To construct a single tetracycline-regulatable plasmid vector based on the double tetracycline-regulatable plasmid vector system for regulating HSV-tk gene expression so as to effectively kill HeLa cells. Two tetracyc...To construct a single tetracycline-regulatable plasmid vector based on the double tetracycline-regulatable plasmid vector system for regulating HSV-tk gene expression so as to effectively kill HeLa cells. Two tetracycline operator（TetO2） was cloned into pcDNA3.1 and a cassette was made for a cytomegalovirus-type 2 tetracycline operator（CMV-TetO2） promoter, and the obtained vector was named pcDNA3.1-CMV-TetO2. Herpes simplex virus thymidine kinase（HSV-tk） gene and tetracycline repressor（TR） gene were cloned into pcDNA3.1-CMV-TetO2 and the two genes were linked with internal ribosome entry site（IRES） to gain a vector named pcDNA3.1-CMV-TetO2- HSV-tk-IRES-TR. The HeLa cells were stablly transfected with pcDNA3.1-CMV-TetO2-HSV-tk-IRES-TR plasmid. The expression of HSV-tk and TR were detected by RToPCR, the tumorcidal activity of HSV-tk/GCV was determined by MTT assay. In Hela cells transfected with the above plasmid vector, HSV-tk gene and TR gene can be expressed lowly and the concentration of GCV producing a 50% decrease in cell viability was about 50 lag/mL without adding deoxycycline; in contrast, the expessions of HSV-tk gene and TR gene increased significantly and the concentration of GCV producing a 50% decrease in cell viability was about 5 μg/mL with adding deoxycycline. Therefore tetracycline can regulate the expression and tumorcidal activity of HSV-tk gene in HeLa cells with this single plasmid vector.展开更多
Objective: To investigate the synergistic anti-tumor effects of murine IL-12 gene and HSV-TK gene therapy in mice bearing liver cancer. Methods: Mouse liver cancer MM45T. Li (H-2d) cells were transfected with retrovi...Objective: To investigate the synergistic anti-tumor effects of murine IL-12 gene and HSV-TK gene therapy in mice bearing liver cancer. Methods: Mouse liver cancer MM45T. Li (H-2d) cells were transfected with retroviral vector containing IL-12 gene or HSV-TK gene insert. Gene-modified liver cancer cells, MM45T. Li/IL-12 and MM45T. Li/TK, with stable expression of IL-12 and TK were obtained. Balb/c mice were inoculated subcutaneously with 2′105 MM45T. Li cells. When the tumor reached a size of 0.5-1.0 cm, a mixture of MM45T.Li/TK cells and 60Co-irradiated MM45T. Li/IL-12 cell were injected intratumoraly. Ganciclovir (GCV) was injected ip (40 mg.kg-1.d-1) for 10 days. Intratumoral injection of 60Co-irradiated MM45T. Li/IL-12 cells was repeated twice in one week apart. Mice with distant tumors were treated according to the same protocol. CTL activity of spleen cells was measured by 51Cr-release assay and phenotype of tumor infiltrating lymphocytes by immunohistochemical staining. Results: In mice treated with MM45T. Li/IL-12 or MM45T. Li/TK+GCV individually led to moderate reduction in tumor growth, but neither could eradicate the tumor completely, while in 60% of mice treated with a mixture of MM45T. Li/IL-12 and MM45T. Li/TK cells plus GCV, complete tumor regression was observed, with no tumor recurrence for two months. The growth of distant tumor was also inhibited significantly in mice similarly treated. Most of the mice received combined gene therapy plus GCV had abundant CD4+, CD8+T lymphocyte infiltration. Their CTL activity was significantly higher than in mice received single gene therapy. Conclusion Combination therapy with IL-12 gene and HSV-TK gene plus GCV is effective for mouse liver cancer.展开更多
