS）, SOD and MDA in cardiomyo- cytes were also measured. Finally, cardiomyocytes apoptosis was determined with flow cytometry. RESULTS： MLB significantly limited infarct size, ameliorated histopathological damages and prevented leakage of CK-MB, cTnl and LDH. Additional- ly, SOD, CAT, GPx and GSH activities were notably increased by MLB, along with the MDA content decreased as compared with the model group in rats. In vitro study, MLB also decreased LDH activity in the cultured supernatant, increased SOD activity in cardiomyocytes, reduced intracellular ROS and MDA levels, and significantly suppressed cardiomyocytes apoptosis. CONCLUSION： MLB possessed remarkably cardioprotective effects on MI/R injury in vivo and in vitro. The protection of MLB may contribute to its antioxidant properties.展开更多
基金Acknowledgment This work was supported by a grant from the National Nature Science Foundation of China （No. 39970374）. The authors wish to thank Professor Yi-Pong Hu, Second Military Medical University of China, for his kindness in providing us the recombinant plasmid （pBR322-HBV）. We wish to thank Mr. Michael Talion of Shantou University Medical College, English Language Training Section for his assistance in proofreading this manuscript. We gratefully acknowledge the support of the leaders of Shantou University Medical College.
文摘Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization （IVF） technique. Methods： Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction （PCR） was used to detect HB S and pre-Core/Core （pre-C/C） coding genes both in one- and two-cell embryos. Reverse transcription-PCR （RT-PCR） analysis was used to study the expression of the two genes. Fluorescence in situ hybridization （FISH） analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Results： Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Conclusion： Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.
文摘AIM: To demonstrate the relationship between H-ras oncogene and hepatocellular carcinoma (HCC) metastasis. METHODS: Activated H-ras oncogene was transfected into SMMC 7721, a cell line derived from human HCC, by calcium phosphate transfection method. Some metastasis-related parameters were detected in vitro, including adhesion assay, migration assay, expression of collagenase IV(c IV ase) and epidermal growth factor receptor (EGFR). RESULTS: The abilities of H-ras-transfected cell clones in adhesion to laminin (LN) or fibronectin (FN), migration, c IV ase secretion increased markedly, and the expression of EGFR elevated moderately. More importantly, these alterations were consistent positively with the expression of p21, the protein product of H-ras oncogene. CONCLUSION: H-ras oncogene could induce the metastatic phenotype of HCC cell in vitro to raise its metastatic potential.
基金supported by a China Pig Modern Industrial Technology System Grant(CARS-36),the ChinaZhejiang province Postdoctoral Science Foundation(518000-X91604,518000-X81601)
文摘Background: Corn and soybean meal(SBM) are two of the most common feed ingredients used in pig feeds.However, a variety of antinutritional factors(ANFs) present in corn and SBM can interfere with the bioavailability of nutrients and have negative health effects on the pigs. In the present study, two-stage fermentation using Bacillus subtilis followed by Enterococcus faecium was carried out to degrade ANFs and improve the nutritional quality of corn and SBM mixed feed. Furthermore, the microbial composition and in vitro nutrient digestibility of inoculated mixed feed were determined and compared those of the uninoculated controls.Results: During the fermentation process, B. subtilis and lactic acid bacteria(LAB) were the main dominant bacteria in the solid-state fermented inoculated feed, and fermentation produced a large amount of lactic acid(170 mmo L/kg),which resulted in a lower pH(5.0 vs. 6.4) than the fermented uninoculated feed. The amounts of soybean antigenic proteins(β-conglycinin and glycinin) in mixed feed were significantly decreased after first-stage fermentation with B. subtilis. Inoculated mixed feed following two-stage fermentation contained greater concentratioin of crude protein(CP), ash and total phosphorus(P) compared to uninoculated feed, whereas the concentrations of neutral detergent fiber(NDF), hemicellulose and phytate P in fermendted inoculated feed declined(P < 0.05) by 38%, 53%, and 46%,respectively. Notably, the content of trichloroacetic acid soluble protein(TCA-SP), particularly that of small peptides and free amino acids(AA), increased 6.5 fold following two-stage fermentation. There was no difference in the total AA content between fermented inoculated and uninoculated feed. However, aromatic AAs(Phe and Tyr) and Lys in inoculated feed increased, and some polar AAs, including Arg, Asp, and Glu, decreased compared with the uninoculated feed. In vitro dry matter and CP digestibility of inoculated feed improved(P < 0.05) compared with the uninoculated feed.Conclusions: Our results s
文摘CRISPR/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.
基金the grant form the Guangdong Science and Technology De-partment (No. 2006B35630009).
