过表达Nkx2.5对骨髓间充质干细胞向心肌细胞分化的影响

Effect of Nkx2.5overexpression on differentiation of bone marrow mesenchymal stem cells into myocardial cells

目的探讨过表达心脏特异性同源盒转录因子(Nkx2.5)对骨髓间充质干细胞(BMSC)向心肌细胞分化的影响。方法将慢病毒过表达Nkx2.5基因质粒和空载体转染到BMSC后,过表达Nkx2.5的BMSC 1×10^(7)个(过表达组)、空载体BMSC 2×10^(7)个(空载体组)、BMSC 1×10^(7)个(BMSC组)分别与小鼠心肌细胞1×10^(7)个共培养24h;BMSC 2×10^(7)个作为对照组。45只小鼠随机均分为五组:假手术组仅做心脏暴露,不做其他处理;其他小鼠建立小鼠心肌梗死(心梗)模型,在模型缝合完毕24h后经尾静脉移植不含细胞的PBS(心梗模型未处理组)、BMSC(心梗模型BMSC组)、空载体BMSC(心梗模型空载体组)和过表达Nkx2.5的BMSC(心梗模型过表达组)各200μL。采用Western blot法检测肌间线蛋白(desmin)、α肌动蛋白(α-actin)、肌钙蛋白T(cTnT)和缝隙连接蛋白43(Cx43)蛋白表达,2,3,5-氯化三苯基四氮唑染色观察小鼠梗死面积变化,免疫组织化学染色检测梗死组织desmin、α-actin、cTnT和Cx43表达。结果与对照组比较,BMSC组、空载体组、过表达组desmin、α-actin、cTnT、Cx43蛋白表达增加(P<0.05);与BMSC组和空载体组比较,过表达组desmin、α-actin、Cx43蛋白表达增加(P<0.05)。心梗模型过表达组梗死面积小于心梗模型未处理组、心梗模型BMSC组和心梗模型空载体组(P<0.05)。心梗模型过表达组desmin、α-actin、cTnT和Cx43表达高于心梗模型未处理组、心梗模型BMSC组和心梗模型空载体组。结论过表达Nkx2.5可以促进BMSC向心肌细胞分化。

Objective To investigate the effect of overexpression of heart-specific homeobox transcription factor(Nkx2.5)on the differentiation of bone marrow mesenchymal stem cells(BMSC)into myocardial cells.Methods The lentivirus overexpressed Nkx2.5gene plasmid and empty vector were transfected into BMSC.BMSC overexpressed Nkx2.5(group A),BMSC with empty vector(group B)and BMSC(group C)were co-cultured with mouse myocardial cells for 24hours,respectively.BMSC was taken as control group without any treatment.Forty-five mice were divided into five groups with 9mice each.The heart was exposed in sham operation group(group S)without other treatment.The mouse myocardial infarction model was established in other mice.The mice in groups of D,E,F and G were injected via caudal vein with PBS,BMSC,BMSC with empty vector and BMSC overexpressed Nkx2.5.The protein expressions of desmin,α-actin,cTnT and Cx43 were detected by Western blot.The infarct area of the mice was observed by 2,3,5-triphenyltetrazole chloride staining.The expressions of desmin,α-actin,cTnT and Cx43in infarct tissues were detected by immunohistochemical staining.Results The protein expressions of desmin,α-actin,cTnT and Cx43were higher in groups of A,B and C than those in control group(P<0.05).The protein expressions of desmin,α-actin and Cx43were higher in group A than those in groups of B and C(P<0.05).The infarct area was smaller in group G than that in groups of D,E and F(P<0.05).The expressions of desmin,α-actin,cTnT and Cx43were higher in group G than those in groups of D,E and F.Conclusion Overexpression of Nkx2.5can promote the differentiation of BMSC into myocardial cells.