AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepa...AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited th展开更多
AIM:To observe the effect of acupuncture and moxibustion on the expression of IL-1beta and IL-6 mRNA in ulcerative colitis rats.METHODS:The SD rat ulcerative colitis model was created by immunological method associate...AIM:To observe the effect of acupuncture and moxibustion on the expression of IL-1beta and IL-6 mRNA in ulcerative colitis rats.METHODS:The SD rat ulcerative colitis model was created by immunological method associated with local stimulation. Colonic mucosa was prepared from human fresh surgical colonic specimens, homogenized by adding appropriate amount of normal saline and centrifuged at 3000r/min. The supernatant was collected for measurement of protein conentration and then mixed with Freund adjuvant. This antigen fluid was first injected into the plantae of the model group rats, and then into their plantae, dorsa, inguina and abdominal cavities (noFreund adjuvant for the last injection) again on the 10th, 17th, 24th and 31st day. When a certain titer of serum anti colonic antibody was reached, 2% formalin and antigen fluid (no Freund adjuvant) were administered separately by enema. The ulcerative colitis rat model was thus set up. The animals were randomly divided into four groups: model control group (MC, n = 8), electro acupuncture group (EA, n = 8), herbs partition moxibustion group (HPM 8), normal control group (NC,n = 8). HPM: Moxa cones made of refined mugwort floss were placed on the medicinal pad (medicinal pad dispensing: Radix Aconiti praeparata, cortex Cinnamomi, etc) for Qihai (RN 6) and Tianshu (ST 25, bilateral) and ignited. Two moxa cones were used for each acupoint once a day and 14 times in all. EA: Tianshu (bilateral) and Qihai were stimulated by the intermittent pulse with 2Hz frequency, 4mA intensity for 20 minutes once a day and 14 times in all. After treatment, rats of all four groups were killed simultaneously. The spleen was separated and the distal colon was dissected. Total tissue RNA was isolated by the guanidinium thiocyanate phenol chloroform extraction method. RT-PCR technique was used to study the expression of IL-1 beta and IL-6 mRNA.RESULTS:IL-1 beta and IL-6 mRNAs were not detected in the spleen and colonic mucosa of the NC rats, whereas they were significantly expressed in 展开更多
Mesoporous silica nanoparticles(MSNs) are attracting increasing interest for potential biomedical applications. With tailored mesoporous structure, huge surface area and pore volume,selective surface functionality, as...Mesoporous silica nanoparticles(MSNs) are attracting increasing interest for potential biomedical applications. With tailored mesoporous structure, huge surface area and pore volume,selective surface functionality, as well as morphology control, MSNs exhibit high loading capacity for therapeutic agents and controlled release properties if modified with stimuli-responsive groups, polymers or proteins. In this review article, the applications of MSNs in pharmaceutics to improve drug bioavailability, reduce drug toxicity, and deliver with cellular targetability are summarized. Particularly,the exciting progress in the development of MSNs-based effective delivery systems for poorly soluble drugs, anticancer agents, and therapeutic genes are highlighted.展开更多
INTRODUCTIONLiver fibrosis or cirrhosis is a common progressively pathological lesion of chronic liver diseases in response to various liver-damaging factors. The main mechanisms of fibrotic or cirrhotic initiation an...INTRODUCTIONLiver fibrosis or cirrhosis is a common progressively pathological lesion of chronic liver diseases in response to various liver-damaging factors. The main mechanisms of fibrotic or cirrhotic initiation and progression at the level of cellular and molecular events have been elucidated in the past two decades[1,2].展开更多
AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cass...AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with co展开更多
