67-kD层粘连蛋白受体在肝纤维化中的作用及其机制研究

Our previous studies have found that matrine derive MASM exhibits potent antifibrotic and antiinflamatory effects and specifically binds to the 67-kD laminin receptor (67LR). The 67LR antibody prevented hepatic stellate cell (HSC) activation and noticeably blocked inhibitory effect of MASM on HSC activation, indicating that MASM may act on extracellular 67LR. This observation was further supported by the 67LR knockdown that efficiently reduced extracelluar 67LR expression. In addition, immunohistochemical analysis of the experimental fibrotic liver revealed that the 67LR expression was strikingly increased and localized exclusively to the α-SMA positive cells. These results suggest that antifibrotic effect of MASM may be mediated by the 67LR and this receptor may represent a novel target for future therapeutic approach in liver fibrosis. The 67LR has been associated with cancer metastasis, bacterial and viral infection and other pathological events that make it a prime target for therapeutic interventions. There are not any reports about matrine and its derivatives as a 67LR ligand and involvement of the 67LR in hepatic fibrosis at present. This project will validate 67LR as cell membrane receptor of MASM using 67LR antibody, siRNA and known ligand EGCG. In addition, we will investigate the dynamic change of 67LR expression during the process of liver fibrosis. Moreover, the role of RPS5 in the pathological process of liver fibrosis will be studied in vitro and in vivo using lentivirus carrying HSC specific glial fibrillary acidic protein （GFAP）GFAP promoter-driver 67LR overexpression or knockdown as well as 67LR knockout mouse. Meanwhile, we will study whether benifical effect of MASM on hepatic fibrosis is lost in such systems. Lastly, we will identify 67LR cytosol binding proteins to investigate the signal transduction process, and investigate how these signal molecules affect MASM’s action. Together，our results will clarify the molecular mechanism of MASM for its antifibrotic effects and point to 67LR as a therapeutic target for liver fibrosis.

我们前期研究发现苦参碱衍生物MASM具有抗肝纤维化作用并特异结合层粘连蛋白受体(67LR)，67LR抗体和基因沉默可抑制肝星状细胞（HSC）活化并阻断MASM的作用，还发现纤维化肝脏中HSC高表达67LR，结果提示67LR 可能是介导MASM的作用靶点，并影响肝纤维化的进程。67LR参与肿瘤和感染等疾病过程使之成为药物干预的潜在靶点，但其与MASM抗肝纤维化作用及肝纤维化病理过程的关系未见报道。本项目将应用67LR抗体和小干扰RNA及其唯一的配体来确证67LR为MASM抗肝纤维化作用靶点；研究67LR在肝纤维化病理过程中的表达变化及其作用；采用基因敲除小鼠最终明确67LR在肝纤维化中的作用及对MASM作用的影响；初步探讨其细胞内信号通路以及通路是否影响MASM作用。研究结果将揭示67LR在肝纤维化中的作用，阐明MASM抗肝纤维化作用的细胞和分子机制，为肝纤维化治疗提供新的靶点和药物。

我们前期研究发现苦参碱衍生物MASM具有抗肝纤维化作用并特异结合层粘连蛋白受体67LR，本项目主要围绕67LR在肝纤维化病理过程中的作用以及是否可以作为肝纤维化治疗靶点进行深入研究。首先，我们探索了67LR对肝星状细胞（HSC）活化以及肝纤维化的影响，结果发现67LR配体对HSC活化无显著影响，而在敲除小鼠构建过程中发现，GFAP-Cre小鼠无法实现67LR肝星状细胞特异性敲除。接着，我们考虑构建67LR全身性敲除杂合子小鼠研究67LR与肝纤维化的联系，结果发现杂合子可能同全身性敲除纯合子一样胚胎致死。进一步，我们利用流式细胞术对HSC的67LR表达情况进行分析，结果显示HSC基本不表达67LR，因此，我们将方向调整为髓系细胞67LR的功能以及在肝纤维化中的作用研究。为此，我们构建了67LR髓系敲除小鼠，体外研究发现67LR髓系敲除能够显著抑制腹腔巨噬细胞以及骨髓来源的巨噬细胞的迁移，但是对炎症细胞因子的分泌没有影响，而MASM的迁移抑制作用在67LR敲除后消失。单核巨噬细胞的迁移在急慢性肝损伤过程中发挥重要作用，因此，我们进一步考察了67LR髓系敲除对急性肝损伤以及肝纤维化的影响。结果发现67LR髓系敲除并未对急性肝损伤或肝纤维化表现出明显影响，表明67LR影响的迁移过程在急慢性肝损伤模型中发挥的作用十分有限。除此之外，我们还考察了67LR髓系敲除对DSS诱导的结肠炎模型、盲肠结扎（CLP）引起的脓毒症模型的影响。结果发现67LR髓系敲除对DSS诱导的结肠炎并没有显著影响，而对盲肠结扎引起的脓毒症模型表现出缓解作用，即67LR髓系敲除能够显著延长CLP模型小鼠的生存率，抑制炎性细胞组织浸润，减轻组织损伤，表现出一定的组织保护作用。目前，相关研究正在深入进行中，拟揭示67LR髓系敲除缓解脓毒症的细胞机制和分子机制。该研究结果有望揭示67LR在脓毒症中的作用，论证67LR作为脓毒症治疗靶点的可能性，具有重要的科学研究价值。