文摘OBJECTIVE To study the inhibitory effect of oridonin on the growth of cancer cells. METHODS Fifteen human cancer cell lines were subjected to various concentrations of oridonin in culture medium. The inhibitory rate of cell growth was measured by the MTT assay, and compared with a negative control and 5-Fu-positive control. RESULTS The 50% inhibiting concentration （IC50） and maximal inhibition （Imax） of oridonin shown by studying the growth of the cancer cell lines were as follows： leukemias （HL60 cells： 3.9 μg/ml and 73.8%, K562 cells： 4.3 μg/ml and 76.2%）; esophageal cancers（SHEEC cells： 15.4 μg/ml and 99.2%, Eca109 cells： 15.1 μg/ml and 84.6%, TEl cells： 4.0 μg/ml and 70.2%）; gastric cancers （BGC823 cells： 7.6 μg/ml and 98.7%, SGC7901 cells： 12.3 μg/ml and 85.7%）; colon cancers （HT29 cells： 13.6 μg/ml and 97.2%, HCT cells： 14.5 μg/ml and 96.5%）; liver cancers （Bel7402 cells： 15.2 μg/ml and 89.2%, HepG2 cells： 7.1 μg/ml and 88.3%）; pancreatic cancer （PC3 cells： 11.3 μg/ml and 68.4%）; lung cancer （A549 cells： 18.6 μg/ml and 98.0% ）; breast cancer （MCF7 cells： 18.4 μg/ml and 84.7%）; uterine cervix cancer （Hela cells： 13.7 μg/ml and 98.5%）. CONCLUSION Oridonin had a relatively wide anti-tumor spectrum, and a relatively strong inhibitory effect on the growth of the 15 human cancer cells. Inhibitory effects were concentration dependent.
基金supported by the Natural Science Foundation of the Jiangsu Province for Young Scholars,China(BK20130694)the Specialized Research Fund for the Doctoral Program of Higher Education,China(20130097120053)+1 种基金the National Natural Science Foundation of China(31502014)the Project of Jiangsu Independent Innovation,China(CX(15)1003)
文摘The aim of the current study was to investigate the effects of a lactic acid bacteria inoculant（Lactobacillus plantarum, LP）, fibrolytic enzyme（EN）, combination of LP and EN（LP＋EN） on fermentation quality, nutritive characteristics and in vitro digestibility of total mixed ration（TMR） silages containing 0, 7.5 and 15.0%（on dry matter basis） of rape（Brassica campestris L.） straw（RS）（denoted as CTMR, LTMR and MTMR, respectively）. After ensiling for 60 days, TMR silages without additives were well preserved, but MTMR had higher p H than CTMR and LTMR. There were no differences in other parameters of fermentation quality, microbial composition, nutrition and in vitro digestibility between CTMR and LTMR except for yeast and mold number and crude protein（CP） content. CTMR and LTMR silage had higher CP content, in vitro neutral detergent fiber digestibility（IVNDFD） and in vitro acid detergent fiber digestibility（IVADFD）, lower acid detergent fiber（ADF） content than MTMR silage. LP and EN decreased p H and increased dry matter（DM） recovery of TMR silages. LP＋EN improved the fermentation quality, nutritive characteristics and in vitro digestibility of TMR silages, showed by lower p H, ADF content, higher lactic acid content, in vitro dry matter digestibility（IVDMD） and IVNDFD. Therefore, It was suggested that TMR silage contained 7.5% RS on a DM basis and treated with LP＋EN can be as a useful feed for ruminant.
文摘Infertility in humans is surprisingly common occurring in approximately 15% of the population wishing to start a family. Despite this, the molecular and genetic factors underlying the cause of infertility remain largely undiscovered. Nevertheless, more and more genetic factors associated with infertility are being identified. This review will focus on our current understanding of the chromosomal basis of male infertility specifically： chromosomal aneuploidy, structural and numerical karyotype abnormalities and Y chromosomal microdeletions. Chromosomal aneuploidy is the leading cause of pregnancy loss and developmental disabilities in humans. Aneuploidy is predominantly maternal in origin, but concerns have been raised regarding the safety of intracytoplasmic sperm injection as infertile men have significantly higher levels of sperm aneuploidy compared to their fertile counterparts. Males with numerical or structural karyotype abnormalities are also at an increased risk of producing aneuploid sperm. Our current understanding of how sperm aneuploidy translates to embryo aneuploidy will be reviewed, as well as the application of preimplantation genetic diagnosis （PGD） in such cases. Clinical recommendations where possible will be made, as well as discussion of the use of emerging array technology in PGD and its potential applications in male infertility.
基金Suppprted by the Mational Natural Science Foundation of China,No.39670672.
文摘AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.
