miRNA介导人间充质干细胞直接转化为肝细胞的系统优化及机制探讨

At the post-transcriptional level, miRNAs are emerging as key players in the control of proliferation and cell fate determination during differentiation. Specific microRNAs can mediate cell reprogram. In a previous study, we elucidated the specific miRNA profile of hepatic differentiation. We found that downregulation of any one of these hepatic differentiation-specific miRNAs can inhibit HGF-induced hepatic differentiation including albumin expression and LDL uptake. Although overexpression of any one of these miRNAs alone or liver-enriched miR-122 cannot initiate hepatic differentiation, ectopic overexpression of seven miRNAs (miR-1246, miR-1290, miR-148a, miR-30a, miR-424, miR-542-5p and miR-122) together can stimulate human MSC conversion into functionally mature induced hepatocytes (iHep). Additionally, after transplantation of the iHep cells into mice with CCL4-induced liver injury, iHep not only can improve liver function but it also can restore injured livers. The findings indicate that miRNAs have the capability of directly converting human MSCs to a hepatocyte phenotype in vitro. To determine the essential factors among the 7 candidate miRNAs, we will examine the effects of withdrawing individual factors from the 7 mix pool in this study by the hepatic function analysis of induced cells in vitro and in vivo. Finally, we hope to obtain a better miRNAs combination that can convert human MSC into hepatocyte directly and efficiently. Additionally, we will try to clarify the mechanism of miRNAs mediated hepatic differentiation in human MSC by bioinformatics analysis and Mass Spectrometry & Protein Analysis. This can provide a theoretical basis for improving hepatic differentiation efficiency of stem cells in vivo and obtaining hepatocyte efficiently in vitro.

miRNAs决定多种细胞的分化命运，并介导细胞的重编程。但miRNA在间充质干细胞（MSC）肝向分化中的作用尚不清楚。本课题组前期筛选并鉴定了一组MSC肝向分化特异性的miRNA谱。发现这一组miRNA在MSC肝向分化中起重要作用，即外源性抑制miRNA的活性可以抑制MSC向肝细胞的分化。并首次证实7个miRNA组合可以更高效更快捷的启动MSC直接转化为在体内外均可以发挥功能的肝细胞。但能否使用更少的miRNA分子启动这一过程，以及miRNA分子启动MSC肝向分化的机制尚不清楚。本项目拟在此基础上，利用筛减方法，逐一减少miRNA分子，通过体内外检测肝细胞特异性基因的表达情况及肝细胞特异性的功能，进一步寻找更优化的促使MSC转化为肝细胞的miRNA 组合，同时，通过蛋白质谱技术及生物信息学分析，探讨miRNA调控的信号通路，以明确miRNA组合调控MSC肝向分化的机制。

课题组前期利用7个miRNA成功地将脐带间充质干细胞转分化为肝细胞样细胞（HLC-7），不仅在体外证实诱导细胞HLC-7具有糖原合成，尿素合成和LDL摄取能力，还在体内证实诱导的HLC-7可以促进急性肝损伤动物模型的修复。那么，能否使用更少的miRNA组合实现这个转分化过程呢？基于此，本项目采用 n-1 方法继续筛减和优化诱导干细胞转分化为HLC的miRNA组合，并对肝损伤小鼠进行细胞移植治疗，观察诱导细胞对裸鼠肝损伤和急性肝衰竭动物模型的修复作用。发现miR-1290和miR-30a为非必需miRNA,而miR-122、miR-1246，miR-542-5p、 miR-424、 miR-148a为必需miRNA,无论减少哪一个均会影响间充质干细胞向肝细胞的分化。诱导得到的HLC-5不仅在体外具备肝细胞的功能，在动物模型体内也可发挥与HLC-7 相似的损伤修复功能，并可显著改善肝衰竭裸鼠的生存情况。随后，利用多个miRNA 靶基因预测的数据库对miRNA组合的靶基因进行预测，取交集获得交集靶基因后，进行生物通路富集分析，提取出最相关靶基因。通过功能注释建立统计模型，分析整个基因集中最显著的功能，计算这些基因在 GO 分类中分布关系。最后，以miRNA-mRNA靶向关系和靶基因总集的蛋白互作关系，构建了包含WNT信号通路AXIN2分子和TGF-β信号通路的TGF-β分子在内的miRNA作用网络。从而初步阐释了miRNA调控MSC转分化为肝细胞的机制，本项目取得的主要核心成果已经发表于2017年J Cell Mol Med 杂志（IF=4.499），发表中文核心期刊论文3篇，参与撰写英文专著1项，培养硕士研究生2名，1名博士生在读，2次获得青年优秀论文奖。基于本项目成果，项目负责人于2017年获邀担任中华医学会组织修复与再生分会干细胞与肝脏损伤修复学组委员。