维生素D活化障碍促进胆道闭锁肝纤维化的分子机制研究

Biliary Atresia (BA) is the most common biliary obstructive disease in infants. Idiopathic liver fibrosis, rather than secondary to cholestasis is one of the most important pathological change in BA patients, which is supported by clinical data. It was reported that Vitamin D (VD) can inhibit the activation of Hepatic stellate cells (HSC) and the development of liver fibrosis. Our previous clinical research has implied that activation disorder of VD might existed in BA, and we have verified that CYP27A1, the key enzyme in the pathway of 25-hydroxy vitamin D (25(OH)D) biosynthesis from VD, was down regulated in the liver of BA. The expression of CYP27A1 is regulated by the methylation level of promoter region. And we also confirmed by pyro-sequencing that the promoter region of CYP27A1 gene was hyper-methylated in the liver of BA patients. Therefore, we hypothesise that a regulatory pathway, “DNA hypermethylation - low expression of CYP27A1 - reduction of 25(OH)D synthesis – HSC over activated – aggravation of liver fibrosis”play a role in development of liver fibrosis in BA. The activation of this regulatory pathway and the markers of liver fibrosis are intended to be measured and compared between the BA patients’ liver and the control in this study. The expression of CYP27A1 is planned to be intervened in LX-2 cell line and BA animal models, and the exogenous activated VD and VDR antibody are also planned to be used, then the liver fibrosis markers and success rate of establishment of BA model will be detected and compared. Thus, this regulatory pathway can be exploded and verified at the level of human tissue, cell and animal model.

胆道闭锁（BA）婴幼儿最常见的胆道梗阻性疾病，临床资料显示肝纤维化是BA原发性致病因素，而不仅是继发性的胆汁性硬化。研究发现维生素D（VD）能抑制肝星形细胞（HSC）活化和肝纤维化的发生。前期研究显示BA患儿可能存在VD活化障碍，且已证实BA肝脏中VD合成25-羟维生素D（25(OH)D）的关键酶CYP27A1表达降低。CYP27A1的表达受启动子区DNA甲基化水平调控，已证实BA肝脏中该启动子区甲基化水平升高。故推测“DNA高甲基化—CYP27A1表达降低—25(OH)D合成减少—HSC过度活化—肝纤维化加重”这一调控通路促进BA肝纤维化。本研究拟比较BA和正常肝脏中该通路的活性和肝纤维化标记的表达情况，采用LX-2细胞系及BA动物模型，干预CYP27A1的表达、加入外源性活性VD或VD受体拮抗剂观察肝纤维化标记的表达及动物模型成模率的变化。 从人体、细胞和动物模型三个层面探讨这一机制。

胆道闭锁（biliary atresia，BA）婴幼儿最常见的胆道梗阻性疾病，大部分患儿存在进行性的肝纤维化。近年研究发现维生素D（VD）在肺、肾等多种组织中具有抗纤维化的作用。最新的研究发现VD受体大量表达于肝星状细胞（HSC）等肝脏非实质细胞，这一发现提示VD可能是肝纤维化的一种调节因子。体内实验和动物实验均显示VD在肝纤维化的发生中发挥重要作用。近年来有研究发现VD可降低体HSC的增殖增殖活性，并抑制Col-1α1、TIMP-1等促纤维化基因的表达，促进BMP7、MMP8、MMP9等抗纤维化基因的表达。.本课题组前期研究已发现BA患儿 VD缺乏率达31.3%，25羟VD（25(OH)D）缺乏率更是高达89.6%，这一差别提示BA患儿可能存在VD向25(OH)D转化障碍。而通过对 BA患者血清25(OH)D与肝纤维化分级的研究发现，25(OH)D与肝纤维化分级呈负相关。本课题通过对胆道闭锁患儿的临床资料、肝脏标本、原代HSC及动物模型等不同层面的研究，进一步揭示VD代谢异常和胆道闭锁肝纤维化之间的内在关系。结果显示：BA患儿术前25(OH)D缺乏现象常见。25(OH)D水平受季节影响。25(OH)D可减少重度肝纤维化的发生率，III型前胶原N端肽的表达降低可能是其机制之一。BA患儿存在VD活化障碍现象（25羟化障碍），肝脏内25羟化酶的低表达是其主要机制。VD可抑制BA原代HSC TGF-β1、Col-1α1、α-SMA等纤维化相关基因的表达，并抑制其增殖和迁移，具有抗纤维化作用。CYP2R1的表达和肝纤维化有关，CYP2R1可以抑制肝纤维化。可能的机制是CYP2R1通过增强VD的生物转化，产生更多具有生物活性的VD代谢产物，并通过抑制TGF-β1信号通路的方式调节MMPs和TIMPs的平衡，从而减少胶原的产生，抑制细胞外基质的形成。.本研究一定程度上揭示了BA患儿VD的代谢障碍与肝纤维化的内在分子生物学机制，为BA抗纤维化治疗提供新的理论依据和治疗靶点。