CD4+辅助性T淋巴细胞对肝纤维化大鼠肝窦内皮细胞及促发肝窦毛细血管化的影响

Hepatic sinusoidal capillarization?is initial pathologic change of hepatic fibrosis and cirrhosis that are great burden to the society.Hepatic sinusoidal capillarization is characterized by gradually decrease fenestrae on the hepatic sinusoidal endothelial cell.Hepatic sinusoidal endothelial cell from chronic liver injury can express vascular adhesion protein 1 (VAP-1) and?α4β1 integrin,which can respectively adhere CD4 positive helper T lymphocyte 1 （TH1）and 2（TH2）.Hepatic fibrosis is relative to inflammation and immunologic derangement.We previously found that antiimflammaiton and immunorepression contributed to immunoregulation of CD4 positive helper T lymphocyte 1 and 2, and subsequent improvement of liver function and hepatic fibrosis of hepatic fibrotic rats.With the result of cocultrue,We considered that TH1 and TH2 could make great influence on hepatic sinusoidal endothelial cell.However, the specific influence remains unknown.In this project, anti VAP-1 and α4β1 integrin antibody will be used and subsequent in vivo and in vitro experiment will be performed to detect changes of cytoskeleton and Rho A/Rho kinase signal pathway in hepatic sinusoidal endothelial cell,which can reveal the mechanism of how TH1 and TH2 influence hepatic sinusoidal endothelial cell and the sinusoidal capillarization.This project will be helpful for blocking sinusoidal capillarization from aspect of immunoregulation.

肝窦毛细血管化是肝纤维化、肝硬化的基础病理改变，肝硬化危害性大。肝窦内皮细胞上窗孔逐渐减少或消失是肝窦毛细血管化的主要特征。肝纤维化与机体炎症免疫状态密切相关。慢性肝损伤的肝窦内皮细胞可表达血管黏附蛋白1(VAP-1)和α4β1整合素，并分别黏附CD4+辅助性T淋巴细胞1（TH1）和2（TH2）。申请人前期研究发现，抗炎和抑制免疫可明显调节肝纤维化大鼠TH1和TH2的功能，并且可明显改善大鼠肝功能及肝纤维化，结合初步的共培养结果，我们认为，TH1和TH2细胞对肝窦内皮细胞具有重要影响。但目前TH1和TH2细胞对肝窦内皮细胞以及肝窦毛细血管化的具体作用尚不明确。本研究将通过VAP-1和α4β1整合素抗体封闭及体内外实验检测肝窦内皮细胞骨架变化以及Rho A/Rho激酶信号通路的激活情况，明确TH1和TH2对肝窦内皮细胞以及肝窦毛细血管化的作用，可望从免疫角度为阻断肝窦毛细血管化提供理论依据。

课题组完成了CD4+辅助性T淋巴细胞对肝纤维化大鼠肝窦内皮细胞毛细血管化的作用及其机制的确立。建立Wistar大鼠肝纤维化模型，确定大鼠分组，通过体内实验：经大鼠尾静脉注射抗受损肝窦内皮细胞表达的VAP-1和α4整合素抗体，以阻断肝窦内皮细胞对CD4+辅助性T淋巴细胞的黏附，我们发现，阻断肝窦内皮细胞对CD4+辅助性T淋巴细胞的黏附后，CD4+T淋巴细胞经肝窦内皮细胞移位明显减少，肝窦内皮细胞窗孔孔径变大，毛细血管化的程度减轻；在体外实验中，肝窦内皮细胞与CD4+辅助性T淋巴细胞共培养后，我们通过对肝窦内皮细胞骨架变化以及Rho A/Rho激酶信号通路的激活情况的检测发现：CD4+辅助性T淋巴细胞可以引起肝窦内皮细胞骨架的变化，引起窗孔缩小，并激活Rho A/Rho激酶信号通路，导致毛细血管化的产生。以上结果表明：CD4+辅助性T淋巴细胞可以促进肝窦内皮细胞毛细血管化进而引发大鼠肝纤维化的产生。