Gene and viral therapies for cancer have shown some therapeutic effects, but there has been a lack of real breakthrough. To achieve the goal of complete elimination of tumor xenograft in animal models, we have develop...Gene and viral therapies for cancer have shown some therapeutic effects, but there has been a lack of real breakthrough. To achieve the goal of complete elimination of tumor xenograft in animal models, we have developed a new strategy called Targeting Gene-Virotherapy of Cancer, which aims to combine the advantages of both gene therapy and virotherapy. This new strategy has produced stronger anti-tumor effects than either gene or viral therapy alone. A tumorspecific replicative adenovirus vector, designated as ZD55, was constructed by deletion of the 55kDa E1B region of adenovirus. The resulting viral construct not only retains a similar function to ONYX-015 by specifically targeting p53 negative tumors, but also allows for the insertion of various therapeutic genes to form appropriate ZD55 derivatives due to the newly introduced cloning site, a task not feasible with the original ONYX-015 virus. We showed that the anti-tumor effect of one such derivative, ZD55-IL-24, is at least 100 times more potent than that of either ZD55 virotherapy or Ad-IL-24 gene therapy. Nevertheless, complete elimination of tumor mass by the use of ZD55-1L-24 was only observed in some but not all mice, indicating that one therapeutic gene was not sufficient to ＂cure＂ these mice. When genes with complementary or synergetic effects were separately cloned into the ZD55 vector and used in combination （designated as the Dual Gene Therapy strategy）, much better results were obtained; and it was possible to achieve complete elimination of all the xenograft tumor masses in all mice if two suitable genes were chosen. More comprehensive studies based on this new strategy will likely lead to a protocol for clinical trial. Finally, the concept of Double Controlled Targeting Virus-Dual Gene Therapy for cancer treatment, and the implication of the recent progress in cancer stem cells are also discussed.展开更多
AIM To further investigate the effect of cyclin D1 on the biologic behavior of cancer cells and its potential role in gene therapy of tumor. METHODS A cyclin D1 subcloning plasmid termed BKSD1 was constructed by su...AIM To further investigate the effect of cyclin D1 on the biologic behavior of cancer cells and its potential role in gene therapy of tumor. METHODS A cyclin D1 subcloning plasmid termed BKSD1 was constructed by subcloning the human cyclin D1 cDNA into Bluescript KS, a plasmid vector with a pair of T7 and T3 promoters, with recombinant DNA technology of molecular biology. So, it is easy to generate digoxigenin (DIG) labeled RNA probes of antisense and sense to cyclin D1 using RKSD1 as a template vector. PDORD1AS, an eukaryotic expression vector containing the full length human cyclin D1 cDNA in its antisense orientation cloned into the retroviral vector pDOR neo, was successfully constructed with BKSD1 to change restriction sites. A gastric cancer cell line, SGC7901/VCR, was transfected with pDORD1AS by Lipofect Amine mediated introduction and a subline termed SGC7901/VCRD1AS, which had stable overexpression of antisense RNA to cyclin D1, was obtained by selection in G418. The subline, control subline transfected pDOR neo and SGC7901/VCR were evaluated by methods of immunohistochemistry, flow cytometry, molecular hybridization, morphology and cell biology. RESULTS Compared with control cell lines, SGC7901/VCRD1AS had a reduced expression of cyclin D1 (inhibition rate was about 36%), increased cell size and cytoplasm to nucleus ratio, increased doubling time (42 2h to 26 8h and 26 4h), decreased saturation density (18 9×10 4 to 4 8×10 5 and 4 8×10 5), increased percentage of cells in the G1/G0 phase (80 9%-64 6% and 63 8%), reacquired serum dependence, and a loss of tumorigenicity in nude mice (0/4 to 4/4 and 4/4). CONCLUSION Stable overexpression of antisense RNA to cyclin D1 can reverse the transformed phenotype of human gastric cancer cells and may provide an approach of gene therapy for gastric cancer.展开更多
INTRODUCTION Human tissue homeostasis is precisely regulated bycellular division,differentiation and death.Normalhuman somatic cells progressively lose telomererestriction fragment(TRF)length with eachsuccessive cell ...INTRODUCTION Human tissue homeostasis is precisely regulated bycellular division,differentiation and death.Normalhuman somatic cells progressively lose telomererestriction fragment(TRF)length with eachsuccessive cell division,eventually leading tocellular quiescence,chromosomal end-degradationand apoptosis.On the contrary,stabilization oftelomere lengths by expressing telomerase,an RNA-dependent DNA polymerase,may be involved incellular immortality and carcinogenesis.展开更多