基金This study was supported by a grant from the Natural Science Foundation of Zhejiang(No.Y2080505).
文摘Background Endometriosis affects natural fertility through various approaches, and in vitro fertilization （IVF） is a good treatment. But the IVF result of endometriosis patients is still under debate. We investigated the effect of endometriosis on IVF by analyzing the data from a single reproductive center. Methods A retrospective, database-searched cohort study was performed. Relevant information was collected from the electronic records of women who underwent IVF/intracytoplasmic sperm injection between January 2006 and December 2010 in the Assisted Reproductive Unit of Sir Run Run Shaw Hospital. Patients with endometriosis were enrolled the study group. The rest of the women formed the control group. The main outcome was the clinical pregnancy rate. Secondary outcomes were oocytes retrieved number, fertilization rate, high-quality embryo rate, number of high-quality embryo for embryo transplantation, and implantation embryo/high-quality embryo ratio （IE/HQE ratio）. Comparisons were performed by the z2-test and independent t-test. Results The endometriosis group （n=177） had a markedly lower oocytes retrieved number, fertilization rate, implantation rate, and clinical pregnancy rate （7.6_＋5.1, 63.6%, 27.7%, and 45.2%, respectively） compared with the non-endometriosis group （n=4267; 11.8＋7.3, 68.4%, 36.2%, and 55.2%, respectively）. Stratified analysis showed that this difference was found in the subgroup younger than 35-years old, while only fertilization rate and implantation rate were different in the elder subgroup. The ratio of high-quality embryos transferred is lower in endometriosis group （53.7% vs. 71.8%, P 〈0.05）, but there is no difference in IE/HQE ratio between two groups. There is no significant difference in fertilization rate, implantation rate, and clinical pregnancy rate between mild and severe endometriosis patients. Conclusions Endometriosis patients suffer a decreasing IVF pregnancy rates mainly caused by reducing oocytes number and fertilization rate, regardless
文摘Background:With the development of assisted reproductive technology(ART)and its increasing success rate in the mainland of China,more attention has been paid to the safety of ART.In this study,we explored the associations between conception by ART and pregnancy/perinatal complications,and neonatal outcomes compared with similar outcomes following spontaneous conception.Methods:This retrospective cohort study of pregnancies over a 3-year period(2013-2015)was performed at Beijing Obstetrics and Gynecology Hospital,Beijing,China.Subjects were divided into two groups:conception by ART(n=2256)or spontaneous conception(n=6768).According to different fertilization modes,the ART group was divided into in vitro fertilization(IVF,n=1873)and intracytoplasmic sperm injection(ICSI,n=383)subgroups.The ART group was also divided into two different embryo transfer methods;fresh embryo transfer(ET,n=1583)and frozen embryo transfer(FET,n=673)subgroups.Pregnancy complications,perinatal complications,and neonatal outcomes of the enrolled subjects were investigated and analyzed by univariate analysis and multivariate logistic regression.Results:After adjustment for maternal age,gravidity,parity,maternal education,smoking,alcohol consumption,and body mass index(BMI),pregnancies conceived by ART were associated with a significantly increased incidence of gestational diabetes mellitus(GDM;OR 1.88,95%CI 1.56-2.27),gestational hypertension(OR 2.18,95%CI 1.83-2.60),and intrahepatic cholestasis of pregnancy(ICP)(OR 2.79,95%CI 2.15-3.64),compared with spontaneous conception.These associations were similar for the singleton group.In the twin group,only the incidence of ICP was significantly higher than in controls.We found that pregnancies conceived by ART were associated with perinatal complications,including placental abruption(OR 2.14,95%CI 1.33-3.45),premature rupture of membranes(PROM;OR 1.24,95%CI 1.06-1.45),postpartum hemorrhage(OR 2.89,95%CI 2.33-3.59)and polyhydramnios(OR 2.01,95%CI 1.29-3.16).The singleton group had a similar result
基金Supported by the Key Projects of the National Science andTechnology Pillar Program during the 11th Five-Year PlanPeriod(No.2006BA104A)
