【作者】 沈海容；

【导师】 张奉学；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2010， 硕士

【摘要】 肝纤维化是多种慢性肝病向肝硬化发展的必经之路。不同原因(炎症、中毒、慢性淤胆、代谢障碍、血液循环障碍、营养障碍等)引起肝细胞(HC)慢性损伤(肝细胞发生炎症坏死,肝内纤维结缔组织异常增生),使枯否细胞(KC)激活,分泌多种细胞因子,随同血小板、窦内皮细胞(SEC)和HC等分泌的细胞因子,和某些化学介质共同激活肝星状细胞(HSC),使其转化为肌成纤维细胞(MFB),再通过旁分泌和自分泌作用,大量合成、增殖以胶原为主的细胞外间质(ECM),而降解相对不足,使ECM在肝内过多沉积,肝窦基底膜形成,肝窦毛细血管化,导致肝纤维化的形成和发展。若进一步引起肝小叶改建,假小叶和结节形成,则致肝硬化。因此,肝纤维化是肝脏对各种原因所致肝损伤的代偿修复反应,又是肝损害的促进因素,这种动态演变过程是各种慢性肝病向肝硬化发展的必经阶段,是肝病慢性化的共同特征。肝纤维化是现代医学病理形态学概念,中医并无此称谓,根据其病因、病机以及临床表现,可归属于“胁痛”、“臌胀”、“黄疸”、“积聚”等病范畴。从中医学角度看,本病多因外感湿热之邪或感染疫毒,或饮酒过度,酿生湿热,羁留不去,再加情志不遂等因素,渐致肝、脾、肾功能失调,气血、津液搏结,以致气滞血瘀,肝络瘀阻。目前,治疗肝纤维化尚无特效药,西医药在针对肝纤维化发生的各个环节进行抗肝纤维化治疗上已取得了一些进展,很多药物在体外和动物实验中均显示有较强的抗纤维化作用,但由于这些药物或疗效不够稳定,或毒副作用较大,临床应用者尚少。因此,从天然药物中发掘抗纤维化药物,已成为国内外研究的热点。应用单味中药、有效单体、复方、中西药联用等多种治疗方法进行抗纤维化的研究表明,中药在抗纤维化方面有广阔的应用前景,中医中药在治疗肝纤维化上面有着多部位、多靶点、毒副作用小的优势。为了挖掘更好的抗肝纤维化的中医药,造福人类,本课题研究以肝纤维化的发病机制为依据,以肝纤维化的中西医结合治疗为基础,以中药复方健脾活血方为实验药物,探讨其体内外抗肝纤维化的作用机制。健脾活血方由太子参、茯苓、白术、赤芍、丹参、田七、珍珠草、红花等组成,具有健脾益气,活血祛瘀,清热解毒的功效。本课题研究体内实验采用DMN肝纤维化大鼠模型。大鼠造模成功后,以健脾活血方为实验药物,给药4个月。通过检测DMN肝纤维化大鼠的肝指数和脾指数、镜下观察肝组织的病理切片,以及放射免疫法测定大鼠血清的肝纤四项,探讨健脾活血方体内抗肝纤维化作用。体外实验以肝星状细胞为细胞模型,以健脾活血方含药血清为实验药物,含药血清作用于HSC-T6后,貂肺上皮细胞(Mv-1-Lu)生长抑制MTT法检测HSC-T6分泌的TGF-β1的活性及其总量；ELISA法检测PDGF诱导的HSC-T6的Ⅰ型胶原含量,RT-PCR法检测PDGF诱导HSC-T6的TGF-β1mRNA,Ⅰ型胶原mRNA的表达水平,以探讨健脾活血方体外抗肝纤维化的作用。体内实验结果表明,健脾活血方高、中剂量组可以显著降低肝脾指数(P<0.01);同时,病理切片结果显示健脾活血方高、中、低剂量组均可减轻DMA肝纤维化大鼠肝脏的病变程度,以高、中剂量组为佳。另外,健脾活血方高、中剂量组还可降低DMN肝纤维化大鼠血清的HA、PCⅢ、LN, IV-C的水平(P<0.01),提示健脾活血方可以改善肝组织的病理形态,降低血清肝纤维化指标,具有良好的抗肝纤维化作用。体外实验结果表明,健脾活血方含药血清高剂量组可明显减少HSC TGF-β1的分泌总量(P<0.01),高剂量组及中剂量组可抑制TGF-β1的活性(P<0.01),高剂量组可降低TGF-β1mRNA的表达；同时健脾活血方含药血清还可降低Ⅰ型胶原的分泌,降低Ⅰ型胶原mRNA的表达,无剂量依赖效应(P<0.01),提示健脾活血方能够抑制HSC-T6增殖活化,干预胶原的合成,具有良好的抗肝纤维化作用。更多还原

