【作者】 张玮；

【导师】 郭顺根；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2003， 硕士

【摘要】 肝纤维化是诸多慢性肝病的基础病理改变。肝纤维化的机制为细胞外基质(extracelluar matrix，ECM)的合成与降解失衡，导致ECM过度沉积所致。肝纤维化进一步发展即肝硬化。此时，肝的正常组织结构已被破坏，进一步发展，肝中央静脉周围和汇管区出现小叶间隔，形成假小叶，即可出现肝功能障碍。因此，积极防治肝纤维化的发生，无论从延缓或阻断肝硬化的形成，还是逆转肝纤维化病理进程，均具有十分重要的意义。近年来，大量资料表明，肝星形细胞(hepatocyte stellate cells ，HSC)是肝脏主要产生ECM的细胞，也是各种病因导致肝纤维化发生的共同中心环节。HSC活化是产生胶原的关键环节。HSC活化后，可出现细胞性状和细胞功能的改变。最突出的表现为ECM的合成与降解的失衡。HSC的活化可分为活化启动和活化持续两个阶段。HSC的活化依靠于细胞内信号转导的同时，还与核转录因子的调节密切相关。目前以HSC为靶向治疗肝纤维化的主要措施包括：①去除病因，减轻肝脏炎症，间接抑制HSC的活化；②选择性阻止HSC合成ECM；③促进HSC对ECM的降解④促进过剩的HSC凋亡，减少其数量。目前，西医对肝纤维化的治疗主要集中在防止肝损伤、抗炎、干预细胞内信号转导通路、拮抗促纤维化细胞因子作用、抑制胶原合成(转录水平或翻译水平)、促进胶原降解和促进HSC凋亡等多个方面。中医药对肝纤维化的治疗通常采用是活血化瘀、益气养阴、软肝散结、清热利湿和调理肝脾等治则。本实验采取的复方鳖甲软肝方由鳖甲、党参等十一味中药组成。其功能为：扶正祛邪，活血化瘀。扶正：党参健脾益气，紫河车益肾填精。驱邪：板蓝根清热解毒。活血化瘀：鳖甲，入肝、肾经，既可软坚散结，又可滋阴潜阳。当归养血活血，莪术行气祛瘀。其临床有效率达81.67%，治愈率达55.23%。本方系国家级中药新药(批准文号：国药准字1999，2-102号)已上市。本研究拟采用复方鳖甲软肝方药物血清对大鼠离体肝星形细胞作用机理开展实验研究。目的在于进一步深入了解复方鳖甲软肝方药物血清对HSC的增殖，活化以及对ECM降解和合成的影响。此外，本研究还从活化的HSC细胞内信号转导及核转录因子方面探讨复方鳖甲软肝方对HSC活化所产生的影响。<WP=4>1.复方鳖甲软肝方药物血清对HSC增殖影响的实验研究本实验从离体的角度观察复方鳖甲软肝方药物血清对肝星形细胞(HSC)增殖的影响，以探讨其抗肝纤维化的作用机制。研究表明，肝纤维化是一种常见的慢性进行性肝病，肝星形细胞的活化增殖以及细胞外基质(ECM)的沉积是肝纤维化的两个重要环节。肝星形细胞的大量增殖，可以合成过多的胶原、蛋白多糖等ECM成分，与肝纤维化的发生、发展过程密切相关。本实验结果显示，不同剂量的复方鳖甲软肝方药物血清作用48 h、72 h均可抑制肝星形细胞的增殖，尤以高、中剂量组明显，提示复方鳖甲软肝方药物血清抑制肝星形细胞增殖的药物效应与药物浓度呈量效依赖关系，其机制可能与药物中的有效物质能阻止HSC分泌血小板衍生生长因子(PDGF)和阻止HSC已分泌的PDGF与HSC受体结合有关，从而起到抑制肝星形细胞的活化增殖，达到治疗肝纤维化的作用。而培养24h，各组无显著性差异，提示HSC的增殖需要一个从接受细胞信号刺激活化到HSC增殖数量发生显著变化的时间过程，从本实验结果提示，该时间过程无疑要大于24h。因此，如何在早期通过抑制HSC的增殖途径达到遏止HSC的迅速增殖，无疑具有重要的临床意义。与本室以往活血化瘀方药物血清的MTT实验结果相比，培养48h、72h，复方鳖甲软肝方药物血清与活血化瘀方药物血清均可显著抑制肝星形细胞的活化增殖，表明复方鳖甲软肝方药物血清与活血化瘀方药物血清均能抑制肝星形细胞的增殖。2.复方鳖甲软肝方药物血清对HSC活化影响的实验研究本实验采用免疫组化技术观察复方鳖甲软肝方药物血清对肝星形细胞(HSC)中α-SMA、desmin、突触素表达的影响，以探讨其抗肝纤维化的作用机制。研究表明，肝星形细胞的活化是肝纤维化的中心环节。活化的主要特征包括肝星形细胞中α-SMA、desmin和突触素的表达。本实验结果显示在时间上desmin、突触素的表达早于α-SMA的表达。突触素作为一种较新的HSC活化指标，可通过自分泌、旁分泌等方式参与肝纤维化发生、发展过程。并为肝纤维化的发生发展过程中神经调节的参与提供了指标依据。实验结果还显示，复方鳖甲软肝方药物血清作用24h、48h、72h均可显著抑制desmin和突触素的表达；复方鳖甲软肝方药物血清作用48h、72h还可显著α-SMA的表达，提示复方鳖甲软肝方药物血清可抑制HSC的活化。由于肝纤维化时α-SMA、desmin和突触素在表达时间上存在差异，而复方鳖甲软肝方药物血清抑制α-SMA、desmin和突触素的表达时间上也同样存在差异，故推测复方鳖甲软肝方药物血清抑制α-SMA、desmin和突触素表达的作用机制可能存在差异，详情有待进一步研究。实验结果还显示，培养24h，各组α-SMA表达并不显著，提示HSC中α-SMA的表达同样需要一个时间阶段，同增殖实验结果提示一样，如在早期加以积极治疗，则可取得更好的疗效。与本室以往<WP=5>活血化瘀方药物血清对α-SMA影响的实验结果相比更多还原