Background Bcl-2, the anti-apoptotic protein is overexpressed in the majority of gastric cancers and associated with its pathogenesis. To better understanding of the role of Bcl-2, RNA interference （RNAi） was used t...Background Bcl-2, the anti-apoptotic protein is overexpressed in the majority of gastric cancers and associated with its pathogenesis. To better understanding of the role of Bcl-2, RNA interference （RNAi） was used to inhibit Bcl-2 expression in the human gastric cancer cells in vitro and in vivo. Methods Bcl-2 small interfering RNA （siRNA） was transfected into human gastric cancer cells $GC-7901, and Bcl-2 expression was monitored by real-time polymerase chain reaction （PCR） and Western blot. Cell proliferation, apoptosis, and telomerase activity were examined by MTT, flow cytometry, and TRAP assay, respectively. Gastric cancer cells treated with 100 nmol/L Bcl-2 siRNA were subcutaneously transplanted into nude mice and tumor growth was assessed. Results Bcl-2 siRNA significantly inhibited the expression of Bcl-2 in human gastric cancer cells at both mRNA and protein levels in a time- and dose-dependent manner. Bcl-2 siRNA also decreased telomerase activity （by 78.76%） and increased the rate of apoptosis （by 37.47%）. SGC-7901 cell growth was also significantly suppressed in vivo and in vitro. Conclusions Bcl-2 expression knockdown suppressed the growth of gastric cancer cells. Thus, Bcl-2 may play a very important role in carcinogenesis of gastric cancer and its knockdown may offer a new potential gene therapy approach for human gastric cancer in future.展开更多
Aim： To determine whether adenoviral gene transfer of insulin like growth factor-1 （IGF-1） to the penis of streptozotocin （STZ）-induced diabetic rats could improve erectile capacity. Methods： The STZ diabetic ra...Aim： To determine whether adenoviral gene transfer of insulin like growth factor-1 （IGF-1） to the penis of streptozotocin （STZ）-induced diabetic rats could improve erectile capacity. Methods： The STZ diabetic rats were transfected with AdCMV-βgal or AdCMV-IGF-1. These rats underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age-matched control rats 1 to 2 days after transfection. In control and transfected STZ diabetic rats, IGF-1 expression were examined by reverse transcription polymerase chain reaction （RT-PCR）, Western blot and histology. The penis β-galactosidase activity and localization of the STZ diabetic rats were also determined. Results： One to two days after transfection, the β-galactosidase was found in the smooth muscle cells of the diabetic rat penis transfected with AdCMV-βgal. One to 2 days after administration of AdCMV- IGF-1, the cavernosal pressure, as determined by the ratio of maximal intracavernous pressure-to-mean arterial pressure （ICP/MAP） and total intracavernous pressure （ICP）, was increased in response to cavernous nerve stimulation. Transgene expression was confirmed by RT-PCR, Western blot and histology. Conclusion： Gene transfer of IGF-1 significantly increased erectile function in the STZ diabetic rats. These results suggest that in vivo gene transfer of IGF- 1 might be a new therapeutic intervention for the treatment of erectile dysfunction （ED） in the STZ diabetic rats.展开更多
With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology,it is anticipated that increasing numbers of therapeutic genes or targets will become ava...With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology,it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies.Despite numerous setbacks,efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases.It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies.There has been a long-lasting interest in using viral vectors,especially adenoviral vectors,to deliver therapeutic genes for the past two decades.Among all currently available viral vectors,adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types.The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development.In fact,among over 2000 gene therapy clinical trials approved worldwide since 1989,a significant portion of the trials have utilized adenoviral vectors.This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors,including adenoviral biology,approaches to engineering adenoviral vectors,and their applications in clinical and preclinical studies with an emphasis in the areas of cancer treatment,vaccination and regenerative medicine.Current challenges and future directions regarding the use of adenoviral vectors are also discussed.It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.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
基金Supported by the Postdoctoral Science Foundation of China(No.1999-10 State Postdoctoral Foundation Commission)
文摘AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited th
基金Supparted by the Ntiona1 Natura1 Science Foundation of China No.39670899.
