Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-B...Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP_2 vector by the optimized retroviral transduction protocol.Fluorescent microscopy's examination was to evaluate the results of the transduction,flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells.Bioactivity test from C_2C_12K_4 cells was to show the expression and bio-activity of the fusion gene.Results Fluorescent microscopy showed the success of the transduction.By flow cytometer's analysis,the mean efficiency of the transduction with EGFP was(42.8±6.1)% SD.Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting,almost of those showed the expression of BMP_2.Fluorescently and strongly bioactivity test for C_2C_12K_4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control.Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting.Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2,the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique,the expressed chimeric protein embraced the double bioactivity of EGFP and BMP_2,and moreover,the expression had not attenuated over time.展开更多
Objective： To establish a stable C6/EGFP glioma cell transfected with the human immunodeficiency virus line for studies on glioma. Methods： The C6 glioma cell line was type I （HIV-1） based lentivirus vector contai...Objective： To establish a stable C6/EGFP glioma cell transfected with the human immunodeficiency virus line for studies on glioma. Methods： The C6 glioma cell line was type I （HIV-1） based lentivirus vector containing two enhancer-promoters CMV and EF1α. Enhanced green fluorescent protein （EGFP）-positive C6 cells were sorted out by fluorescence-activated cell sort. Expression of EGFP was observed by fluorescent microscopy. EGFP gene in C6 genome was assessed by Polymerase chain reaction （PCR） and DNA sequencing. Original and transfected cells were compared biologically and cytomorphologically. Results： Lentivirus vector transfection produced up to 40% EGFP-positive cells. After fluorescence-activated cell sort selection, a pure cell line C6/EGFP was established. PCR and DNA sequencing revealed integration of EGFP gene in C6 cell genome. Analysis of cell characteristics revealed no difference between transfected and original cells. Conclusion： A C6/EGFP cell line expressing EGFP as a marker is established, in which the EGFP gene is integrated into the genome. This cell line can be served as a promising tool for further basic research and gene therapy studies.展开更多
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
目的研究腺病毒携带目的基因经完整圆窗膜途径耳蜗转导的可行性及安全性,为内耳基因治疗提供实验基础和理论依据。方法20只白色红目豚鼠,术前及术后分别行听性脑干反应(ABR)检查。实验组(12只)以重组腺病毒携带的增强型绿色荧光蛋白基因...目的研究腺病毒携带目的基因经完整圆窗膜途径耳蜗转导的可行性及安全性,为内耳基因治疗提供实验基础和理论依据。方法20只白色红目豚鼠,术前及术后分别行听性脑干反应(ABR)检查。实验组(12只)以重组腺病毒携带的增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP),对照组(8只)以人工外淋巴液注入豚鼠圆窗龛内。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片,耳蜗冰冻切片观察。结果于圆窗龛内注入腺病毒携带目的基因的转导方法对听力无明显影响。转染耳蜗及对侧耳蜗内目的基因呈广泛表达。5天组表达产物最高,14天组逐渐降低。对照组耳蜗未见EPFP表达。结论于圆窗龛内注入腺病毒携带目的基因转导的方法对耳蜗无明显毒害作用,且能够将目的基因成功转导至双侧耳蜗组织并广泛表达。展开更多
构建增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)和IgG4基因的融合蛋白真核表达载体pMM-EGFP-IgG4/WG,转染至中华仓鼠卵巢细胞(Chinese HamsterOvary,CHO)中成功表达,并发出绿色荧光,证明pMM-EGFP-IgG4/WG是一种良好...构建增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)和IgG4基因的融合蛋白真核表达载体pMM-EGFP-IgG4/WG,转染至中华仓鼠卵巢细胞(Chinese HamsterOvary,CHO)中成功表达,并发出绿色荧光,证明pMM-EGFP-IgG4/WG是一种良好的生产分泌型外源融合蛋白的阳性对照.展开更多
目的:构建pIRES2-绿色荧光蛋白(EGFP)-骨形成蛋白2(bone morphogenetic protein 2,BMP-2)真核表达质粒,并检测其表达活性。方法:采用逆转录聚合酶链式反应(RT-PCR)技术从人骨肉瘤组织中扩增出人骨形态发生蛋白2(hBMP-2)的基因片段,构建...目的:构建pIRES2-绿色荧光蛋白(EGFP)-骨形成蛋白2(bone morphogenetic protein 2,BMP-2)真核表达质粒,并检测其表达活性。方法:采用逆转录聚合酶链式反应(RT-PCR)技术从人骨肉瘤组织中扩增出人骨形态发生蛋白2(hBMP-2)的基因片段,构建成pIRES2-EGFP-BMP-2重组质粒,经酶切、测序检测其构建的正确性,通过转染3T3细胞后分别采用RT-PCR、免疫组化及Western免疫印迹(Western blotting,WB)法检测其转录、表达活性。结果:构建的pIRES2-EGFP-BMP-2质粒经酶切、测序、RT-PCR、免疫组化、EGFP表达鉴定、WB等检验表明质粒构建成功并具有转录活性。结论:本实验成功构建了具有表达活性的hBMP-2真核表达质粒,为进一步研究用BMP-2基因转染的方法来促进实验动物骨折的愈合奠定了基础。展开更多
基金This work was supported by the National Natural Science Foundation of China(No.30400163).
