肝星状细胞诱导生成调节T细胞在肝纤维化机制中的研究

Objective: to study the role of retinoic acid (RA)-producing hepatic stellate cells (HSC) in liver-associated Treg induction as a result of liver fibrosis..Backgrounds: Liver fibrosis and cirrhosis result from the majority of chronic liver insults and represent a common and difficult clinical challenge of worldwide importance. In China, hepatitis and schistosomiasis are the two most common cause of cirrhosis. At present, the only curative treatment for end stage cirrhosis is transplantation. Recent developments in our understanding confirm that the process is dynamic with respect to the activation of HSCs to differentiate to myofibroblasts for production of extracellular matrix. HSCs within liver lobules are also called vitamin A-storing cells. Under physiological conditions, these cells play pivotal roles in the regulation of vitamin A homoeostasis, liver fibrosis and regulation of hepatic blood flow. Recent studies suggest the RA-producing HSC may be involved in TGF-β-dependent Foxp3+ Treg induction in pathological conditions as the part of the liver tolerance mechanism (1). However, several key issues are not addressed here. First, it is still controversial whether the antigen presenting cell function of HSC is involved in the induction of iTreg. Second, it is unclear whether the liver-associated iTreg induction favors fibrogenesis in liver injury. By understanding the mechanisms of how iTreg is induced in liver and further regulates the fibrosis, it may seem to provide a new insight into the treatment of advanced fibrosis..Based on preliminary work, our central hypothesis states accordingly that ① activated HSC respond to vasoactive substances in peripheral venous blood, such as nitric oxide (NO); ② the process of HSC activation triggers vitamin A metabolism and releases the highly bioactive form RA; ③ in the presence of RA, HSC may act as an immune cells and differentiate na?ve CD4+T cells into the Foxp3 expressing T cells; ④ iTreg themselves secrete large amounts of TGF-β and act reciprocally on HSCs. Thus, an increased local concentration of TGF-β may induce HSC to fully differentiate into myofibrolasts and provide a positive feedback loop in hepatic fibrosis; ⑤ simultaneously, iTreg may further mediate suppression of cellular immunity in the liver. This hypothesis will be studied under in vitro and in vivo conditions:.1..Conduct the in vitro test to understand whether the pre-activation of HSC is required by vasoactive substances such as NO releasers to regulate Foxp3 level in T cells..2..Using eGFP-Foxp3 reporter mice and adoptive transfer the GFP- na?ve CD4+T cells into a congenic recipient to examine the generation of GFP+iTreg in 3 different murine models of liver injury.

肝纤维化是慢性肝病发展到肝硬化的必经之路，是我国常见疾病和主要死亡病因之一。近年來, 淋巴细胞及细胞因子介导的免疫学机制成为肝纤维化病研究的热点。最新的研究表明，在肝纤维化进程中起核心作用的肝星状细胞（HSC）可直接诱导适应性调节性T细胞（iTreg）的产生，但其在慢性肝病进展中的作用尚不明确。本课题将拟研究iTreg主动参与肝纤维化进程的免疫学机制。将重点阐明肝损伤时，当HSC受血管活性递质一氧化氮激活后诱导产生iTreg，其分泌的TGF-β等炎症因子能反馈活化HSC、诱导淋巴比例失衡，从而加重肝纤维化进程。申请人将利用增强型绿色荧光蛋白(eGFP)－Foxp3转染小鼠模型，动态和准确追踪、并比较在不同病因所致的肝纤维化宿主体内，诱导而生的eGFP阳性iTreg的功能性和差异性。这将为寻找到干预靶点，更有效延缓并阻断肝纤维化提出一套新的细胞免疫学依据。

项目的背景： 最新的研究表明，在肝纤维化进程中起核心作用的肝星状细胞（HSC）可直接诱导适应性调节性T细胞（iTreg）的产生，但其在慢性肝病进展中的作用尚不明确。本课题将拟研究iTreg主动参与肝纤维化进程的免疫学机制。目的在于阐明肝损伤时，当HSC受血管活性递质一氧化氮激活后诱导产生iTreg，其分泌的TGF-β等炎症因子能反馈活化HSC、诱导淋巴比例失衡，从而加重肝纤维化进程。.主要研究内容：利用增强型绿色荧光蛋白(eGFP)－Foxp3转染小鼠模型，动态和准确追踪、并比较在不同病因所致的肝纤维化宿主体内，诱导而生的eGFP阳性iTreg的功能性和差异性。研究内容包括三个方面：1）完善了小鼠纤维化模型，2）探索肝纤维化机制，3）探索iTreg在肝纤维化的作用机制。.重要结果、关键数据：1）我们发现四氯化碳（CCl4）诱导肝损伤组里，部分幼稚T细胞（eGFP－Foxp3－）已转变为eGFP－Foxp3＋；2）通过免疫组化法，我们发现幼稚T细胞虽然已转变为eGFP－Foxp3＋，但于对照组相比，宿主肝纤维化特征并未有明显改变；3）通过用CD45.1、CD45.2等分子标记物由流式细胞仪提取移植到受体内的已转变为eGFP－Foxp3＋细胞，对新宿主的肝纤维化是有一定保护作用的。4）通过全基因的甲基化芯片对正常肝脏组织，及肝纤维化组织进行扫描,探索可以作为潜在的肝纤维化诊断的生物标记。 5）我们发现肝损伤、肝纤维化的组织中具有独特的全甲基化谱，有成千上万的显著高甲基化或低甲基化基因被分析出来。但通过iTreg治疗后密切相关基因差异甲基化基因不多。通过对特异基因进行疾病差异显 著性功能分析、信号通路分析和信号网络分析,我们挑选了12个基因进行进一步筛选。通过临床样本中这些基因的表达量和启动子甲基化验证,发现 IFITM1、CHST4、TBX15、SMAD6等的启动子高甲基化，低基因表达量和iTreg的植入密切相关,他们可能作为一个独立的预测肝脏损失及肝纤维化预后的因素。.科学意义：当前工作从而论证了这种精确的小鼠模型，能准确及动态的探索局部肝组织中iTreg的扩增、HSC的激活与肝纤维化临床特征的相关性。新发现异常甲基化基因主要富集的TGF-β、ERK信号通路和细胞增值相关，可能为肝纤维化发生机制相关，以及影响肝纤维化的预后。

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