【作者】 韩海啸；

【导师】 杨晋翔；

【作者基本信息】 北京中医药大学 ， 中医内科， 2007， 博士

【摘要】 中医药在防治酒精性肝病方面具有潜在的优势和鲜明的特点,本研究运用临床流行病学的方法和原则,探讨酒精性肝硬化的中医证候特点,为临床更好地防治酒精性肝硬化做有益的尝试。酒精性肝纤维化是肝硬化发展的必经过程,由于具有可逆性,故研究肝纤维化的形成机制是当前研究的热点之一。酒精性肝纤维化是以肝星状细胞(HSC)为主的细胞激活增殖,使细胞外基质各成分合成明显增多,降解相对减少的动态可逆病理过程。本课题运用细胞生物学和分子生物学技术在两个层面上研究赤芍水提物抗酒精性肝纤维化的细胞与分子机制。本课题由理论研究、临床研究及实验研究三部分组成:理论研究部分,一部分内容总结了中医对酒精性肝病病因病机的认识,对当前国内有关中药方剂和有效成分治疗酒精性肝病以及酒精性肝病流行病学的研究进展进行了综述;另一部分系统论述了酒精性肝纤维化形成机制的研究成果。临床研究部分,为多中心前瞻性研究。以符合临床流行病学要求的全国6家医院病例资料为基础,对酒精性肝硬化患者的临床资料进行调查,探讨酒精性肝硬化患者中医证候分布的特点;应用因子分析的方法,研究了100例酒精性肝硬化患者中医证候特点的分布,发病的特点。实验研究部分,主要进行了离体实验。本实验以肝星状细胞为靶细胞,以乙醛为刺激因子,研究灌服赤芍水提物后犬血清对肝星状细胞HSC-T6增殖抑制及促凋亡作用的影响,同时通过观察药物对细胞基因蛋白表达的变化,探讨该药抗酒精性肝纤维化的作用机理。研究结果:(1)临床研究:实验一:男性为主要的饮酒和酒精性肝硬化患病人群;酒精性肝硬化的发病高峰年龄在50岁左右。不同职业人群酒精性肝硬化发生率有所不同,干部、商人最高,分别为32%和38%;日饮酒量160g/d,饮酒年限20年,累计酒精摄入量1500kg左右时,具有较高的患病率。酒精性肝硬化患者中谷氨酰转肽酶(γ-GT)值(100～250U/L)、AST/ALT比值>2、碱性磷酸酶(ALP)值(220～550U/L)、总胆红素(TBIL)(>51umol/l)、白蛋白(ALB)(<28g/l)、凝血酶原百分活动度PTA(50%～70%)的出现率较高。实验二:酒精性肝硬化患者中医证候特点为虚实夹杂;中医病位主要在肝胆脾肾;病机主要为气滞、血瘀、湿(热)浊内蕴,兼有肝肾阴虚,脾肾阳虚。γ-GT、AST/ALT比值、ALB、PTA、Child-pugh分级对酒精性肝硬化患者中医证候特点的分布有显著性的影响。(2)实验研究:实验一:MTT法分别检测乙醛浓度在17.5μM～375 mM对HSC-T6的存活率和增殖率,结果表明乙醛浓度在17.5μM～3.5mM可明显促进HSC-T6的增殖,并确定乙醛浓度在87.5um建立了大鼠酒精性肝纤维化离体HSC模型。实验二:乙醛造模后肝星状细胞HSC-T6为体外模型,以赤芍水提取物给彼格犬一次性灌胃,取给药前的血清及给药后不同时间点的血清作为实验药物血清。以四甲基固氮唑盐(MTT)法分别检测每个采血时间点含药血清对HSC-T6作用72h后的抑制情况,找出最佳含药血清时点。结果表明,2h和3h时点药物血清对HSC-T6细胞生长的抑制率最高,分别达到79.8%,77.9%。实验三:以流式细胞仪分别检测给药后2h,3h药物血清对HSC-T6作用72h后的促凋亡作用。PI染色流式细胞分析: 2h和3h时点药物血清作用HSC-T6,细胞在G0/G1,APOP期比例较模型组明显上升,S期比例明显下降。实验四:给药后2h的血清作为实验药物血清。以免疫细胞化学法检测药物血清对HSC-T6作用72h后Bcl-2、Bax、Caspase-3三种基因蛋白表达情况。结果表明,乙醛造模后的HSC-T6中Bcl-2的基因蛋白表达较正常培养组增加,Bax、Caspase-3表达降低;药物血清作用后的HSC-T6中Bcl-2的基因蛋白表达较造模组显著降低,Bax、Caspase-3的表达较造模组显著增加。结论:酒精性肝硬化患者多发于男性,年龄在50岁左右,干部或商人居多;日饮酒量160g/d,饮酒年限20年,累计酒精摄入量1500kg左右。患者中医证候特点为虚实夹杂;中医病位主要在肝胆脾肾;病机主要为气滞、血瘀、湿(热)浊内蕴,兼有肝肾阴虚,脾肾阳虚。赤芍水提物入血后可通过影响乙醛造模后的肝星状细胞(HSC-T6) Bcl-2、Bax、Caspase-3等基因蛋白异常表达,来实现对HSC-T6的增殖抑制及促凋亡作用,这可能为赤芍水提物抗酒精性肝纤维化的细胞和分子作用机制。更多还原

