载脂蛋白-J质粒转染大鼠BMSCs及其在靶细胞中的表达

The expression of Apo-J plasmid of target cells in rat bone marrow mesenchymal stem after transfection

目的对比pEGFP-N1-apoJ质粒在不同DNA浓度条件下转染骨髓间充质干细胞的转染率,并测定载脂蛋白-J的表达情况。方法 0.8、1.2、1.6、2.0μg pEGFP-N1-apoJ质粒分别在脂质体的介导下转染大鼠骨髓间充质干细胞,并在荧光显微镜下观察,确定24、48、72h时转染率,筛选出能获得最高转染率的质粒的量。设置pEGFP-N1-apoJ质粒组、空pEGFP-N1质粒组、空白对照组,用蛋白免疫印迹法检测载脂蛋白-J在24、48、72h3个时间点的表达情况。结果 4个不同质粒浓度组转染率分别为:(16.7±1.0)%、(19.7±1.4)%、(30.1±3.6)%、(21.7±5.5)%,比较4个组间转染效率差异有统计学意义(P<0.05),且1.6μg pEGFP-N1-apoJ质粒组转率最高。通过蛋白免疫印迹法检测后,3个目的蛋白灰度值与内参甘油醛-3-磷酸脱氢酶(GAPDH)灰度值相比较,蛋白质相对表达水平为:pEGFP-N1-apoJ质粒组1.91±0.12,空pEGFP-N1质粒组0.56±0.11,空白对照组0.57±0.17,差异有统计学意义(P<0.05)。结论质粒pEGFP-N1-apoJ可在脂质体的介导下转染入大鼠骨髓间充质干细胞,携带载脂蛋白-J基因的靶细胞可表达载脂蛋白-J。

Objective To compare the transfection efficiency of BMSCs transfected with pEGFP-N1-apoJ plasmid in different DNA concentration,and the expression of Apo-J was determined.Methods The pEGFP-N1-apoJ plasmid was transfected into BMSCs by Liposome which the quality respective is 0.8,1.2,1.6,2.0μg respectively,and we observed by fluorescence microscope,determined the transfection efficiency at 24,48,72 h,and screened out the amount of plasmid that could get the highest transfection efficiency.Then we set pEGFP-N1-apoJ plasmid group,empty pEGFP-N1 plasmid group,blank control group.The expression of Apo-J at 3time points was detected by Western blot.Results The transfection efficiency of 4different plasmid concentration groups were：（16.7±1.0）%,（19.7±1.4）%,（30.1±3.6）%,（21.7±5.5）%.There was significant difference in transfection efficiency among 4groups（P〈0.05）.And the transfection rate of the 1.6μg pEGFP-N1-apoJ plasmid group was the highest.After the detection by Western blot,the relative expression level of protein was as follow：PEGFP-N1-apoJ plasmid group 1.91±0.12,empty pEGFP-N1 plasmid group 0.56±0.11,blank control group 0.57±0.17,the difference was statistically significant（P〈0.05）.Conclusion Plasmid pEGFP-N1-apoJ might be transfected into rat BMSCs by liposome.The target cells carrying the Apo-J gene can express Apo-J.