慢病毒载体介导的NEP1-40在人脐血间充质干细胞中的表达

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。

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

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.