【Abstract】 Hepatic fibrosis is a response to chronic liver injury, which is the outcome of deposition of hepatic fibrosis tissue. The mechanism of hepatic fibrosis is the imbalance of synthesis and degradation of extracellular matrix. The progress of hepatic fibrosis is sclerosis. There are pseudo leaflets in hepatic veins and the common structure is destructed. cirrhosis of liver can lead to hepatic malfunction. So active treatment of hepatic fibrosis can postpone and block formation of cirrhosis of liver, sometimes it can inversio the process of hepatic.Recently, lots of data indicate HSC is the key cell of formation of ECM and HSC is also the core link of occurrenc of hepatic fibrosis. Activation of HSC is the main step of formation of collagen. Change in character and function of cell arise after the activation of HSC. The mostly manifestation is the imbalance of synthesis and degradation of extracellular matrix. Activation of HSC is made up of start of activation and continue of activation. And it is closely related to signal pathway in cell and regulation of nuclear transcription factor. The measure in aim of HSC include: ①remove the etiological factor, relieve the hepatic inflammation. ②selectivity prevent the formation of ECM by HSC. ③Facilitate decomposition of ECM by HSC. ④Facilitate apoptosis of excess HSC.At present western medical doctor are concentrate on the some aspects about treatment of hepatic fibrosis which include prevention of hepatic injury; intervention of signal conduct pathway in cell ; decrease the formation of collagen; increase the degradation of collagen; facilitate the apoptosis of HSC.The law of tradition medicine on hepatic fibrosis include huoxuehuayu;ruangansanjie;qingrelishi;tiaolipiwei et al. Our study investigate the mechanism of treatment of hepatic fibrosis by fufangbiejiaruanganfang. The content of fufangbiejiaruanganfang include biejia; dangseng; et al. The aim of study is to know the influence on proliferation of HSC; activation of HSC; synthesis and degradation of extracellular matrix; signal conduct pathway; nuclear transcript factor.1.the study of effect on proliferation of HSC by fufangbiejiaruanganfangIn view of in vitro，our experiment observe the effect of proliferation of<WP=8>HSC by influence of different serum. The result indicate that hepatic fibrosis is a common chronic hepatic disease. The two main points of hepatic fibrosis are proliferation of HSC and deposition of ECM. Proliferation of HSC can lead to synthesis of ECM including collagen and proteoglycan and so on. So proliferation of HSC is closely related to occurrence and progress of hepatic fibrosis. Our experiment indicate that different doze of fufangbiejiaruanganfang drug serum can decrease the proliferation of HSC. And high doze and medium doze have more effective result. So it may be the effect of fufangbiejiaruanganfang drug serum is depend on the concentration of fufangbiejiaruanganfang doze. The mechanism of treatment may be related to the content in fufangbiejiaruanganfang drug serum can block the excretion of PDGF by HSC and binding of PDGF and receptor of PDGF. So the mechanism of fufangbiejiaruanganfang may be related to decreasing the proliferation of HSC.2．the study of effect on activation of HSC by fufangbiejiaruanganfangOur experiment on effect on expression of α-SMA、desmin、synaptophysin by technology of immunohistochemistry. Our finding indicates that the main point of hepatic fibrosis is activation of HSC. Expression of desmin、synaptophysin is earlier than expression of α-SMA. The outcome of experiment indicates that fufangbiejiaruangajfang drug serum can decrease the amount and area of desmin、synaptophysin andα-SMA in different time. And we estimate the mechanism of effect on expression of α-SMA、desmin、synaptophysin may be different. More investigation should be done. 3. the study of effect on balance of synthesis and degradation of extracellular matrix by fufangbiejiaruanganfangActivatived HSC is the main source of excessive ECM in hepatic fibros更多还原