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

WU Ming; XIAO Yong-qiang; LIU Ming-zhuo; LI Yu-cong; LI Ming-li; Peng Ling-hua; FENG Yong-wen; ZHU Feng

Abstract：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.