外源表达Nkx2.5诱导骨髓间充质干细胞向心肌分化

目的 探讨转染外源性Nkx2.5基因对大鼠骨髓间充质干细胞(BMSCs)向心肌细胞分化的作用.方法 全骨髓法分离大鼠BMSCs,经多次传代培养扩增纯化,流式细胞术鉴定细胞表面抗原CD90、CD45.脂质体法将pEGFP-N1-Nkx2.5质粒转染BMSCs,观察细胞形态变化;转染48h后,免疫细胞化学检测Nkx2.5的表达.转染4周后,Western blotting检测心肌肌钙蛋白(cTnT)的表达;免疫细胞化学检测cTnT、GATA4的表达,鉴定细胞分化.结果 第3代生长良好的细胞中表达CD90而不表达CD45的占细胞总数的99%.转染48h后,实验组可见部分细胞表达绿色的Nkx2.5-pEGFP融合蛋白,免疫细胞化学结果 表明,Nkx2.5蛋白只在实验组有表达(n=3).转染4周后,Western blotting、免疫细胞化学结果 表明,cTnT、GATA4表达在实验组显著高于pEGFP-N1空质粒转染组和空白对照组(n=3).结论 外源表达Nkx2.5基因能够增强BMSCs向心肌分化.

Differentiation of rat bone marrow mesenchymal stem cells into cardiomyocytes induced by exogenous Nkx2.5 gene

Objective To investigate the feasibility of transfecting bone marrow mesenchymal stem cells (BMSCs) with Nkx2.5 to enhance cardiomyogenic differentiation. Methods Rat BMSCs were isolated by the whole bone marrow culture and amplified by serial subcultivation. CD90 and CD45 of the third passage cells were identified by flow cytometry. Using the cationic liposome reagent, Lipofectamine 2000, the plasmid pEGFP-N1-Nkx2.5 was transfected into BMSCs. The transfected cells were observed under an inverted fluorescence microscope and expression of Nkx2.5 was examined with immunocytochemistry. Expression of cardiac troponin T(cTnT) and GATA4 were determined with Western blotting and immunocytochemistry. Results 99% of the third passage cells were positive for CD90 and negative for CD45 on flow cytometry. After 48 hours of transfection, some cells in the transfected group expressed the green Nkx2.5-EGFP fusion protein and Nkx2.5 was expressed only in pEGFP-N1-Nkx2.5 transfected group (n =3). After 4 weeks, Western blotting results indicated that the expression of cTnT in pEGFP-N1-Nkx2.5 transfected group was significantly higher than those in pEGFP-N1 vector transfected group and in control one (EM>n/EM> =3). Immunocytochemistry results showed that the expressions of cTnT and GATA4 in pEGFP-N1-Nkx2.5 transfected group were respectively the highest in the three groups ( EM>n/EM> =3). Conclusion Exogenous expression of Nkx2.5 gene may enhance the cardiomyogenic differentiation of BMSCs.