肿瘤坏死因子相关凋亡诱导配体基因转染骨髓间充质干细胞后基因表达情况及其对C6胶质瘤细胞作用的体外研究

Gene expression in bone mesenchymal stem cells transfered by tumor necrosis factor-related apoptosis-inducing ligand and these cells＇effects on C6 glioma cells in vitro

目的 探讨转染肿瘤坏死因子相关凋亡诱导配体（TRAIL）基因的骨髓间充质干细胞（BMSC）基因表达情况及其功能。方法 实验分三组，即转染TRAIL基因组、转染空载体组及空白对照组。用脂质体法将TRAIL转入绿色荧光蛋白（GFP）-BMSC中，反转录PCR法检测BMSC的TRAIL mRNA水平，Western blot、免疫荧光法检测TRAIL蛋白的表达；将携带有TRAIL的GFP-BMSC同大鼠C6胶质瘤细胞共培养，通过四甲基偶氮唑蓝比色法检测其对肿瘤细胞的旁观者效应，Hochest-PI双染色法观察TRAIL转染的BMSC对C6细胞凋亡的影响。结果 免疫荧光检测显示，转染TRAIL 24、48 h的GFP-BMSC细胞质和细胞膜有TRAIL蛋白的表达，24 h比48 h荧光强，空白对照组及空载体组细胞未见表达。反转录PCR、Western blot显示转染TRAIL基因组细胞TRAIL mRNA及蛋白高表达，空白对照组及空载体组未见表达。转染TRAIL的GFP-BMSC明显抑制C6细胞存活，抑制率为（62.7±0.1）%，高于空载体组的（16.7±0.1）%（P＜0.05），同时转染TRAIL基因的BMSC可促进C6细胞的凋亡。结论 转染TRAIL的BMSC能够稳定表达目的基因，且能促进大鼠C6胶质瘤细胞凋亡，具有明显的旁观者效应。

Objective To investigate the gene expression in bone mesenchymal stem cells transferred by tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） and its effect on C6 glioma cells in vitro. Methods The experiment was divided into three groups, the test group transfected with TRAIL gene to BMSC, control group of BMSC transfected with empty liposomal vector, and blank control of BMSC alone. After transfering the TRAIL into GFP-BMSC with Liposomes, the expression of TRAIL was detected by RT-PCR, Western blot, and immunofluorescence. After co-culturing C6 glioma cells with GFP-BMSC-TRAIL, the bystander effect of TRAIL was detected by MTT assay, and C6 cells apoptosis was detected by immunohistochemical method. Results GFP-BMSC-TRAIL vector was successfully constructed, with stable expression of TRAIL. The immunofluorescence showed the increased expression of TRAIL in GFP-BMSC cells at 48 h than 24 h. Western blot and PCR further revealed TRAIL expression in transfected cells, but no expression was found in control cells. In addition, compared with the blank control group and control group, GFP-BMSC-TRAIL significantly inhibited the C6 cell proliferation, with a lower survival rate （62.7 ± 0.1） % and higher apoptosis rate. Conclusion BMSC transfected with TRAIL gene can inhibit the proliferation of C6 glioma cell, suggesting BMSC may be an effective vector for gene therapy.