肿瘤坏死因子相关凋亡诱导配体修饰的人羊水间充质干细胞

Tumor necrosis factor-related apoptosis-inducing ligand modified human amniotic fluid-derived mesenchymal stem cells

背景:以间充质干细胞为载体的基因治疗新方法具有广阔的应用价值。目的:利用慢病毒感染方法将肿瘤坏死因子相关凋亡诱导配体导入人羊水来源的间充质干细胞,以期获得稳定表达肿瘤坏死因子相关凋亡诱导配体的人羊水间充质干细胞。方法:首先利用多位点Gateway技术构建慢病毒表达载体pLVpuro/EF1α-肿瘤坏死因子相关凋亡诱导配体,将该表达载体与慢病毒包装质粒同时转染293FT细胞,从而获得携带肿瘤坏死因子相关凋亡诱导配体基因的慢病毒颗粒。利用重组慢病毒颗粒感染人羊水间充质干细胞,并通过抗生素筛选的方法获得稳定表达目的基因-肿瘤坏死因子相关凋亡诱导配体的羊水间充质干细胞,并对其稳定性进行鉴定。结果与结论:酶联免疫吸附法和Westernblot检测结果表明,肿瘤坏死因子相关凋亡诱导配体蛋白在感染的人羊水间充质干细胞内呈高表达,其表达量可达到72μg/L。说明实验成功制备了肿瘤坏死因子相关凋亡诱导配体修饰的人羊水间充质干细胞。

BACKGROUND： The new gene therapy with mesenchymal stem cells as carriers has wide application value. OBJECTIVE： To import tumor necrosis factor-related apoptosis-inducing ligand into human amniotic fluid-derived mesenchymal stem cells using lentiviral vector, in order to obtain human amniotic fluid-derived mesenchymal stem cells that stably express tumor necrosis factor-related apoptosis-inducing ligand. METHODS： The lentiviral expression vector pLVpuro/EF1 a-tumor necrosis factor-related apoptosis-inducing ligand was constructed by multisite Gateway technique. The expression vector and the lentiviral packaging plasmid were ce-transfected with 293FT cells to obtain the lentiviral plasmid carrying tumor necrosis factor-related apoptosis-inducing ligand. The human amniotic fluid-derived mesenchymal stem cells were transfected with recombinant lentivirus particles, and then the human amniotic fluid-derived mesenchymal stem cells that stably express target gene-tumor necrosis factor-related apoptosis-inducing ligand were obtained by using antibiotic screening methods. The stability of human amniotic fluid-derived mesenchymal stem cells was detected. RESULTS AND CONCLUSION： The enzyme-linked immunosorbent assay and western blot results indicated that the tumor necrosis factor-related apoptosis-inducing ligand protein could be highly expressed in the trensfected human amniotic fluid-derived mesenchymal stem cells, and the concentration of tumor necrosis factor-related apoptosis-inducing ligand protein could reach 72 μg/L. These results suggest that the tumor necrosis factor-related apoptosis-inducing ligand modified human amniotic fluid-derived mesenchymal stem cells are established successfully.