为探索建立持续稳定表达 h BD- 2的哺乳类动物型工程细胞的可能性 ,作者研究构建真核重组表达载体 p CMV.tag2 B/h BD- 2以进行 COS- 7细胞转染表达实验。将本室克隆获得的 h BD- 2全长 c DNA片段插入带有报告基因的真核表达质粒 p CMV....为探索建立持续稳定表达 h BD- 2的哺乳类动物型工程细胞的可能性 ,作者研究构建真核重组表达载体 p CMV.tag2 B/h BD- 2以进行 COS- 7细胞转染表达实验。将本室克隆获得的 h BD- 2全长 c DNA片段插入带有报告基因的真核表达质粒 p CMV.tag2 B构建出 h BD- 2的重组表达载体 p CMV.tag2 B/h BD- 2 ,并经 DNA测序证明h BD- 2 c DNA片段的插入方向和其全长 c DNA的碱基组成顺序均准确无误。通过脂质体转染法将 p CMV.tag2 B/h BD- 2导入 COS- 7细胞。RT- PCR法和免疫印迹法分别从 m RNA水平和蛋白质水平检测到被转染的 COS- 7细胞系能有效表达 h BD- 2。展开更多
Objective To investigate the effect of U14 vaccine transfected with the B7 gene in inducing antitumor immune response to murine cervical carcinoma in Chinese 615-strain mice.Methods A recombinant retroviral plasmid ...Objective To investigate the effect of U14 vaccine transfected with the B7 gene in inducing antitumor immune response to murine cervical carcinoma in Chinese 615-strain mice.Methods A recombinant retroviral plasmid vector expressing mouse B7-1 gene (pLNSX-mB7) was transfected into 615-strain mouse cervical carcinoma cell line No. 14 (U14) by electroporation to set up a highly-expressed mB7-1 U14 cell clonal strain (B7+U14). In vivo experiments: (1) B7+U14 vaccine was primed to protect the 615-strain mice against U14 re-challenge. (2) B7+U14 vaccine was injected into tumor-bearing mice with different tumor sizes. Lifetimes and tumor sizes were recorded. In vitro cytotoxicity assay: Mice were immunized with B7+U14 or U14 vaccine and 2 weeks later, spleen cells of those mice were cultured for 2 days. The cytotoxicity of these cells against U14 was detected by 5-diphenyl tetrazolium bromide assay.Results We obtained several B7-1 high expression clonal U14 lines. In vivo experiment, we did not find tumor growing in 3 of the 6 mice primed by B7+U14 vaccine during their entire life after re-challenge with U14. The other 3 mice developed tumors and their average survival time was longer than that of the control group (P<0.01). All 6 mice grew tumors in the control group. When the transplanted tumors became palpable, the mice were randomly divided into 3 groups to be injected with B7+U14 vaccine. It was effective for tumor-bearing mice only when the tumor diameters were <3?mm. When the diameters were ≥3?mm, it was not efficacious to inject B7+U14 vaccine (P<0.05). In vitro cytotoxicity assay, cytotoxic T lymphocytes induced by B7+U14 vaccine had a higher cytotoxicity against U14 than that induced by U14 vaccine (F=310.8, P<0.001).Conclusions Vaccines of cervical cancer cells transfected with the costimulatory molecule B7 gene can induce antitumor immune protection in host mice against U14 re-challenge. This treatment may cure part of the tumor-bearing mice but be restricted by tumor size. The results suggest that transfecting the B7 gene into cervical cancer as a cell vaccine may be an efficient supplementary method to treat cervical cancer after operation.展开更多
文摘为探索建立持续稳定表达 h BD- 2的哺乳类动物型工程细胞的可能性 ,作者研究构建真核重组表达载体 p CMV.tag2 B/h BD- 2以进行 COS- 7细胞转染表达实验。将本室克隆获得的 h BD- 2全长 c DNA片段插入带有报告基因的真核表达质粒 p CMV.tag2 B构建出 h BD- 2的重组表达载体 p CMV.tag2 B/h BD- 2 ,并经 DNA测序证明h BD- 2 c DNA片段的插入方向和其全长 c DNA的碱基组成顺序均准确无误。通过脂质体转染法将 p CMV.tag2 B/h BD- 2导入 COS- 7细胞。RT- PCR法和免疫印迹法分别从 m RNA水平和蛋白质水平检测到被转染的 COS- 7细胞系能有效表达 h BD- 2。
基金theNationalNaturalScienceFoundation ofChina (No 3970 0 14 6 )
文摘Objective To investigate the effect of U14 vaccine transfected with the B7 gene in inducing antitumor immune response to murine cervical carcinoma in Chinese 615-strain mice.Methods A recombinant retroviral plasmid vector expressing mouse B7-1 gene (pLNSX-mB7) was transfected into 615-strain mouse cervical carcinoma cell line No. 14 (U14) by electroporation to set up a highly-expressed mB7-1 U14 cell clonal strain (B7+U14). In vivo experiments: (1) B7+U14 vaccine was primed to protect the 615-strain mice against U14 re-challenge. (2) B7+U14 vaccine was injected into tumor-bearing mice with different tumor sizes. Lifetimes and tumor sizes were recorded. In vitro cytotoxicity assay: Mice were immunized with B7+U14 or U14 vaccine and 2 weeks later, spleen cells of those mice were cultured for 2 days. The cytotoxicity of these cells against U14 was detected by 5-diphenyl tetrazolium bromide assay.Results We obtained several B7-1 high expression clonal U14 lines. In vivo experiment, we did not find tumor growing in 3 of the 6 mice primed by B7+U14 vaccine during their entire life after re-challenge with U14. The other 3 mice developed tumors and their average survival time was longer than that of the control group (P<0.01). All 6 mice grew tumors in the control group. When the transplanted tumors became palpable, the mice were randomly divided into 3 groups to be injected with B7+U14 vaccine. It was effective for tumor-bearing mice only when the tumor diameters were <3?mm. When the diameters were ≥3?mm, it was not efficacious to inject B7+U14 vaccine (P<0.05). In vitro cytotoxicity assay, cytotoxic T lymphocytes induced by B7+U14 vaccine had a higher cytotoxicity against U14 than that induced by U14 vaccine (F=310.8, P<0.001).Conclusions Vaccines of cervical cancer cells transfected with the costimulatory molecule B7 gene can induce antitumor immune protection in host mice against U14 re-challenge. This treatment may cure part of the tumor-bearing mice but be restricted by tumor size. The results suggest that transfecting the B7 gene into cervical cancer as a cell vaccine may be an efficient supplementary method to treat cervical cancer after operation.