【Abstract】 The hepetic fibrosis is the road taken by many kinds of chronic liver diseases that develop into liver cirrhosis. Different reasons (inflammation, toxicant, chronic silt gallbladder, metabolic block, blood circulation barrier, trophopathy, et al) cause chronic damage of the hepetic cell (HC) (HC have inflammation necrosis; fibrous connective tissue in liver proliferates abnormaly), makes Kupffer Cell (KC) activate, and secrete many kinds of cytokines which accompany cytokines of the blood platelet, sinusoidal endothelial cell (SEC) and HC to activate hepatic stellate cell (HSC) together with certain chemical mediator, causes it to transform into myofibroblast (MFB), then through paracrine and autocrine, the extracellular matrix(ECM), primarily the collagen, synthesize, proliferate massively, while degrading relatively insufficient, thus ECM in the liver excessively depositing, basilar membrane in hepetic sinusoid appearing, capillarity in hepetic sinusoid vascularizing, causes the formation and developmen of hepetic fibrosis. If it further causes the hepatic lobe reconstruction, pseudolobuli and tubercle take shape, then comes the liver cirrhosis. Obviously, the liver fibrosis is not only the liver’s compensation and restoration reaction to the damages caused by different reasons but also the contributing factor to the liver injure. this kind of dynamic successional variation process is the stage that each kind of chronic liver disease must pass through in order to develop into liver cirrhosis, and also the common characteristic for chronic liver diseases. The hepetic fibrosis is the modern medicine’s pathological and morphological concept, the Chinese medicine does not have this name. According to its cause, the pathogenesis as well as the clinical manifestation, we can clarify it to the sickness such as "costal pleurisy", "the abdominal distension", "the jaundice", "the agglomeration" and so on. Looking from the traditional Chinese medicine angle, this sickness is mostly caused by hot and damp evil or infected by epidemic disease, or drinking wine excessively, hot and damp ferments, which cannot stop over, plus other factors such as bad mood, gradually makes the function of liver, spleen and kidney out of balance, the vitality, the body fluids wrestles the knot, so that the stagnation of vital energy and blood stasis, stagnation of the liver channels take shape.At present, the treatment of hepetic fibrosis still have no specific medicine. West medicine, aiming at each link which the hepetic fibrosis occurs, has made some progress in the anti-liver fibrosis treatment, many medicines demonstrate strong anti-fibrosis function in vitro and the animal experimentation.But these medicines are still few in the clinical practice because their curative effect is not very stable, or the poisonous side effect is big. Therefore, excavateing the anti-fibrosis medicine from natural medicine, has become the hot spot of the domestic and foreign research. The research, using the single traditional Chinese medicine, the effective component monomer, the compound prescription, the combination of Chinese and western drugs, many such kinds of methods of treatment carrying on the anti-fibrosis, indicates that the traditional Chinese medicine has the broad application prospect in the anti-fibrosis aspect, the traditional Chinese medicine has superiorities such as multi-spots, multi-targets, small poisonous side effect in treating hepetic fibrosis.In order to unearth better anti-liver fibrosis Chinese medicine, benefit the humanity, this topic research, taking the hepetic fibrosis’s pathogenesis as the basis, the hepetic fibrosis’s cooperation treatment of Chinese and Western medicine as the foundation, the traditional Chinese medicine compound prescription:spleen-tonifying and blood-invigorating compound as the tested medicine, investigates its anti-liver fibrosis function mechanism in vivo and vitro. Spleen-tonifying and blood-invigorating compound which is mainly composed of radix pseudostellariae, poria cocos, white atractylodes rhizome, radix paeoniae rubra, salvia miltiorrhiza, radix notoginseng, phyllanthus urinaria, safflower, is able to strengthen the spleen and replenish qi, promote blood circulation and remove blood stasis, clearing away heat and toxic material.This topic research in vivo experiment use the DMN hepetic fibrosis rats as animal model, the spleen-tonifying and blood-invigorating compound as the tested medicine. After making the mold successfully, through examining the DMN hepetic fibrosis rats’liver index and spleen index, observing the pathological section of hepetic tissue with electron microscope, as well as determineing the rat blood serum’s liver filament four items with radioimmunoassay, we explore spleen-tonifying and blood-invigorating compound’s anti-liver fibrosis function in vivo.For the purpose of investigating spleen-tonifying and blood-invigarating compound’s anti-liver fibrosis function in vitro, experimens in vitro take the hepetic stellate cell as the cell model, spleen-tonifying and blood-invigorating compound blood serum as the tested medicine. After the blood serum acting on HSC-T6, the TGF-β1’s activity and total content in the supernatant of culture was measured by mink lung epithelial cell(Mv-1-Lu) proliferation inhibition MTT assay. And type I collagen was measured by ELISA.the RT-PCR assay examines TGF-β1mRNA, type I collagen mRNA of HSC-T6 induced by PDGF. Experimental results in vivo indicate that the high-dosed and mid-dosed group of spleen-tonifying and blood-invigorating compound can obviously reduce the liver and spleen index (P<0.01). At the same time, the pathological section result shows that all the high-dosed, mid-dosed and low-dosed group of spleen-tonifying and blood-invigorating compound can reduce the DMA hepetic fibrosis rat liver’s pathological change, especially the high and middle dose group. Besides, the high-dosed and mid-dosed group of spleen-tonifying and blood-invigorating compound also can reduce DMN hepetic fibrosis rat blood serum’s HA, PCIII, LN, IV-C level (P<0.01), suggesting that spleen-tonifying and blood-invigorating compound can not only improve the pathomorphism of hepetic tissue, but also reduce the sera markers for heptic fibrosis, and has good anti-liver fibrosis function.Experimental results in vitro indicate that the high-dosed group of spleen-tonifying and blood-invigorating compound blood serum can reduce the HSC TGF-β1 secretion total quantity obviously (P<0.01), the high-dosed and mid-dosed group can suppress TGF-β1 activeness (P<0.01), and the high-dosed group can reduce TGF-β1 mRNA expression; Simultaneously spleen-tonifying and blood-invigorating compound blood serum also can reduce the secretion of type I collagen, and reduce type I collagen mRNA the expression, without dosage dependence effect (P<0.01). The results suggest the spleen-tonifying and blood-invigorating compound blood serum is able to suppress the multiplication and activation of HSC-T6, intervene type I collagen’s synthesis, and has good anti-liver fibrosis function.更多还原