目的：观察慢病毒介导 shRNA 沉默非肌肉球蛋白重链ⅡA（MYH9）对骨髓间充质干细胞（BMSCs）旁分泌功能的影响。方法采用脂质体法将含 MYH9干扰序列的 pHBLV －U6－ZsGreen －Puro（实验组）质粒转染到293T 细胞，以转染 pHBLV －U6－ZsGreen －Puro 空载质粒的细胞为空载组，以正常未转染细胞为对照组，包装产生的病毒液感染 BMSCs。用荧光显微镜观察荧光蛋白表达；以β－actin 为内参，分别用实时荧光定量聚合酶链反应（qRT －PCR）和免疫印迹（Western blot）检测 MYH9 mRNA 及蛋白表达；酶联免疫吸附测定法（ELISA）检测其培养基上清液中血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）、促血管生成素1（ANG －1）和促血管生成素2（ANG －2）的含量。结果成功构建重组质粒 pHBLV －U6－MYH9－ZsGreen －Puro，转染293 T 细胞产生高滴度慢病毒，荧光显微镜下可见被慢病毒感染的 rBMSCs 表达强绿色荧光蛋白，qRT －PCR 和 Western blot结果显示2条慢病毒感染 MYH9 mRNA 表达水平明显下调，显著低于对照组和空载组（P ＜0．05）；沉默 MYH9基因后，实验组的 ANG －1和 bFGF 在增殖末期较对照组和空载组有明显上升（P ＜0．05），ANG －2和 VEGF 未见明显差异（P ＞0．05）。结论pHBLV －U6－MYH9－ZsGreen －Puro 慢病毒包装质粒转染 BMSCs 后，BMSCs 旁分泌能力未受明显影响。%Objective To observe the effects of lentiviral vector with shRNA silencing non -muscle myosin heavychain A (MYH9) on the paracrine of bone marrow mesenchymal stem cells (BMSCs).Methods The plasmid of pHBLV -U6 -ZsGreen -Puro, which contained the shRNA targeting non -muscle myosin heavy chain -A gene sequence, wasused to transfect 293T cells with lipofectamine.The 293T cells transfected by the empty vector pHBLV -U6 -ZsGreen -Puro were assigned as blank group, and the one without transfection as control group.The recombinant plasmid were transfectedinto BMSCs cells.Fluorescent protein expression was observed by fluorescence microscope, and β-actin was usedas a reference.The expression of MYH9 mRNA and protein were assessed by real -time fluorescence quantitative PCR(qRT -PCR) and Western blot, respectively.The contents of vascular endothelial growth factor (VEGF), basic fibroblastgrowth factor (bFGF), angiopoietin -1 (ANG -1) and angiopoietin -2 (ANG -2) in the culture medium were assessedby ELISA.Results The recombinant plasmid pHBLV -U6 -MYH9 -ZsGreen -Puro was successfully constructed.The transfected 293 T cells produced high titer of lentivirus and rBMSCs infected by lentiviral vector expressed stronggreen fluorescent protein.According to q -PCR and Western blot, the expression levels of MYH9 mRNA and protein ininfected group were significantly lower than those in the blank and the control group (P <0.05).After MYH9 gene wassilenced, the expression levels of ANG -1 and bFGF in the experimental group were significantly higher than those in theblank and the control group (P <0.05).Conclusion The paracrine of BMSCs is not affected by pHBLV -U6 -MYH9 -ZsGreen -Puro recombinant plasmid mediated with lentivirus transfection.
展开▼
