摘要
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.
出处
《广东医学》
CAS
北大核心
2016年第16期2377-2383,共7页
Guangdong Medical Journal
基金
国家自然科学基金资助项目(编号:81360292)