文摘AIM:To observe the effect of acupuncture and moxibustion on the expression of IL-1beta and IL-6 mRNA in ulcerative colitis rats.METHODS:The SD rat ulcerative colitis model was created by immunological method associated with local stimulation. Colonic mucosa was prepared from human fresh surgical colonic specimens, homogenized by adding appropriate amount of normal saline and centrifuged at 3000r/min. The supernatant was collected for measurement of protein conentration and then mixed with Freund adjuvant. This antigen fluid was first injected into the plantae of the model group rats, and then into their plantae, dorsa, inguina and abdominal cavities (noFreund adjuvant for the last injection) again on the 10th, 17th, 24th and 31st day. When a certain titer of serum anti colonic antibody was reached, 2% formalin and antigen fluid (no Freund adjuvant) were administered separately by enema. The ulcerative colitis rat model was thus set up. The animals were randomly divided into four groups: model control group (MC, n = 8), electro acupuncture group (EA, n = 8), herbs partition moxibustion group (HPM 8), normal control group (NC,n = 8). HPM: Moxa cones made of refined mugwort floss were placed on the medicinal pad (medicinal pad dispensing: Radix Aconiti praeparata, cortex Cinnamomi, etc) for Qihai (RN 6) and Tianshu (ST 25, bilateral) and ignited. Two moxa cones were used for each acupoint once a day and 14 times in all. EA: Tianshu (bilateral) and Qihai were stimulated by the intermittent pulse with 2Hz frequency, 4mA intensity for 20 minutes once a day and 14 times in all. After treatment, rats of all four groups were killed simultaneously. The spleen was separated and the distal colon was dissected. Total tissue RNA was isolated by the guanidinium thiocyanate phenol chloroform extraction method. RT-PCR technique was used to study the expression of IL-1 beta and IL-6 mRNA.RESULTS:IL-1 beta and IL-6 mRNAs were not detected in the spleen and colonic mucosa of the NC rats, whereas they were significantly expressed in
基金financial support from the National Natural Science Foundation of China (81473155)the Natural Science Fund Project of Guangdong Province (Grant No. 2016A030312013)+1 种基金the Science and Technology Plan Projects of Guangdong Province (Grant No. 2015B020232010)the Science and Technology Foundation Guangzhou (201707010103)
文摘Mesoporous silica nanoparticles(MSNs) are attracting increasing interest for potential biomedical applications. With tailored mesoporous structure, huge surface area and pore volume,selective surface functionality, as well as morphology control, MSNs exhibit high loading capacity for therapeutic agents and controlled release properties if modified with stimuli-responsive groups, polymers or proteins. In this review article, the applications of MSNs in pharmaceutics to improve drug bioavailability, reduce drug toxicity, and deliver with cellular targetability are summarized. Particularly,the exciting progress in the development of MSNs-based effective delivery systems for poorly soluble drugs, anticancer agents, and therapeutic genes are highlighted.
文摘INTRODUCTIONLiver fibrosis or cirrhosis is a common progressively pathological lesion of chronic liver diseases in response to various liver-damaging factors. The main mechanisms of fibrotic or cirrhotic initiation and progression at the level of cellular and molecular events have been elucidated in the past two decades[1,2].