文摘Objective： To compare the clinical effect of Chinese medicine （CM） and Western medicine （WM） for controlling the recurrence of pelvic endometriosis after a conservative operation. Methods： The study was a multi-center, randomized, parallel controlled and prospective clinical trial. Patients were randomly divided into two groups： CM group （106 cases） and WM group （102 cases）. Drugs were given to patients during 1--5 days of the first menstruation after a conservative operation in both groups. Patients with stages I and I1 （revised American Fertility Society） were treated for 3 months, while the patients with stages Ill and IV were treated for 6 months. The patients in the CM group were treated using three types of Chinese herbal medicine based on syndrome differentiation. Patients in the WM group were treated using gonadotropin releasing hormone agonist （GnRH-a） or gestrinone. Patients treated with GnRH-a received add-back therapy of Tibolone Tablets once a day after 4 months of treatment. Any cases of dysmenorrheal chronic pelvic pain, menstruation and any adverse reactions of patients were recorded once a month during the preoperative and postoperative periods and once every 3 months during the follow-up period. During the preoperative, postoperative and the follow-up periods, patients underwent type B ultrasonography of the pelvis and measurements of serum CA125 levels, gynecologic examination, routine evaluations of blood, urine, hepatic function （glutamate pyruvate transaminase）, renal function （blood urea nitrogen） and electrocardiograms. Dudng the follow-up period they underwent type B pelvic ultrasonography, measurement of serum CA125 levels and further gynecologic examinations. The two treatments were compared for clinical recurrence rates, pregnancy rates and the incidence of adverse reactions. Results： The incidence and timing of recurrence of endometriosis were not significantly different between the two groups. The first pregnancy achieved by the patient in the
文摘This is a retrospective,observational study to evaluate the effect of maternal age on the outcomes of in vitro fertilization and embryo transfer(IVF-ET).11830 IVF-ET cycles from 10268 women were included.Four groups of different maternal age periods were compared.The groups were 21 30 years old group(4549 cycles),31-35 years old group(4424 cycles),36-40 years old group(2429 cycles),and over 40 years old group(428 cycles).The mean starting dose of Gn and mean total dose of Gn in each cycle were significantly higher(P<0.01),while the mean retrieved oocyte number was significantly lower(P<0.01) in groups of higher maternal age period than those in each of the lower groups.The biochemical pregnancy rate and the clinical pregnancy rate were significantly lower(P<0.01),while the miscarriage rate was significantly higher(P<0.01) in groups of higher maternal age period than those in the lower groups.No difference was found in two-pronuclear zygotes(2PN) rate and good quality embryo rate among different groups.Birth defect rate was also comparable in the born babies in different groups.In the group with patients' age over 40 years old,the pregnancy rate was 26.87%,the clinical pregnancy rate was 19.39%,while the miscarriage rate after clinical pregnancy was 36.14%.To draw the conclusion,patients with higher maternal age had worse IVF outcomes.In women of fertile age,patients between 20 and 30 years old have the best IVF outcomes.Patients over 40 years old have poor IVF outcome and high miscarriage rate,which suggested the necessity of preimplantation genetic screening(PGS).
基金Supported by the National Postdoctoral Science Foundation of China,No.199711.
文摘AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.
基金Supported by the National Natural Science Foundation of China (No. 81173514,No.81001673)the Xijing Research Boosting Program (No. XJZT10D02)the Excellent Civil Service Training Fund of Fourth Military Medical University(No. 4138C4IDK6)
文摘OBJECTIVE： To determine the cardioprotective ef- fect of magnesium lithospermate B （MLB） on myo- cardial ischemia/reperfusion （MI/R） injury and to in- vestigate the antioxidant potential in vivo and in vitro. METHODS： MI/R injury was induced by the occlu- sion of left anterior descending coronary artery for 30 min followed by reperfusion for 3 h in rats. After reperfusion, hearts were harvested to assess infarct size, histopathological damages, the levels of superoxide dismutase （SOD）, catalase （CAT）, glutathione peroxidase （GPx）, reduced glutathione （GSH） and malondialdehyde （MDA）. Blood samples were collected to determine serum levels of creatine kinase-MB （CK-MB）, cardiac troponin （cTnl） and lactate dehydrogenase （LDH）. Furthermore, simulatedischemia/reperfusion （SI/R） injury in vitro was established by oxygen and glucose deprivation （OGD） for 2 h followed by 24-hour recovery period in cardiomyocytes. The activity of LDH in the cultured su- pernatant and the levels of intracellular reactive oxygen species （ROS）, SOD and MDA in cardiomyo- cytes were also measured. Finally, cardiomyocytes apoptosis was determined with flow cytometry. RESULTS： MLB significantly limited infarct size, ameliorated histopathological damages and prevented leakage of CK-MB, cTnl and LDH. Additional- ly, SOD, CAT, GPx and GSH activities were notably increased by MLB, along with the MDA content decreased as compared with the model group in rats. In vitro study, MLB also decreased LDH activity in the cultured supernatant, increased SOD activity in cardiomyocytes, reduced intracellular ROS and MDA levels, and significantly suppressed cardiomyocytes apoptosis. CONCLUSION： MLB possessed remarkably cardioprotective effects on MI/R injury in vivo and in vitro. The protection of MLB may contribute to its antioxidant properties.