To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatoc...To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatocellular carcinoma cell line SMMC 7721 by using liposome mediated transfection technique,and then gene expression was detected by RT PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model,the antitumor effects of pDR2 TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2 TK /GCV had cytotoxic effect and about 70 % SMMC 7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2 TK gene was significantly smaller than that in control group . On the 10th day the tumor in 3 mice (60 %) disappeared completely after GCV treatment. It is concluded that the pDR2 TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.展开更多
Objective： The aim of this study was to observe the affection of targeted therapy to plasmid AF-pGL3-hTERT-TK in HCC cell line HepG2. Methods： We constructed therapeutic plasmid pGL3-hTERT-TK （containing suicide ge...Objective： The aim of this study was to observe the affection of targeted therapy to plasmid AF-pGL3-hTERT-TK in HCC cell line HepG2. Methods： We constructed therapeutic plasmid pGL3-hTERT-TK （containing suicide gene TK that promoted by promoter of hTERT） and was conjugated with AF-liposome （a protein that can combine with the receptor ASPGR on HCC cell surface）. Then we transfected HCC cell line HepG2 and hepatic cell L02 with AF-pGL3-hTERT-TK, observed the effects of therapeutic plasmid AF-pGL3-hTERT-TK for HCC cell line growth and apoptosis in vitro by Flow cytometry and Tun- nel method. Results： Our results showed that TK gene was 1100 bp in plasmid pGL3-hTERT-TK. Plasmid pGL3-hTERT-TK can effectively transfect HCC cell HepG2 and the transfection rate was 8.91%. By recognizing and combining effects of recep- tor protein ASPGR on HCC cell surface the therapeutic plasmid AF-pGL3-hTERT-TK could easily enter into HCC cell HepG2 and induce its apeptosis. The apoptosis rate was 85.87% while only 8.65% in L02 cell. Four days after AF-pGL3-hTERT-TK transfected HepG2 was intervention by ganciclovir （GCV）, a lot of apeptotic bodies were found by Tunnel analysis, while little apoptotic body was found in hepatic cell L02. Conclusion： AF-pGL3-hTERT-TK can target to HCC cell line and induce it to apoptesis, almost has no influence on hepatic cell L02. AF-pGL3-hTERT-TK has the potential therapeutic effects for HCC.展开更多
Objective To investigate the effects of combination adenovirusmediated HSVtk/GCV system and antisense IGF1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV kill...Objective To investigate the effects of combination adenovirusmediated HSVtk/GCV system and antisense IGF1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV killing effeciency after combined gene transfer of HSVtk and antisense IGF1 was observed in vitro.Rat glioma was treated with HSVtk/GCV and antisense IGF1 and the survival rate of rats was observed.Results C6 cells transfected with tk and antisense IGF1 gene were more sensitive to GCV than that transfected with tk gene alone.The survival of the combination gene therapy group was prolonged significantly and large amounts of CD+ 4,CD+ 8 lymphocytes were detected in the tumor tissues.Conclusion Antisense IGF1 gene may enhance the tumorkilling effects of HSVtk/GCV.展开更多
Porcine interleukin-2 and porcine interleukin-6 cDNA sequences were cloned into the expressing vectors pET-28a and pGEX-KG respectively. They were expressed in E. coli BL21(DE3)with high-level production. The gene del...Porcine interleukin-2 and porcine interleukin-6 cDNA sequences were cloned into the expressing vectors pET-28a and pGEX-KG respectively. They were expressed in E. coli BL21(DE3)with high-level production. The gene deleted vaccine of pseudorabies virus Ea strain(TK-/gG-/LacZ+)was mixed with the two different purified recombinant proteins each, or both, with the doses of 2, 5 or 10 μg ml-1. Ten groups of pseudorabies negative antibody swines were immuned twice with tested vaccines with different doses, or control vaccine, respectively. The antibody liters of the test groups were detected by neutralization test, and the daily weight gains of swines were calculated and analyzed statistically. In the study, all the neutralizing antibody ti-ters in test groups were higher than the control group, and the recombinant proteins appeared a dose dependent adjuvant effect. The tested vaccines with 2 μg ml-1 pIL-2 and with 10 μg ml-1 pIL-2/pIL-6 got significant and extremely significant differences, compared with the vaccines without pILs. The difference of the daily weight gain indicated the potential positive influence of pIL-2 and pIL-6 on immune protection.展开更多
