转染isl1基因促进骨髓间充质干细胞向心肌样细胞分化  

isl1gene transfection promotes BMSCs to diffenentiate into cardiomyocytes

作　　者：李杏肖[1] 李晓红[1] 林秋雄[1] 肖定璋[1] 刘晓颖[1] 单志新[1] 朱杰宁[1] 麦丽萍[1] 夏慧苏[1] 余细勇[1] 

机构地区：[1]广东省心血管病研究所,广东省人民医院,广东省医学科学院,广东广州510080

出　　处：《中国病理生理杂志》2012年第1期131-135,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金重大国际合作项目(No.81120108003);国家自然科学青年基金资助项目(No.30900610);广东省自然科学基金资助项目(No.9451008002003467)

摘　　要：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。AIM： To investigate the role of Isl1 protein in the differentiation of bone marrow mesenchymal stem cells（BMSCs） into cardiomyocyte in cardiac microenvironment.METHODS： BMSC-isl1 stable cell line was constructed using lentivirus vector.BMSCs,BMSC-vehicle and BMSC-isl1 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-isl1 stable cell line by real-time PCR,Western blotting and immunofluorescence analysis.RESULTS： A BMSC-isl1 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-isl1 stable cell line than those in BMSCs and BMSC-vehicle.The results of Western blotting and immunofluorescence showed that the expression levels of cardiac troponin T（cTnT）,cardiac troponin I（cTnI） and α-actinin significantly increased in BMSC-isl1 compared with BMSCs and BMSC-vehicle.CONCLUSION： The differentiation efficiency of BMSCs into cardiomyocytes is higher in BMSC-isl1 stable cell line than that in the primary BMSCs.

关 键 词：骨髓间充质干细胞 慢病毒 Isli蛋白 微环境 心肌样细胞 

分 类 号：R363[医药卫生—病理学]