基金Project supported by National Natural Science Foundation of China,No.863 Z2001-04
文摘AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with co
文摘Gene and viral therapies for cancer have shown some therapeutic effects, but there has been a lack of real breakthrough. To achieve the goal of complete elimination of tumor xenograft in animal models, we have developed a new strategy called Targeting Gene-Virotherapy of Cancer, which aims to combine the advantages of both gene therapy and virotherapy. This new strategy has produced stronger anti-tumor effects than either gene or viral therapy alone. A tumorspecific replicative adenovirus vector, designated as ZD55, was constructed by deletion of the 55kDa E1B region of adenovirus. The resulting viral construct not only retains a similar function to ONYX-015 by specifically targeting p53 negative tumors, but also allows for the insertion of various therapeutic genes to form appropriate ZD55 derivatives due to the newly introduced cloning site, a task not feasible with the original ONYX-015 virus. We showed that the anti-tumor effect of one such derivative, ZD55-IL-24, is at least 100 times more potent than that of either ZD55 virotherapy or Ad-IL-24 gene therapy. Nevertheless, complete elimination of tumor mass by the use of ZD55-1L-24 was only observed in some but not all mice, indicating that one therapeutic gene was not sufficient to ＂cure＂ these mice. When genes with complementary or synergetic effects were separately cloned into the ZD55 vector and used in combination （designated as the Dual Gene Therapy strategy）, much better results were obtained; and it was possible to achieve complete elimination of all the xenograft tumor masses in all mice if two suitable genes were chosen. More comprehensive studies based on this new strategy will likely lead to a protocol for clinical trial. Finally, the concept of Double Controlled Targeting Virus-Dual Gene Therapy for cancer treatment, and the implication of the recent progress in cancer stem cells are also discussed.
文摘AIM To further investigate the effect of cyclin D1 on the biologic behavior of cancer cells and its potential role in gene therapy of tumor. METHODS A cyclin D1 subcloning plasmid termed BKSD1 was constructed by subcloning the human cyclin D1 cDNA into Bluescript KS, a plasmid vector with a pair of T7 and T3 promoters, with recombinant DNA technology of molecular biology. So, it is easy to generate digoxigenin (DIG) labeled RNA probes of antisense and sense to cyclin D1 using RKSD1 as a template vector. PDORD1AS, an eukaryotic expression vector containing the full length human cyclin D1 cDNA in its antisense orientation cloned into the retroviral vector pDOR neo, was successfully constructed with BKSD1 to change restriction sites. A gastric cancer cell line, SGC7901/VCR, was transfected with pDORD1AS by Lipofect Amine mediated introduction and a subline termed SGC7901/VCRD1AS, which had stable overexpression of antisense RNA to cyclin D1, was obtained by selection in G418. The subline, control subline transfected pDOR neo and SGC7901/VCR were evaluated by methods of immunohistochemistry, flow cytometry, molecular hybridization, morphology and cell biology. RESULTS Compared with control cell lines, SGC7901/VCRD1AS had a reduced expression of cyclin D1 (inhibition rate was about 36%), increased cell size and cytoplasm to nucleus ratio, increased doubling time (42 2h to 26 8h and 26 4h), decreased saturation density (18 9×10 4 to 4 8×10 5 and 4 8×10 5), increased percentage of cells in the G1/G0 phase (80 9%-64 6% and 63 8%), reacquired serum dependence, and a loss of tumorigenicity in nude mice (0/4 to 4/4 and 4/4). CONCLUSION Stable overexpression of antisense RNA to cyclin D1 can reverse the transformed phenotype of human gastric cancer cells and may provide an approach of gene therapy for gastric cancer.
基金the Natural Science Foundation of Gansu Province,China,No.ZS981-A23-086-Y
文摘INTRODUCTION Human tissue homeostasis is precisely regulated bycellular division,differentiation and death.Normalhuman somatic cells progressively lose telomererestriction fragment(TRF)length with eachsuccessive cell division,eventually leading tocellular quiescence,chromosomal end-degradationand apoptosis.On the contrary,stabilization oftelomere lengths by expressing telomerase,an RNA-dependent DNA polymerase,may be involved incellular immortality and carcinogenesis.