基金Supported Partly by the Program of Jilin Provincial Science & Technology Department,China(Nos.200705335 and 200504057)the Program of Development and Reform Commission of Jilin City,China(No.2004987)
文摘To construct a single tetracycline-regulatable plasmid vector based on the double tetracycline-regulatable plasmid vector system for regulating HSV-tk gene expression so as to effectively kill HeLa cells. Two tetracycline operator（TetO2） was cloned into pcDNA3.1 and a cassette was made for a cytomegalovirus-type 2 tetracycline operator（CMV-TetO2） promoter, and the obtained vector was named pcDNA3.1-CMV-TetO2. Herpes simplex virus thymidine kinase（HSV-tk） gene and tetracycline repressor（TR） gene were cloned into pcDNA3.1-CMV-TetO2 and the two genes were linked with internal ribosome entry site（IRES） to gain a vector named pcDNA3.1-CMV-TetO2- HSV-tk-IRES-TR. The HeLa cells were stablly transfected with pcDNA3.1-CMV-TetO2-HSV-tk-IRES-TR plasmid. The expression of HSV-tk and TR were detected by RToPCR, the tumorcidal activity of HSV-tk/GCV was determined by MTT assay. In Hela cells transfected with the above plasmid vector, HSV-tk gene and TR gene can be expressed lowly and the concentration of GCV producing a 50% decrease in cell viability was about 50 lag/mL without adding deoxycycline; in contrast, the expessions of HSV-tk gene and TR gene increased significantly and the concentration of GCV producing a 50% decrease in cell viability was about 5 μg/mL with adding deoxycycline. Therefore tetracycline can regulate the expression and tumorcidal activity of HSV-tk gene in HeLa cells with this single plasmid vector.
基金the National Natural Science Foundation of China! (No. 39730440).
文摘Objective: To investigate the synergistic anti-tumor effects of murine IL-12 gene and HSV-TK gene therapy in mice bearing liver cancer. Methods: Mouse liver cancer MM45T. Li (H-2d) cells were transfected with retroviral vector containing IL-12 gene or HSV-TK gene insert. Gene-modified liver cancer cells, MM45T. Li/IL-12 and MM45T. Li/TK, with stable expression of IL-12 and TK were obtained. Balb/c mice were inoculated subcutaneously with 2′105 MM45T. Li cells. When the tumor reached a size of 0.5-1.0 cm, a mixture of MM45T.Li/TK cells and 60Co-irradiated MM45T. Li/IL-12 cell were injected intratumoraly. Ganciclovir (GCV) was injected ip (40 mg.kg-1.d-1) for 10 days. Intratumoral injection of 60Co-irradiated MM45T. Li/IL-12 cells was repeated twice in one week apart. Mice with distant tumors were treated according to the same protocol. CTL activity of spleen cells was measured by 51Cr-release assay and phenotype of tumor infiltrating lymphocytes by immunohistochemical staining. Results: In mice treated with MM45T. Li/IL-12 or MM45T. Li/TK+GCV individually led to moderate reduction in tumor growth, but neither could eradicate the tumor completely, while in 60% of mice treated with a mixture of MM45T. Li/IL-12 and MM45T. Li/TK cells plus GCV, complete tumor regression was observed, with no tumor recurrence for two months. The growth of distant tumor was also inhibited significantly in mice similarly treated. Most of the mice received combined gene therapy plus GCV had abundant CD4+, CD8+T lymphocyte infiltration. Their CTL activity was significantly higher than in mice received single gene therapy. Conclusion Combination therapy with IL-12 gene and HSV-TK gene plus GCV is effective for mouse liver cancer.
