肝纤维化逆转中胶原结构及交联的动态变化及LOXL-1对其调控机制

Liver fibrosis is the net result of dynamic changes between fibrogenesis and fibrolysis. Histological regression will only occur in a subset of patients even after effective treatment of liver disease. Our preliminary data showed that collagen structure and cross-links changed after fibrosis regression. LOXL-1 expression was strongly associated with liver fibrosis progression. Therefore, we hypothesized that collagen structure and cross-links play ctirical role in fibroiss regression. Blocking LOXL-1may suppress collagen cross-linking, and therefore contribute to collagen remodeling and fibrosis regression.. Firstly, we will use second harmonic generation/two photon excitation fluorescence (SHG/TPEF) to quantify the dynamic changes of collagen structures in fibrosis regression, and to elucidate the specific collgen structures that resulting in fibrosis irreversible; Secondly, we will extract insoluble collagen and perform high performance liquid chromatography to quantify the dynamic changes of cross-links in carbon tetrachloride induced liver fibrosis mice and spontaneous regression mice; Finally, we will conditionally knockout LOXL-1 in hepatic stellate cells to elucidate the regulatory effects of LOXL-1 on collgen crosslinking and on the reversibility of liver fibrosis.This study provides a potential intervention target for liver fibrosis regression.

肝纤维化是动态可逆的过程，但临床研究发现即使去除致病因素后仍有部分患者难以逆转。我们前期工作提示，纤维化消退时伴有胶原微细物理结构及交联变化，交联调控分子LOXL-1与纤维化关系密切。据此我们提出假设：胶原物理结构及交联影响肝纤维化逆转，通过阻断LOXL-1减少胶原交联，可以使胶原重构促进纤维化逆转。本课题首先借助二次谐波/双光子激发荧光技术，明确纤维化逆转中胶原物理结构动态变化，阐明引起肝纤维化难以逆转的胶原结构特征；其次应用CCl4小鼠肝纤维化自发逆转模型，通过不可溶性胶原及高效液相色谱分析明确胶原交联在纤维化逆转中的变化；最后通过对LOXL-1的分子调控机制研究，深入探讨LOXL-1对纤维化逆转的调控作用，进一步验证胶原交联对肝纤维化可逆性的影响。本研究为肝纤维化逆转提供可能的干预靶点，为临床应用提供依据。

肝纤维化是动态可逆的过程，但临床研究发现即使去除致病因素后仍有部分患者难以逆转 。我们前期工作提示，纤维化消退时伴有胶原微细物理结构及交联变化，交联调控分子LOXL-1 与纤维化关系密切。据此我们提出假设：胶原物理结构及交联影响肝纤维化逆转，通过阻断LOXL-1减少胶原交联，可以使胶原重构促进纤维化逆转。我们首先收集慢乙肝患者抗病毒治疗前后肝穿病理切片并染色，利用“北京标准”从病理形态角度评估纤维化逆转，发现慢乙肝抗病毒治疗78周肝纤维化率达70%以上；然后借助二次谐波/双光子激发荧光（SHG/TPEF）技术，提取了病理切片中胶原部位（汇管区、纤维间隔及窦周）、胶原结构特征及相应特征的胶原数量，通过比较肝纤维化逆转与不逆转的胶原特征及交联差异，发现逆转组治疗后胶原交联、胶原数量、结构等参数下降显著多于非逆转组，且这些特征参数在纤维间隔和窦周区域变化最为显著，汇管区变化不明显；最后在CCl4注射6周的小鼠肝纤维化模型中，特异性敲除Loxl1Gfap-Cre小鼠星状细胞LOXL-1，可以明显缓解肝纤维化，进一步验证交联对肝纤维化逆转的影响。