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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。
OBJECTIVE: Isli protein plays an important role in the regulation of cardiac and pluripotent cardiac progenitor cells. The aim of this study was to investigate whether transfection of isl1 gene can enhance the ability of BMSCs to differentiate into cardiomyocyte-like cells. Methods: Human bone marrow-derived mesenchymal stem cells (MSCs) were infected with lentivirus carrying isl1 gene (LVS-isl1). The stable cell lines were screened by Puromycin and identified by RT-PCR and Western blotting. On the basis of co-culture, real-time PCR, Western blotting and immunofluorescence were used to detect the myocardial inducing capacity of Isl1 protein. Results: The stable cell line BMSC-isl1 was successfully obtained by RT-PCR and Western blotting. The results of real time-PCR showed that GATA-Binding protein 4, NK2 transcription factor 5 and myocyte increased in BMSC-isl1 group compared with BMSC- The expression of factor 2C and ryanodine receptor 2 were increased by 2.3, 2.7, 2.6 and 3.2 times, respectively. Western blotting and immunofluorescence indicated that the expression of cardiac troponin T, cardiac troponin I and α-actinin expression than BMSCs group and BMSC-vehicle group was higher. CONCLUSION: BMSCs transfected with isl gene can not only express Isli protein stably, but also improve the ability of BMSCs to differentiate into cardiomyocytes under co-culture of microenvironment.