人前脑啡肽原基因修饰人骨髓间充质干细胞系的构建

Construction of human bone marrow mesenchymal stem cell line genetically modified with human proenkephalin gene

目的 构建人前脑啡肽原（PENK）基因修饰的并可稳定分泌脑啡肽蛋白的人骨髓间充质干细胞系（hMSCs）.方法 采用脂质体法将PENK基因逆转录病毒载体质粒（pBABE-PENK）转染至Phoenix-293T细胞,收集病毒上清液感染hMSCs细胞,经过嘌呤霉素筛选得到稳定表达PENK基因的hMSCs细胞株（hMSC-PENK细胞）.以转染空载体细胞作为对照,即hMSC-pBABE细胞.采用RT-PCR法检测PENK mRNA的表达,免疫荧光法测定亮氨酸脑啡肽（LEK）的表达,ELISA法测定细胞培养上清液LEK浓度.结果 与hMSCs细胞和hMSC-pBABE细胞比较,hMSC-PENK细胞PENK mRNA和LEK表达上调,细胞培养上清液中LEK浓度升高（P＜0.05或0.01）.结论 PENK基因修饰的hMSCs可表达PENK基因并分泌脑啡肽蛋白,成功构建了稳定分泌镇痛物质的细胞系.

Objective To construct h n bone marrow mesenchymal stem cell line genetically modified with human proenkephalin gene. Methods The packaging cell line Phoenix-293T was transfected with the recombinant pBABE-PENK vector to aquire virus. The recombinant virus was then collected and used to infect hMSCs. Stable expression of proenkephalin gene and leucine enkephalin protein and the concentration of leucine enkephalin protein were detected by RT-PCR, immunofluorescence and ELISA respectively. Results The expression of proenkephalin gene and leucine enkephalin protein were significantly up-regulated in the hMSC-PENK cells, and the concentration of leucine enkephalin protein was also increased in the culture medium. Conclusion A human mesenchymal stem cell line that expresses proenkephalin gene and secrets enkephalin was successfully established.