文摘To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatocellular carcinoma cell line SMMC 7721 by using liposome mediated transfection technique,and then gene expression was detected by RT PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model,the antitumor effects of pDR2 TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2 TK /GCV had cytotoxic effect and about 70 % SMMC 7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2 TK gene was significantly smaller than that in control group . On the 10th day the tumor in 3 mice (60 %) disappeared completely after GCV treatment. It is concluded that the pDR2 TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.
基金Supported by a grant from the National Natural Sciences Foundation of China (No. 30672405)
文摘Objective： The aim of this study was to observe the affection of targeted therapy to plasmid AF-pGL3-hTERT-TK in HCC cell line HepG2. Methods： We constructed therapeutic plasmid pGL3-hTERT-TK （containing suicide gene TK that promoted by promoter of hTERT） and was conjugated with AF-liposome （a protein that can combine with the receptor ASPGR on HCC cell surface）. Then we transfected HCC cell line HepG2 and hepatic cell L02 with AF-pGL3-hTERT-TK, observed the effects of therapeutic plasmid AF-pGL3-hTERT-TK for HCC cell line growth and apoptosis in vitro by Flow cytometry and Tun- nel method. Results： Our results showed that TK gene was 1100 bp in plasmid pGL3-hTERT-TK. Plasmid pGL3-hTERT-TK can effectively transfect HCC cell HepG2 and the transfection rate was 8.91%. By recognizing and combining effects of recep- tor protein ASPGR on HCC cell surface the therapeutic plasmid AF-pGL3-hTERT-TK could easily enter into HCC cell HepG2 and induce its apeptosis. The apoptosis rate was 85.87% while only 8.65% in L02 cell. Four days after AF-pGL3-hTERT-TK transfected HepG2 was intervention by ganciclovir （GCV）, a lot of apeptotic bodies were found by Tunnel analysis, while little apoptotic body was found in hepatic cell L02. Conclusion： AF-pGL3-hTERT-TK can target to HCC cell line and induce it to apoptesis, almost has no influence on hepatic cell L02. AF-pGL3-hTERT-TK has the potential therapeutic effects for HCC.
文摘Objective To investigate the effects of combination adenovirusmediated HSVtk/GCV system and antisense IGF1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV killing effeciency after combined gene transfer of HSVtk and antisense IGF1 was observed in vitro.Rat glioma was treated with HSVtk/GCV and antisense IGF1 and the survival rate of rats was observed.Results C6 cells transfected with tk and antisense IGF1 gene were more sensitive to GCV than that transfected with tk gene alone.The survival of the combination gene therapy group was prolonged significantly and large amounts of CD+ 4,CD+ 8 lymphocytes were detected in the tumor tissues.Conclusion Antisense IGF1 gene may enhance the tumorkilling effects of HSVtk/GCV.
基金supported by a grant from the National High Tech R&D Program(863 Program)of China(2001AA213051).
文摘Porcine interleukin-2 and porcine interleukin-6 cDNA sequences were cloned into the expressing vectors pET-28a and pGEX-KG respectively. They were expressed in E. coli BL21(DE3)with high-level production. The gene deleted vaccine of pseudorabies virus Ea strain(TK-/gG-/LacZ+)was mixed with the two different purified recombinant proteins each, or both, with the doses of 2, 5 or 10 μg ml-1. Ten groups of pseudorabies negative antibody swines were immuned twice with tested vaccines with different doses, or control vaccine, respectively. The antibody liters of the test groups were detected by neutralization test, and the daily weight gains of swines were calculated and analyzed statistically. In the study, all the neutralizing antibody ti-ters in test groups were higher than the control group, and the recombinant proteins appeared a dose dependent adjuvant effect. The tested vaccines with 2 μg ml-1 pIL-2 and with 10 μg ml-1 pIL-2/pIL-6 got significant and extremely significant differences, compared with the vaccines without pILs. The difference of the daily weight gain indicated the potential positive influence of pIL-2 and pIL-6 on immune protection.
