相关蛋白在携带载脂蛋白-J基因大鼠BMSCs中的表达及意义

The expression of associated proteins in rat BMSCs with ApoJ gene

目的检测重组质粒pEGFP-N1-apoJ在不同量、不同时间条件下转染大鼠骨髓间充质干细胞的瞬时转染效率,并测定载脂蛋白-J(ApoJ)在靶细胞中能否高表达。方法贴壁培养细胞,并用脂质体LipofectaminTM2000分别介导0.8、1.2、1.6、2.0μg重组质粒pEGFP-N1-apoJ转染,置荧光显微镜下观察转染结果,测定在24、48、72h时间点的瞬时转染效率。蛋白免疫印迹法分别检测pEGFP-N1-apoJ质粒转染组、空pEGFP-N1质粒转染组、空白对照组ApoJ的表达情况。结果不同的质粒量中1.6μg pEGFP-N1-apoJ质粒与3.0μL脂质体制备的复合物瞬时转染效率最高,分别作用24、48、72h可达27.8%、34.3%、28.2%。蛋白免疫印迹法检测到pEGFP-N1-apoJ质粒组大量ApoJ表达,空质粒及空白对照组见微量ApoJ表达;并且pEGFP-N1-apoJ质粒组Apo-J的表达明显高于空载体及空白对照组(P<0.05)。结论利用脂质体介导可将重组质粒pEGFP-N1-apoJ成功转染入大鼠骨髓间充质干细胞,ApoJ在大鼠骨髓间充质干细胞中得到大量表达。

Objective To detect the transfection efficiency of recombinant plasmid pEGFP-NI-apoJ in rat bone marrow mesenchymal stem cells （BMSCs） at different DNA concentrations and time points and to detect the expression of apoJ in target ceils. Methods We used bone marrow adherent culture method to culture rat BMSCs. LipofectaminTM 2000 mediated respectively 0. 8, 1. 2, 1. 6 and 2 μg of pEGFP-NI-apoJ recombinant plasmid to transfect BMSCs. The transfection results were observed under the fluorescence microscope. We determined the transient transfection efficiency at 24 h, 48 h and 72 h. Western blot was used to detect the expression of ApoJ in pEGFP-NI-apoJ plasmid transfection group, empty pEGFP-N1 plasmid transfection group and control group. Results In different DNA concentrations, when 1.6μg of pEGFP-NI-apoJ plasmid was transfected with 3.0μL of liposome in rat BMSCs, the transient transfection efficiency was the highest; it reached 27.8%, 34.3% and 28.2% at 24 h, 48 h and 72 h. Western blot results showed that pEGFP-NI-apoJ recombinant plasmid expressed large amounts of ApoJ; small amounts of protein expression of ApoJ was found in the empty plasmid and the control groups. The expression of ApoJ was higher in PEGFP-NI-apoJ group than in the empty carrier and the control groups （P 〈0. 05）. Conclusion Recombinant plasmid PEGFP-NI-apoJ can be transfected into rat BMSCs successfully via liposome. ApoJ is expressed in large amounts in rat BMSCs.