LncRNA-Braveheart促进骨髓间充质干细胞在体外向心肌样细胞分化  

LncRNA-Braveheart promotes the differentiation of bone marrow mesenchymal stem cells in vitro into cardiomyocyte-like cells

作　　者：侯婧瑛[1] 龙会宝[1] 周长青[1] 吴浩[1] 郭天柱[1] 钟婷婷[1] 伍权华[1] 汪蕾[1] 郑韶欣[1] 王彤[1] Hou Jing-ying;Long Hui-bao;Zhou Chang-qing;Wu Hao;Guo Tian-zhu;Zhong Ting-ting;Wu Quan-hua;Wang Lei;Zheng Shao-xin;Wang Tong(Department of Emergency, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University,Guangzhou 510120, Guangdong Province, China)

机构地区：[1]中山大学孙逸仙纪念医院急诊科,广东省广州市510120

出　　处：《中国组织工程研究》2017年第29期4593-4599,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81070125)"抗凋亡与促血管生成miRNA-378干预MSCs治疗心梗后心衰的机制研究";国家自然科学基金(81270213)"ANGⅡ/AT1/SMAD/CX43通路在心肌干细胞提高心梗大鼠心电生理学稳定性和室颤阈值的作用机制研究";国家自然科学基金(81670306)"心肌干细胞经IGF-1/STAT/miR-155通路下调AT1R改善心肌梗死后心电生理稳定性机理研究";广东省科技计划项目(2014A020211002)"LncRNA-Bvht/MESP1/N-cadherin通路调控MSCs向心肌细胞定向分化的机制研究";广东省科技计划项目(2010B031600032)"抗凋亡与促血管生成miRNA-378干预MSCs治疗心梗后心衰的机制研究";高校基本科研业务费中山大学青年教师重点培育项目(13ykzd16)"PPAR-γ/TGF-β1/Smad/CX43通路在PPAR-γ干预MSCs治疗心梗后心衰的疗效及机制研究";广东省医学科研基金(A2016264)"心肌干细胞经由HIF-1α/Apelin/APJ/ACE2通路下调ANGII改善心肌梗死大鼠心电生理学稳定性的机制研究"~~

摘　　要：背景:课题组前期研究发现骨髓间充质干细胞移植能够改善大鼠心肌梗死后心功能,但整体效果并不太理想,骨髓间充质干细胞在体内局部梗死微环境中的分化效率低下,其向心肌细胞分化的能力极其有限。目的:采用lncRNA-Bvht体外转染骨髓间充质干细胞,观察其对骨髓间充质干细胞向心肌样细胞分化的影响。方法:构建含lncRNA-Bvht的病毒载体p LVX-IRES-Zs Green1-lncRNA-Bvht,对骨髓间充质干细胞进行lncRNA-Bvht转染并检测转染效率。分离培养C57BL/6小鼠的骨髓间充质干细胞,并将第3代细胞分为3组:骨髓间充质干细胞组、空载体组和lncRNA-Bvht组。各组细胞培养48 h后用5-氮杂胞苷进行诱导分化24 h再进行正常培养2周,荧光显微镜观察骨髓间充质干细胞向心肌细胞分化情况,免疫荧光染色、Western blot、qRT-PCR检测心肌分化标记物肌钙蛋白T和肌节蛋白的表达。结果与结论:①诱导分化后细胞形态发生改变,细胞之间形成连接,排列方向趋于一致;②免疫荧光检测结果显示lncRNA-Bvht组肌钙蛋白T和肌节蛋白表达呈现强阳性;肌钙蛋白T阳性细胞的比例显著增加;③qRT-PCR和Western blot检测结果显示lncRNA-Bvht组肌钙蛋白T和肌节蛋白的表达较空载体组和骨髓间充质干细胞组均明显升高(P<0.01);④结果提示,lncRNA-Bvht转染能够有效促进骨髓间充质干细胞在体外向心肌样细胞发生分化。BACKGROUND:Our previous work demonstrated that bone marrow mesenchymal stem cells(BMSCs)transplantation could improve cardiac function in rats with myocardial infarction.However,the overall efficacy wasunsatisfactory,and there was a low efficiency of BMSCs differentiating into cardiomyocytes in the local infarctmyocardium.OBJECTIVE:To transfect long non-coding RNA-Braveheart(lncRNA-Bvht)into BMSCs in order to observe whether itcould promote cardiomyocyte differentiation of BMSCs in vitro.METHODS:pLVX-IRES-ZsGreen1-lncRNA-Bvht vector was constructed and applied to transfect lncRNA-Bvht intobMSCs,and then,the transfection efficiency was detected.BMSCs were obtained from C57BL/6mice and cultured invitro.Passage3cells were divided into three groups:BMSCs group,null vector group and lncRNA-Bvht group.Allcells in the three groups were cultured in the normal condition for48hours and cardiomyocytes differentiation wasinduced by5-azacytidine for another24hours followed by2-week culture under normal conditions.Cardiomyocytedifferentiation of BMSCs was observed under fluorescence microscopy and expression of cardiac specific cellmarkers including troponin T and myosin were examined using immunofluorescent staining,western blot assay,andqRT-PCR.RESULTS AND CONCLUSION:Cell morphological changes could be observed in all the groups2weeks after theinduction.Interconnected cells arranged consistently in all the three groups.Immunofluorescent staining resultsshowed that the expression of troponin T and myosin was notably positive,and the proportion of troponin T positivecells was significantly increased.qRT-PCR and western blot assay results indicated that there were significantlyincreased levels of troponin T and myosin in the lncRNA-Bvht group as compared with the BMSCs and null vectorgroups(P<0.01),suggesting that lncRNA-Bvht could efficiently promote cardiomyocyte differentiation of BMSCs invitro.

关 键 词：心肌梗塞 骨髓 间质干细胞 肌细胞 心脏 组织工程 

分 类 号：R394.2[医药卫生—医学遗传学]