The Role of Stem Cell Derived Microvesicles in Cholestatic Liver Injury

 描述（由申请人提供）：人体肝脏的功能对生命至关重要，因为肝脏是唯一能够再生的器官。人类肝脏干细胞（HLSC）的修复、再生和免疫调节特性已被广泛研究。使用不同疾病动物模型的几项研究表明，用外源性HLSC治疗可改善急性器官损伤，包括肝脏疾病。其机制可能涉及旁分泌因子促进存活的固有上皮细胞的增殖。虽然干细胞疗法已在临床前用于治疗肝脏疾病，但对干细胞衍生的微泡（MV）及其相关的非编码RNA（ncRNA）知之甚少，所述微泡及其相关的非编码RNA可介导促进肝脏疾病的进展和恢复的遗传变化。许多ncRNA以组织特异性方式表达，在人类酒精性肝损伤中发生异常改变。大多数ncRNA在肝脏疾病中的生物学功能尚不明确。在我们的初步研究中，我们已经表明选定的ncRNA基因在胆汁淤积性肝损伤后发生改变，并在人肝干细胞及其衍生的MV中异常表达，这些基因可以调节对肝损伤的反应以及细胞抗衰老和重塑潜力。基于这些令人信服的数据，我们提出了中心假设，即干细胞衍生的微泡中的ncRNA通过诱导肝胆组织和细胞的生长和抗衰老/抗纤维化而有助于胆汁淤积性肝损伤的恢复。为了验证这一假设，我们建立了ncRNA基因操作，功能和相互作用分析的技术，并产生稳定转染或敲低细胞系以及胆汁淤积性肝损伤的动物模型。我们的长期目标是鉴定和分离干细胞衍生的微泡，并表征其组织修复的功能特性。在本申请中，我们提出了系统评价干细胞依赖性ncRNA作为干细胞衍生的MV中具有治疗胆汁淤积性肝损伤潜力的标志物。我们将通过关注以下具体目标来解决我们的中心假设：首先，我们将定义肝胆细胞中参与组织修复相关细胞功能的功能性干性调节的ncRNA信号传导。其次，我们将鉴定在胆汁淤积性肝损伤期间参与存活和抗衰老的功能性LPS/TGF-β依赖性miRNA。第三，我们将确定干性相关的ncRNA富集的微泡对加速使用BDL或CCl 4治疗的体内胆汁淤积性肝损伤的形态和功能恢复的作用，以及在Mdr 2缺陷小鼠（PSC的遗传小鼠模型）中的作用。将评价来源于具有抗miRNA或过表达T-UCR的干细胞的MV对肝胆细胞生长、抗衰老和抗纤维化的治疗作用。这些研究结果可能为人类胆汁淤积性肝病提供新的治疗策略。同时，所获得的关于干细胞衍生的MV的基础新知识有望推进干细胞生物学的一般领域。

 DESCRIPTION (provided by applicant): The functions of the human liver are essential to life since the liver is the only organ capable of regeneration. Human liver stem cells (HLSCs) have been extensively studied for their reparative, regenerative and immunomodulatory properties. Several studies, using different animal models of diseases, showed that treatment with exogenous HLSCs ameliorates acute organ injury including hepatic disorders. The mechanisms may involve paracrine factors promoting proliferation of surviving intrinsic epithelial cells. Whil stem cell therapies have been in pre-clinical use for the treatment of liver diseases, very little s known about the stem cell derived microvesicles (MVs) and their related non-coding RNAs (ncRNAs), which can mediate genetic changes that promote the progression and recovery of liver disorders. Many ncRNAs are expressed in a tissue-specific manner that is aberrantly altered in human alcoholic liver injuries. The biological function of the majority of ncRNAs in livr diseases is undefined. In our preliminary studies, we have shown that selected ncRNA genes are altered after cholestatic liver injuries, and aberrantly expressed in human liver stem cells and their derived MVs that can modulate the response to liver injury as well as cell anti-senescence and remodeling potentials. Based on these compelling data, we propose the central hypothesis that ncRNAs in stem cell derived microvesicles contribute to the recovery of cholestatic liver injury through induction of growth and anti-senescence/anti-fibrosis of hepatobiliary tissues and cells. To test this hypothesis, we have established techniques for ncRNA gene manipulation, functional and interaction analysis, and generated stably transfected or knockdown cell lines as well as animal models of cholestatic liver injury. Our long-term objective is to identify and isolate stem cell derived microvesicles and to characterize their functional properties of tissue repair. In this application, we propose the systematic evaluation o stemness dependent ncRNAs as markers in stem cell derived MVs with the therapeutic potentials for cholestatic liver injury. We will address our central hypothesis by focusing on the following specific aims: First, we will define the functional stemness regulated ncRNAs signaling involved in tissue repair-related cellular functions in hepatobiliary cells. Second, we will identiy functional LPS/TGF-ß dependent miRNAs involved in survival and anti-senescence during cholestatic liver injury. Third, we will determine the effects of stemness related ncRNAs enriched microvesicles on accelerating the morphologic and functional recovery of cholestatic liver injury in vivo using BDL or CCl4 treatment and in Mdr2 deficient mice, the genetic mouse model of PSC. Therapeutic effects of MVs derived from stem cells with anti-miRNAs or over-expression of T-UCRs on hepatobiliary cell growth, anti- senescence and anti-fibrosis will be evaluated. The results of the proposed studies may lead to new therapeutic strategies for human cholestatic liver diseases. Meanwhile, the acquired fundamental new knowledge about stem cell derived MVs is expected to advance the general field of stem cell biology.