慢病毒载体介导的NEP1-40在人脐血间充质干细胞中的表达

Expression of lentiviral vectors mediated NEP1-40 in mesenchymal stem cells from human umbilical cord blood

背景:近年来研究证明间充质干细胞具有高度增殖和多向分化潜能,是一种理想的组织工程种子细胞,能高效转染表达外源性目的基因,在基因治疗领域具有广阔的应用前景。目的:将含有NEP1-40基因的慢病毒载体转染脐血间充质干细胞,评价基因转染后脐血间充质干细胞生物学功能变化,观察NEP1-40在脐血间充质干细胞中的表达。方法:体外分离和培养人脐血间充质干细胞,流式细胞仪检测细胞表面标记,并对其生物学特性进行鉴定。同时将NEP1-40基因克隆入慢病毒载体,包装出病毒上清,以不同拷贝数转染脐血间充质干细胞。结果与结论:实验通过密度梯度离心法成功在体外分离和培养脐血间充质干细胞,诱导其向脂肪细胞分化,流式细胞仪检测结果显示脐血间充质干细胞中CD90、CD73及CD105蛋白阳性,不表达CD14、CD34、CD45、CD19、HLA-DR、Stro-1及CD106蛋白;real-timePCR检测发现其NEP1-40mRNA表达水平与转染拷贝数有关,转染拷贝数越高,NEP1-40的表达量越高,此外转染NEP1-40后的脐血间充质干细胞中可检测到NEP1-40蛋白,提示NEP1-40基因转染后脐血间充质干细胞原有的生物学功能无明显影响。

BACKGROUND： In recent years, studies have shown that mesenchymal stem cells are ideal seed cells used fortissue engineering because of their strong proliferation and multi-differentiation potential. Mesenchymal stem cells can be efficiently transfected and expressed exogenous gene, and therefore they have broad application prospects in gene therapy. OBJECTIVE： NEP1-40 gene-containing lentiviral vectors were transfected into umbilical cord blood-derived mesenchymal stem cells to evaluate the biological function changes of mesenchymal stem cells and detect NEP1-40 expression in umbilical cord blood-derived mesenchymal stem cells.METHODS： Human umbilical cord blood-derived mesenchymal stem cells were isolated and cultured in vitro. Cell surface markers were detected by flow cytometry, and their biological characteristics were identified. NEP1-40 gene was cloned into the lentiviral vector and lentiviral supernatant was packaged. Then umbilical cord blood-derived mesenchymal stem cells were transfected with different multiplicities of infection.RESULTS AND CONCLUSIONS： We successfully isolated and cultured human umbilical cord blood-derived mesenchymal stem cells in vitro by density gradient centrifugation method, and the cells could be induced to differentiate into adipocytes. Flow cytometry results showed that umbilical cord blood-derived mesenchymal stem cells were positive for CD90, CD73 and CD105 protein, but they were negative for CD14, CD34, CD45, CD19, HLA-DR, Stro-1 and CD106 protein. Real-time PCR detection showed that the NEP1-40 mRNA expression level was positively correlated with multiplicity of infection. Higher multiplicity of infection yielded higher NEP1-40 expression. In addition, NEP1-40 protein expression could be seen in human umbilical cord blood-derived mesenchymal stem cells after transfected with NEP1-40. These findings suggest that the transfection of NEP1-40 gene has little impact on biological function of human umbilical cord blood-derived mesenchymal stem cells.