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目的 :建立表达IL 18基因的大鼠胶质瘤细胞C6 /IL 18,并探讨外源性IL 18基因对C6细胞生长的影响。方法 :应用逆转录病毒载体 ,将IL 18基因导入C6细胞。经G4 18筛选后 ,获得表达IL 18分子的细胞克隆C6 /IL 18。用RT PCR法检测目的基因mRNA的表达 ;用流式细胞和免疫细胞化学染色法检测目的蛋白的表达。用ELISA法检测C6 /IL 18细胞培养上清诱导脾细胞分泌IFN γ的能力 ,以确定IL 18的生物学活性。用MTT比色法检测细胞体外增殖的状况 ,用流式细胞技术检测细胞的增殖活性 ,并建立大鼠胶质瘤模型 ,观察C6 /IL 18细胞体内致瘤性的改变。结果 :外源IL 18基因于mRNA水平和蛋白水平可获得稳定表达 ,并可诱生大鼠脾细胞分泌IFN γ。同时 ,该细胞系的体外增殖率及体内致瘤性 ,均较亲代C6胶质瘤细胞明显下降。结论 :外源性IL 18基因能部分地抑制C6细胞的体外增殖率和体内致瘤性。建立了可进一步用于相关肿瘤基因免疫和基因治疗研究的大鼠胶质瘤细胞系C6 /IL 18。
OBJECTIVE: To establish C6 / IL-18 cells expressing IL-18 and investigate the effect of exogenous IL-18 on the growth of C6 cells. Methods: IL 18 gene was introduced into C6 cells by retroviral vector. After G418 selection, cell clones C6 / IL18 expressing IL18 molecules were obtained. The mRNA expression of the target gene was detected by RT PCR. The expression of the target protein was detected by flow cytometry and immunocytochemical staining. The ability of the culture supernatant of C6 / IL18 cells to induce IFN? Secretion by splenocytes was examined by ELISA to determine the biological activity of IL-18. MTT assay was used to detect the proliferation of cells in vitro. Flow cytometry was used to detect the proliferative activity of the cells. The rat glioma model was established and the tumorigenicity of C6 / IL-18 cells was observed. Results: Exogenous IL 18 gene was stably expressed at both mRNA and protein levels, and IFNγ was induced in splenocytes of rats. At the same time, the in vitro proliferation rate and in vivo tumorigenicity of this cell line were significantly lower than that of the parental C6 glioma cells. Conclusion: Exogenous IL 18 gene can partially inhibit the proliferation rate of C6 cells in vitro and tumorigenicity in vivo. A rat glioma cell line C6 / IL18 that can be further used for the study of related tumor gene immunity and gene therapy was established.