慢病毒载体介导的shRNA沉默非肌肉肌球蛋白重链ⅡA对骨髓间充质干细胞旁分泌的影响

Lentiviral vector mediated shRNA silencing non- muscle myosin heavy chain- A does not affect the paracrine of bone marrow mesenchymal stem cells

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 heavy chain 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,was used to transfect 293 T cells with lipofectamine. The 293 T 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 transfected into BMSCs cells. Fluorescent protein expression was observed by fluorescence microscope,and β- actin was used as 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 fibroblast growth factor（ bFGF）,angiopoietin- 1（ ANG- 1） and angiopoietin- 2（ ANG- 2） in the culture medium were assessed by 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 strong green fluorescent protein. According to q- PCR and Western blot,the expression levels of MYH9 mRNA and protein in infected group were significantly lower than those in the blank and the control group（ P 〈0. 05）. After MYH9 gene was silenced,the expression levels of ANG- 1 and bFGF in the experimental group were significantly higher than those in the blank 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.