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目的研究重组人RGD-FasL对3株胶质瘤细胞U138/U343/U373的杀伤作用及机制。方法采用RT-PCR方法、MTT比色法、DNA倍体分析、流式细胞术检测融合蛋白对3株胶质瘤细胞的杀伤作用;利用Western blot法探讨其作用机制。结果FasmRNA在U138和U343细胞中有表达,在U373细胞中未有可见表达;DcR3mRNA在3株细胞中均有表达,在U373细胞中强表达。采用流式细胞术检测肿瘤细胞表面两种受体的表达情况,结果与RT-PCR相符。U138细胞株对RGD-FasL敏感并呈剂量依赖性;U343细胞株对RGD-FasL相对敏感;U373细胞对RGD-FasL不敏感。用PI检测细胞周期与凋亡表明RGD-FasL能使U138/U343细胞停留在G1期,推迟进入S期,抑制细胞生长并诱导细胞发生凋亡。Western blot实验表明RGD-FasL作用细胞后caspase-8/3/9的表达升高,Bcl-2的表达降低。结论重组人RGD-FasL可以不同程度的诱导胶质瘤细胞凋亡,其机制与其受体的表达和caspase-8/3/9、Bcl-2的表达有关。
Objective To study the killing effect of recombinant human RGD-FasL on three glioma cells U138 / U343 / U373 and its mechanism. Methods RT-PCR, MTT colorimetric assay, DNA ploidy analysis and flow cytometry were used to detect the cytotoxicity of the fusion protein on 3 glioma cells. Western blot was used to investigate the mechanism. Results Fas mRNA was expressed in U138 and U343 cells but not in U373 cells. DcR3 mRNA was expressed in all three cells and strongly expressed in U373 cells. Flow cytometry was used to detect the expression of two receptors on the surface of tumor cells. The results were consistent with RT-PCR. U133 cell line was sensitive and dose-dependent to RGD-FasL; U343 cell line was relatively sensitive to RGD-FasL; U373 cell line was not sensitive to RGD-FasL. Using PI to detect cell cycle and apoptosis showed that RGD-FasL could arrest U138 / U343 cells in G1 phase, defer S phase, inhibit cell growth and induce cell apoptosis. The result of Western blot showed that the expression of caspase-8/3/9 increased and the expression of Bcl-2 decreased after RGD-FasL treatment. Conclusion Recombinant human RGD-FasL can induce glioma cell apoptosis to some extent. The mechanism is related to the expression of its receptor and the expression of caspase-8/3/9 and Bcl-2.