The Role of Stem Cell Derived Microvesicles in Cholestatic Liver Injury

 描述(申请人提供)：人类肝脏的功能对生命是必不可少的，因为肝脏是唯一能够再生的器官。人肝干细胞(HLSCs)具有修复、再生和免疫调节等特性，已被广泛研究。几项使用不同疾病动物模型的研究表明，外源性HLSCs治疗可以缓解包括肝脏疾病在内的急性器官损伤。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。虽然干细胞疗法已被用于肝病的临床前治疗，但S对干细胞来源的微泡(MVS)及其相关的非编码RNA(NcRNAs)知之甚少，它们可以介导促进肝病进展和恢复的基因变化。许多ncRNAs以组织特异性的方式表达，这种方式在人类酒精性肝损伤中发生了异常变化。大多数ncRNAs在LIVR疾病中的生物学功能尚不明确。在我们的初步研究中，我们已经发现，在胆汁淤积性肝损伤后，选定的ncRNA基因发生了改变，并在人肝干细胞及其衍生的MVs中异常表达，这些细胞可以调节对肝损伤的反应，以及细胞的抗衰老和重塑能力。基于这些有说服力的数据，我们提出了一个中心假设，即干细胞来源的微泡中的ncRNAs通过诱导肝胆组织和细胞的生长和抗衰老/抗纤维化来促进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，并建立了稳定转染或击倒的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来源的微泡，并表征它们在组织修复方面的功能特性。在这一应用中，我们建议系统评价干细胞来源的MVS中干细胞依赖的ncRNAs作为标志物对胆汁淤积性肝损伤的治疗潜力。我们将通过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干调控的ncRNAs信号。其次，我们将鉴定功能性的依赖于内毒素/转化生长因子的miRNAs，这些miRNAs参与了胆汁淤积性肝损伤的存活和抗衰老。第三，我们将在BDL或CCl4治疗的体内以及在mdr2缺陷小鼠(PSC的遗传性小鼠模型)中，确定茎相关ncRNAs丰富的微囊在促进胆汁淤积性肝损伤的形态和功能恢复方面的作用。将评估抗miRNAs或高表达T-UCRs的干细胞来源的MVS对肝胆细胞生长、抗衰老和抗纤维化的治疗作用。拟议的研究结果可能会为人类胆汁淤积性肝病带来新的治疗策略。同时，获得的干细胞来源的MVS的基础新知识有望推动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.