目的MiRNAs筛选、转染及其对人脐带间充质干细胞向心肌细胞转化的调节研究

Umbilical cord mesenchymal stem cells (UCMSCs) have been one of the important sources of stem cell for transplant therapy. Now, studies on UCMSCs transplants for treating myocardial infarction have also been a research hot spots in the worldwide. But, the therapeutic efficiency of UCMSC transplants for cardiac infarction is unsatisfying, because UCMSCs transplanted into infarcted tissues can not generate enough cardiomyocytes to replace the damaged cardiomyocytes. MicroRNAs (miRNAs) are endogenous and small RNAs that play a key role in regulation of physiological processes and genesis of many diseases. Recent studies showed that a part of miRNAs regulate the differentiation of stem cells to many cell lineages. So, we hypothesize that some miRNAs may participate in regulation of cardiomyocyte differentiation of UCMSCs. In the present study, we will find several miRNAs that regulate cardiomyocyte differentiation of UCMSCs by using miRNA microarray and gene transfection combined methods, check the effects of overexpression and inhibition of the selected target miRNAs on cardiomyocyte differentiation of UCMSCs, and compare the advantages and disadvantages of transfection of the selected miRNAs and the general chemical inductive method on induction of UCMSCs differentiating into cardiomyocytes. Finally, we will use heart infarcted mice models to check the efficiency of UCMSCs transfected with miRNAs transforming into cardiomyocytes in vivo, and assess the therapeutic efficiency of UCMSC transplants on heart infarction and heart function recovery .

脐带间充质干细胞(UCMSCs)是临床干细胞移植的主要来源细胞之一。近年来，有关其移植治疗心肌梗死的研究已成为国内外研究的热点。但目前疗效并不理想，主要原因是移植到体内的干细胞并不能产生有效治疗数量的心肌细胞。MiRNAs是新近发现的一类内源性小片段RNA，对机体正常生理功能发挥和疾病的发生、发展都起着重要的调节作用。研究显示，部分miRNAs具有调节干细胞向多种细胞分化的功能。我们推测：可能有部分miRNAs参与调控UCMSCs心肌细胞分化。本项目拟通过miRNA芯片和基因转染相结合的方法筛选出数种能调节UCMSCs心肌细胞分化的miRNAs，检测其过表达或抑制对UCMSCs向心肌分化的影响，并比较其与常规化学诱导法对调节MSCs心肌细胞分化上的优劣性。最后，将经转染miRNAs的UCMSCs移植到小鼠模型心肌梗死部位，观察其在体内向心肌细胞转化的效率以及对心肌梗死的治疗效果。

脐带间充质干细胞（UCMSCs）是临床干细胞移植的主要来源细胞之一。近年来，很多课题组均尝试利用UCMSCs移植治疗心肌梗死，但其疗效并不理想。其主要原因是移植到体内的干细胞并不能产生有效数量心肌细胞。因此，寻求调控MSCs向心肌细胞分化的新途径已成为相关领域内研究的热点问题。相关研究显示，部分miRNAs具有潜在调节干细胞分化的功能。按照课题设计，我们首先完成了UCMSCs分离、培养、生理活性检测和干细胞特征鉴定等工作。同时，利用氧化型低密度脂蛋白（ox-LDL）及ox-LDL联合5-氮杂胞苷（5-Aza）诱导UCMSCs心肌分化，并鉴定上述方法对诱导UCMSCs心肌细胞分化的效率，为利用芯片筛选目的miRNAs奠定基础。在此基础上，我们利用miRNA芯片技术筛选出UCMSCs（经ox-LDL联合5-氮杂胞苷诱导）向心肌样细胞分化过程中发生明显变化的miRNAs，而后利用PCR进一步验证上述miRNAs，最后结合生物信息学分析筛选出具有潜在调节心肌分化的miRNA-1a、miRNA-322和miRNA-155-3p作为目的miRNAs。我们转染上述3种miRNAs的mimics或inhibitors，通过检测β-MHC、Ctnt、α-cardiac actin和myosin等心肌标记分子在UCMSCs中的表达分析干预上述miRNAs表达对UCMSCs向心肌分化的影响。结果发现，过表达miRNA-1a 和抑制miRNA-155-3p 表达具有促心肌分化功能。我们将未经处理的UCMSCs 、干预miRNA-1a 和miRNA-155-3p表达的UCMSCs 及经ox-LDL联合5-Aza诱导的UCMSCs分别经静脉输入到心肌梗死模型小鼠体内，检测移植上述UCMSCs对小鼠心脏功能的影响及其在体内分化情况。结果发现，抑制上述3种处理后UCMSCs均具有改善心肌梗死小鼠模型心功能的作用，并发现干预miRNA-1a表达具有较强的促进UCMSCs向心肌样细胞分化的功能。我们还检测了转染miRNA-1a 和miRNA-155-3p mimics或inhibitors对UCMSCs增殖和凋亡的影响，并发现干预上述miRNAs表达并不影响细胞增殖和凋亡。项目尝试通过调控miRNAs的表达探讨调控UCMSCs向心肌样细胞转化的新方法，为临床UCMSCs移植治疗心肌梗死提供参考。