外泌体对过表达GATA-4小鼠骨髓间充质干细胞向心肌细胞分化能力促进作用的研究

目的　探讨过表达GATA-4小鼠骨髓间充质干细胞（BMSCs）通过外泌体促进BMSCs向心肌细胞分化的能力。方法　2016年1—6月选取健康4周龄SPF级C57BL/6小鼠10只，通过慢病毒载体GV308携带GATA-4转染小鼠BMSCs，构建过表达GATA-4的BMSCs，并加入基因开启剂多西环素（DOX）。采用ExoQuick-TC提取分泌的外泌体。将GATA-4-BMSCs-外泌体与BMSCs共同培养（A组）、空载体-BMSCs-外泌体与BMSCs共同培养（B组）、BMSCs-外泌体与BMSCs共同培养（C组）、BMSCs单独培养（D组）及小鼠心肌细胞单独培养（E组）72 h，采用荧光染色定性检测和反转录聚合酶链式反应（qPCR）定量检测肌钙蛋白T（cTnT）、α肌动蛋白（α-actin）、Connexin 43、肌间线蛋白（Desmin）的表达。结果　免疫荧光定性检测结果显示，A组GATA-4-BMSCs-外泌体与BMSCs共同培养72 h可见细胞核被DAPI染为蓝色，表达出绿色的cTnT、α-actin、Connexin 43、Desmin；B、C、D组可见细胞核被DAPI染为蓝色，没有表达出绿色的cTnT、α-actin、Connexin 43、Desmin；E组可见细胞核被DAPI染为蓝色，表达出绿色的cTnT、α-actin、Connexin 43、Desmin。qPCR法检测结果显示，5组cTnT、α-actin、Connexin 43、Desmin表达水平比较，差异均有统计学意义（P<0.05）；其中E组cTnT、α-actin、Connexin 43、Desmin表达水平较A组升高；A组和E组cTnT、α-actin、Connexin 43、Desmin表达水平较B组、C组和D组升高（P<0.05）。结论　过表达GATA-4的BMSCs分泌的外泌体可以有效促进BMSCs向心肌细胞分化。

Effects of GATA-4 Overexpressed Mouse Bone Marrow Mesenchymal Stem Cells on the Promotion of Cardiomyocyte Differentiation through Exosomes

Objective　To explore the effect of GATA-4 overexpressed mouse bone marrow mesenchymal stem cells（BMSCs） on the promotion of the differentiation of BMSCs into myocardial cells through exosomes.Methods　GATA-4 overexpressed BMSCs of 10 healthy 4-week-old SPF grade mice selected from January to June in 2016，were constructed by transfecting these cells with the lentiviral vector GV308 carrying GATA-4.Doxycycline（DOX） was added to induce this gene.SBI ExoQuick-TC was then applied to extract the secreting exosomes.Co-culturing was performed between GATA-4-BMSCs-exosome and BMSCs for group A，between GATA-4-free-vector-BMSCs-exosome and BMSCs for group B，and between BMSCs-exosome and BMSCs for group C for 72 h.BMSCs were cultured for group D and myocardial cells were cultured for group E，for 72 h，respectively.Fluorescence staining was conducted to qualitatively determine the expression of cTnT，α-actin，Connexin 43，and Desmin.qPCR was performed to quantitatively identify the expression of these specific antigens in the myocardial cells.Results　The results of immunofluorescence assay showed that the nucleus was stained blue with DAPI in group A，and the expression of green cTnT，α-actin，Connexin 43，Desmin；the nuclei were stained blue with DAPI in group B，C，D，no expression of green cTnT，α-actin，Connexin 43，Desmin；the nucleus was stained blue with DAPI in group E，and the expression of green cTnT，α-actin，Connexin 43，Desmin.The results of qPCR assay showed that the expression levels of cTnT，α-actin，Connexin 43 and Desmin in the 5 groups differed significantly（P<0.05），specifically，the expression levels of cTnT，α-actin，Connexin 43 and Desmin in group E were higher than those in group A（P<0.05）；the expression levels of cTnT，α-actin，Connexin 43 and Desmin in groups A and E were higher than those in groups B，C and D（P<0.05）．Conclusion　The exosomes secreted by GATA-4-overexpressed-BMSCs can effectively promote the differentiation of BMSCs into myocardial cells.