基金the grants from Shanghai Medical Key Discipline (No.05Ⅲ005)the National High Technology and Development Program of China(863 Program,No.2002BA7111A06).
文摘Background Bcl-2, the anti-apoptotic protein is overexpressed in the majority of gastric cancers and associated with its pathogenesis. To better understanding of the role of Bcl-2, RNA interference （RNAi） was used to inhibit Bcl-2 expression in the human gastric cancer cells in vitro and in vivo. Methods Bcl-2 small interfering RNA （siRNA） was transfected into human gastric cancer cells $GC-7901, and Bcl-2 expression was monitored by real-time polymerase chain reaction （PCR） and Western blot. Cell proliferation, apoptosis, and telomerase activity were examined by MTT, flow cytometry, and TRAP assay, respectively. Gastric cancer cells treated with 100 nmol/L Bcl-2 siRNA were subcutaneously transplanted into nude mice and tumor growth was assessed. Results Bcl-2 siRNA significantly inhibited the expression of Bcl-2 in human gastric cancer cells at both mRNA and protein levels in a time- and dose-dependent manner. Bcl-2 siRNA also decreased telomerase activity （by 78.76%） and increased the rate of apoptosis （by 37.47%）. SGC-7901 cell growth was also significantly suppressed in vivo and in vitro. Conclusions Bcl-2 expression knockdown suppressed the growth of gastric cancer cells. Thus, Bcl-2 may play a very important role in carcinogenesis of gastric cancer and its knockdown may offer a new potential gene therapy approach for human gastric cancer in future.
文摘Aim： To determine whether adenoviral gene transfer of insulin like growth factor-1 （IGF-1） to the penis of streptozotocin （STZ）-induced diabetic rats could improve erectile capacity. Methods： The STZ diabetic rats were transfected with AdCMV-βgal or AdCMV-IGF-1. These rats underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age-matched control rats 1 to 2 days after transfection. In control and transfected STZ diabetic rats, IGF-1 expression were examined by reverse transcription polymerase chain reaction （RT-PCR）, Western blot and histology. The penis β-galactosidase activity and localization of the STZ diabetic rats were also determined. Results： One to two days after transfection, the β-galactosidase was found in the smooth muscle cells of the diabetic rat penis transfected with AdCMV-βgal. One to 2 days after administration of AdCMV- IGF-1, the cavernosal pressure, as determined by the ratio of maximal intracavernous pressure-to-mean arterial pressure （ICP/MAP） and total intracavernous pressure （ICP）, was increased in response to cavernous nerve stimulation. Transgene expression was confirmed by RT-PCR, Western blot and histology. Conclusion： Gene transfer of IGF-1 significantly increased erectile function in the STZ diabetic rats. These results suggest that in vivo gene transfer of IGF- 1 might be a new therapeutic intervention for the treatment of erectile dysfunction （ED） in the STZ diabetic rats.
基金Research in the authors’laboratories was supported in part by research grants from the National Institutes of Health(AT004418,DE020140 to TCH and RRR)the US Department of Defense(OR130096 to JMW)+3 种基金the Scoliosis Research Society(TCH and MJL)the 973 Program of the Ministry of Science and Technology(MOST)of China(#2011CB707906 to TCH)The reported work was also supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430.
文摘With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology,it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies.Despite numerous setbacks,efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases.It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies.There has been a long-lasting interest in using viral vectors,especially adenoviral vectors,to deliver therapeutic genes for the past two decades.Among all currently available viral vectors,adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types.The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development.In fact,among over 2000 gene therapy clinical trials approved worldwide since 1989,a significant portion of the trials have utilized adenoviral vectors.This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors,including adenoviral biology,approaches to engineering adenoviral vectors,and their applications in clinical and preclinical studies with an emphasis in the areas of cancer treatment,vaccination and regenerative medicine.Current challenges and future directions regarding the use of adenoviral vectors are also discussed.It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.
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.