文摘Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP_2 vector by the optimized retroviral transduction protocol.Fluorescent microscopy's examination was to evaluate the results of the transduction,flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells.Bioactivity test from C_2C_12K_4 cells was to show the expression and bio-activity of the fusion gene.Results Fluorescent microscopy showed the success of the transduction.By flow cytometer's analysis,the mean efficiency of the transduction with EGFP was(42.8±6.1)% SD.Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting,almost of those showed the expression of BMP_2.Fluorescently and strongly bioactivity test for C_2C_12K_4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control.Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting.Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2,the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique,the expressed chimeric protein embraced the double bioactivity of EGFP and BMP_2,and moreover,the expression had not attenuated over time.
基金supported by the National Natural Science Foundation of China(No.30640073)Beijing Municipal Natural Science Foundationand the Scientific Research Foundation for Returned Scholars,from Ministry of Education of china.
文摘Objective： To establish a stable C6/EGFP glioma cell transfected with the human immunodeficiency virus line for studies on glioma. Methods： The C6 glioma cell line was type I （HIV-1） based lentivirus vector containing two enhancer-promoters CMV and EF1α. Enhanced green fluorescent protein （EGFP）-positive C6 cells were sorted out by fluorescence-activated cell sort. Expression of EGFP was observed by fluorescent microscopy. EGFP gene in C6 genome was assessed by Polymerase chain reaction （PCR） and DNA sequencing. Original and transfected cells were compared biologically and cytomorphologically. Results： Lentivirus vector transfection produced up to 40% EGFP-positive cells. After fluorescence-activated cell sort selection, a pure cell line C6/EGFP was established. PCR and DNA sequencing revealed integration of EGFP gene in C6 cell genome. Analysis of cell characteristics revealed no difference between transfected and original cells. Conclusion： A C6/EGFP cell line expressing EGFP as a marker is established, in which the EGFP gene is integrated into the genome. This cell line can be served as a promising tool for further basic research and gene therapy studies.
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.
文摘目的研究腺病毒携带目的基因经完整圆窗膜途径耳蜗转导的可行性及安全性,为内耳基因治疗提供实验基础和理论依据。方法20只白色红目豚鼠,术前及术后分别行听性脑干反应(ABR)检查。实验组(12只)以重组腺病毒携带的增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP),对照组(8只)以人工外淋巴液注入豚鼠圆窗龛内。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片,耳蜗冰冻切片观察。结果于圆窗龛内注入腺病毒携带目的基因的转导方法对听力无明显影响。转染耳蜗及对侧耳蜗内目的基因呈广泛表达。5天组表达产物最高,14天组逐渐降低。对照组耳蜗未见EPFP表达。结论于圆窗龛内注入腺病毒携带目的基因转导的方法对耳蜗无明显毒害作用,且能够将目的基因成功转导至双侧耳蜗组织并广泛表达。
文摘构建增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)和IgG4基因的融合蛋白真核表达载体pMM-EGFP-IgG4/WG,转染至中华仓鼠卵巢细胞(Chinese HamsterOvary,CHO)中成功表达,并发出绿色荧光,证明pMM-EGFP-IgG4/WG是一种良好的生产分泌型外源融合蛋白的阳性对照.
文摘目的:构建pIRES2-绿色荧光蛋白(EGFP)-骨形成蛋白2(bone morphogenetic protein 2,BMP-2)真核表达质粒,并检测其表达活性。方法:采用逆转录聚合酶链式反应(RT-PCR)技术从人骨肉瘤组织中扩增出人骨形态发生蛋白2(hBMP-2)的基因片段,构建成pIRES2-EGFP-BMP-2重组质粒,经酶切、测序检测其构建的正确性,通过转染3T3细胞后分别采用RT-PCR、免疫组化及Western免疫印迹(Western blotting,WB)法检测其转录、表达活性。结果:构建的pIRES2-EGFP-BMP-2质粒经酶切、测序、RT-PCR、免疫组化、EGFP表达鉴定、WB等检验表明质粒构建成功并具有转录活性。结论:本实验成功构建了具有表达活性的hBMP-2真核表达质粒,为进一步研究用BMP-2基因转染的方法来促进实验动物骨折的愈合奠定了基础。
