转染isl1基因促进骨髓间充质干细胞向心肌样细胞分化

摘要

AIM:To investigate the role of Isll protein in the differentiation of bone marrow mesenchymal stem cells ( BMSCs ) into cardiomyocyte in cardiac micro environment. METHODS:BMSC - isll stable cell line was constructed using lentivirus vector. BMSCs, BMSC - vehicle and BMSC - isll with neonatal rat cardiomyocytes were co - cultured in a ratio of 1:10 and separated by Transwell chambers for 1 week. The expression profiles of the myocardial intra - markers were tested to determine the differentiation efficiency of BMSC - isll stable cell line by real - time PCR, Western blotting and immunofluorescence analysis. RESULTS: A BMSC - isll stable cell line was obtained. After co - cultured for 1 week, the results of real -time PCR demonstrated that the expression levels of GATA binding protein 4 ( GATA4 ), NK2 transcription factor related, locus 5 ( Nkx2. 5 ), myocyte enhancer factor 2C ( MEF2C ) and ryanodine receptor 2 ( RyR2 ) were higher (2.3 - ,2.7 - ,2.6- and 3. 2 -fold, respectively ) in BMSC - isll stable cell line than those in BMSCs and BMSC - vehicle. The results of Western blotting and immunofluorescence showed that the expression levels of cardiac tropo-nin T ( cTnT ), cardiac troponin I ( cTnl) and a - actinin significantly increased in BMSC - isll compared with BMSCs and BMSC -vehicle. CONCLUSION: The differentiation efficiency of BMSCs into cardiomyocytes is higher in BMSC - isll stable cell line than that in the primary BMSCs.%目的:Isli蛋白在心脏发生和多能心脏祖细胞分化调节中发挥着重要作用,本文旨在探寻转染isl1基因是否能提高骨髓间充质干细胞(BMSCs)向心肌样细胞分化的能力.方法:利用携带isl1基因的慢病毒(LVS-isl1)感染人骨髓间充质干细胞,通过Puromycin筛选获得稳定细胞株,并对其进行RT-PCR和Western blotting鉴定.以共培养为基础,用real-time PCR、Western blotting以及免疫荧光法检测Isl1蛋白的心肌诱导能力.结果:RT-PCR和Western blotting表明成功获得稳定细胞株BMSC-isl1,real time-PCR结果显示BMSC-isl1组较之BMSCs组和BMSC-vehicle组GATA 结合蛋白4、NK2转录因子5、肌细胞增强因子2C和兰尼碱受体2的表达量分别提高了2.3、2.7、2.6和3.2倍;Western blotting以及免疫荧光法结果均提示BMSC-isl1组内心肌肌钙蛋白T、心肌肌钙蛋白I和α-辅肌动蛋白的表达均比BMSCs组和BMSC-vehicle组要高.结论:转染isl基因的BMSCs不仅可以稳定表达Isli蛋白,而且在微环境共培养条件下,可以提高BMSCs向心肌细胞分化的能力.