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目的:探讨反转录病毒介导的肿瘤坏死因子α(TNF-α)基因对C6胶质瘤的抗瘤效应,从而为肿瘤的基因治疗提供基础研究数据。方法:将重组TNF-α反转录病毒载体PLJ+TNF转染大鼠C6胶质瘤细胞,ELISA法检测该细胞中目的基因的表达,MTT法检测体外增殖能力;将实验大鼠随机分为实验组和对照组,每组10只,实验组大鼠右股内侧接种转基因2×106C6细胞,对照组同法同部位接种未转基因C6细胞,常规饲养5周,每周测量肿瘤大小,绘制肿瘤生长曲线,比较两组皮下肿瘤大小并进行统计学分析。结果:C6胶质瘤细胞与C6细胞的体外增殖能力比较,差异无显著性意义(P>0.05);转基因前后TNF-α分泌情况:1×106个C6细胞体外培养24h每毫升上清液分泌TNF-α(2.42±0.76)U;1×106个C6胶质瘤细胞体外培养24h每毫升上清液分泌TNF-α(17.53±2.31)U,两者比较,差异有显著性意义(P<0.01);动态TNF-α检测显示:转基因C6细胞能稳定地表达高水平的TNF-α;大鼠皮下接种肿瘤细胞5周后,实验组3只未见肿瘤生长,其余肿瘤直径为(1.5±0.3)cm,对照组肿瘤直径为(2.4±0.2)cm,两组比较,差异有显著性意义(P<0.01)。结论:TNF-α基因转染大鼠C6胶质瘤细胞后能持续分泌高水平的TNF-α,对大鼠C6胶质瘤皮下肿瘤具有明显的抗瘤效应。
Objective: To investigate the anti-tumor effect of retrovirus-mediated tumor necrosis factor α (TNF-α) gene on C6 glioma and to provide basic data for the gene therapy of tumors. Methods: The recombinant TNF-α retrovirus vector PLJ + TNF was transfected into rat C6 glioma cells. The expression of the target gene was detected by ELISA, and the proliferation ability was assayed by MTT assay. The rats were randomly divided into Experimental group and control group, with 10 rats in each group. Transgenic 2 × 106C6 cells were inoculated medially in the right femoral head of rats in experiment group. Non-transgenic C6 cells were inoculated in the same way in the control group and routinely housed for 5 weeks. Tumor size Growth curve, the size of subcutaneous tumors were compared and statistically analyzed. Results: There was no significant difference in proliferation between C6 glioma cells and C6 cells (P> 0.05). The secretion of TNF-α before and after transfection: 1 × 106 C6 cells were cultured in vitro for 24 h, and secreted per ml of supernatant TNF-α (2.42 ± 0.76) U; 1 × 106 C6 glioma cells cultured in vitro 24h per ml of supernatant secreted TNF-α (17.53 ± 2.31) U, the difference between the two groups was significant (P < 0.01). The results of dynamic TNF-α showed that the transgenic C6 cells stably expressed high levels of TNF-α. After 5 weeks of subcutaneous inoculation of tumor cells in rats, no tumor growth was found in the experimental group and the remaining tumor diameter was (1.5 ± 0.3) cm in the control group, and (2.4 ± 0.2) cm in the control group. There was significant difference between the two groups (P <0.01). CONCLUSION: TNF-α gene can consistently secrete high level of TNF-α after transfection into rat C6 glioma cells, and has obvious anti-tumor effect on subcutaneous tumors of C6 glioma in rats.