重组质粒pEGFP-N1-apoJ的构建及在大鼠骨髓间充质干细胞中的表达

Construction of recombinant plasmid pEGFP-N1-apoJ and its expression in rat bone marrow mesenchymal stem cells in vitro

目的体外构建重组质粒pEGFP-N1-apoJ,转染大鼠骨髓间充质干细胞,并检测其在大鼠骨髓间充质干细胞中的表达。方法通过脂质体介导重组质粒pEGFP-N1-apoJ转染大鼠骨髓间充质干细胞,确定转染效率,并通过Real-time PCR、免疫细胞化学法、Western blot法检测载脂蛋白-J(apoJ)的表达。结果增强型绿色荧光蛋白在大鼠骨髓间充质干细胞中瞬时转染效率达35%。转染后Real-time PCR、免疫细胞化学法和Western blot法检测证实重组质粒pEGFP-N1-apoJ在大鼠骨髓间充质干细胞中的表达。结论重组质粒p EGFPN1-apoJ成功转染大鼠骨髓间充质干细胞,并在大鼠骨髓间充质干细胞中得到表达,有助于应用apoJ行基因治疗,为进一步研究apoJ作为新的脑出血治疗措施奠定实验基础。

[Objective] To construct recombinant plasmid pEGFP-Nl-apolipoprotein J （apoJ）, transfect it into rat bone marrow mesencbymal stem cells （MSCs）, and detect its expression in rat bone marrow mesenchy- mal stem cells in vitro. [Methods] Rat bone marrow MSCs were transiently transfected with the recombinant plasmid pEGFP-NI-apoJ and the transfection efficiency of green fluorescent protein （GFP） was determined. Real-time PCR, Western blot and immunocytochemistry analysis were also performed for detection of apoJ ex- pression in rat bone marrow MSCs. [Results] Transient transfection efficiency reached 35%. After transfection, the transcription of apoJ was detected in MSCs and the expression of apoJ protein was also verified. [Conclusions] Recombinant plasmid pEGFP-NI-apoJ has been successfully constructed, transfected into rat bone marrow MSCs and expressed in rat bone marrow MSCs. This procedure may be helpful for the applica- tion of apoJ to gene therapy and provide the experimental basis for the further study of apoJ as a new treat- ment of cerebral hemorrhage.