GFP-BMSC-TRAIL载体的构建及其生物学活性的检测

Construction of green fluorescent protein-bone mesenchymal stem cell-TNF-related apoptosis- inducing ligand vector and detection of its biology characteristics

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（BMSCs）基因表达情况。方法实验分3组：实验组转染TRAIL基因至绿色荧光蛋白（GFP）标记的BMSCs，空载体组转染空载体脂质体，对照组即空白对照的BMSCs。采用脂质体法将TRAIL转入GFP—BMSC中，RT-PCR法检测豫A／LmRNA水平的表达，Western blotting、免疫荧光检测TRAIL蛋白的表达，四甲基偶氮唑蓝（MTT）法检测转染后BMSCs的增殖活性。结果免疫荧光显示．转染豫A儿后24h和48h在胞浆和胞膜可见到TRAIL蛋白的表达，24h比48h荧光明显增强，对照组及空载体组未见表达；RT．PCR、Western blotting显示实验组TRIAL高表达，对照组及空载体组未见高表达；MTT法证实转染后BMSCs细胞的增殖活性无明显影响。结论构建的BMSC—TRAIL载体能够稳定表达目的基因，BMSCs可做为生物载体。

Objective To explore the gene expression in bone mesenchymal stem cells （BMSCs） carried TNF-related apoptosis-inducing ligand （TRAIL） vector. Methods GFP-BMSCs were used in our study and divided into three groups： the experimental group, being transfected TRAIL gene to BMSCs, the empty vector group, being transfected with empty vector, and the blank control group, being transfected with lipidosome. Liposomes method was used to transfect TRAIL into GFP-BMSCs in the experimental group. The mRNA expression of TRAIL gene was detected by real time-PCR; Western blotting and immunofluorescence were used to detect the protein expression of TRAIL gene; the proliferation activity of BMSC cells was detected by MTT method. Results GFP-BMSC-TRAIL vectors were successfully constructed. Immunofluorescence indicated that, 24 and 48 h after TRAIL transfection, TRAIL protein expression was noted in the cytoplasm and membrane, and that at 24 h was obviously stronger than that at 48 h; no expression was noted in the empty vector group and blank control group. Real time PCR and Western blotting indicated TRAIL high expression in the experimental group, while not in the blank control and empty vector group. MTT showed no significant changes of BMSCs activity after TRAIL transfection. Conclusion BMSC-TRAIL vectors can stably express the target gene, indicating that BMSCs may be an effective vector for gene therapy.