【Abstract】 Alcoholic liver disease(ALD) includes mild alcoholic liver disease, alcoholic liver Fatties(ALF),alcoholic hepatitis,alcoholic fibrosis(AF),alcoholic liver cirrhosis(AC). Traditional Chinese Medical (TCM) therapy has potential superiority and distinct characteristics in the treatment of alcoholic liver cirrhosis. By means of the methods and doctrines of Clinical Epidemiological Research, we aim at the investigation of the characteristics of traditional Chinese medical syndrome of alcoholic liver cirrhosis, attempt to explore a better way for the treatment of AC. Alcoholic fibrosis is the most phase on the way to alcoholic liver cirrhosis.As its reversibility, Alcoholic fibrosis has been recognized as one of the most exciting domains in the studies on the forming mechanisms of alcoholic liver fibrosis. The forming mechanisms of Alcoholic liver fibrosis base on the activation of hepatic stellate cell, and is the reversible dynamic pathologic process of the increased synthesis and the relatively decreased degradation of ECM. On the grounds of techniques of Cytobiology and Molecular Biology, we aim at the research in the cellular and molecular mechanisms of Radix Paeoniae Rubra Distilled Substances compound anti-AF .The paper is composed of three parts :the theoretical study ,the clinical research and the experimental study.In the theoretical part,I summarized the cognition of the cause and mechanisms of alcoholic liver disease (fibrosis / cirrhosis) by the angle of traditional Chinese medicine; summarized succeedly the development of the ALD treatment by the way of Chinese traditional medical compounds and effective components,and of the clinical epidemiology in the inland studies on ALD. On the other hand, expounded systemically the pathogenesis of alcoholic liver disease (fibrosis / cirrhosis)by the angle of western medicine.In the clinical investigation, our group studied clinical cases in multi-center prospective method. In virtue of factor Analysis, we explored the characteristics of traditional Chinese medical syndrome of alcoholic liver cirrhosis, and the forming characteristics of alcoholic liver cirrhosis in inland.In the experimental research , cellar culture was mainly taken, rat hepatic stellate cells(HSC-T6) were regarded as the study object, acetaldehyde was used as the stimulating factor, Radix Paeoniae Rubra Distilled Substances compound was the experimental drug. Through cells cultivation, we studied the inhibiting proliferation effects and the apoptosis-promoting effects of dog serum after being fed with Radix Paeoniae Rubra Distilled Substances on hepatic stellate cell (HSC-T6)builded。Through analyze the protein levels of Bcl-2 , Bax , Caspase3 in HSC-T6,we find that exposure Radix Paeoniae Rubra medicated serums can change the expression of apoptosis -related genes , partly opened out the molecular mechanisms of Radix Paeoniae Rubra Distilled Substances compound anti-AF .Result and Conclusions(1) clinical investigationExperiment one: Male was the major case among the patients of alcoholic liver cirrhosis, the mean age of AC patients was approximate to 50 years old. The incidence of AC was different with the kinds of profession, the most was businessman(38%), the second was officers(32%); The prevalence rate of AC was relatively higher when the drinkers consumed the equivalent of 160g per day with a mean duration of 20 years, which was approximate to a total alcoholic consumption of 1500kg. With increase of alcoholic consumption, The incidence of the serum levels ofγ-GT、AST/ALT、ALP、TBIL、ALB、PTA rosed.Experiment two: Among the subjects, the characteristics of TCM syndrome of AC mainly manifested excess syndrome with the common manifestations of deficiency and excess; The visceral position was mainly in liver,gallbladder,kidney and spleen; The result suggested that stagnation of qi, blood stasis, damp heat, with deficiency of yin genuine of liver and kidney and with deficiency of yang genuine of spleen and kidney were the interpretation of the causes of AC. The total alcoholic consumption,dringking time were important facts,which effected on the distribution of the characteristics of TCM syndrome of AC, so was the total alcoholic consumption,dringking time when the patients had a alcohol abuse family.γ-GT、AST/ALT、ALB、PTA、Child-pugh had mostly significant effect on the distribution of the characteristics of TCM syndrome,(2)Experiment research:1. Experiment one: acetaldehyde (87.5um) in vitro cell model stimulating hepatic stellate cell- T6 (HSC-T6) proliferation was made.2. Experiment two: We adopted HSC-T6 builded model by acetaldehyde, for study in vitro. We took the serum of a dog before and at different points of time after feeding the dog with Radix Paeoniae Rubra Distilled Substances for experiment one time.The HSC-T6 cells were cultured in mediums with different concentrations of the medicated serum at different points of time for 72 hours. The results showed that HSC-T6 inhibition rate is the highest at the containing 2% 2-hour and 3 - hour medicated serum.3. Experiment three: By means of flow cytometry, we examined the apoptosis-promoting effects of the serums on HSC-T6.. The medicated serums has significant effects of promoting HSC-T6 apoptosis at these 2h,3h points of time.Compared with non-medicated serums under the same conditions, the proportion of apoptosis had a significant increase. HSC-T6 cell’s proportion was increased in G0/G1 period and decreased in the S period.4. Experiment four: we took the serum at 2-hour point after feeding as the medicated serum for experiment. By immunohistochemistry method, we analyze the protein levels of Bcl-2 , Bax , Caspase-3 in HSC-T6 . The results showed that Compared with common HSC-T6, builded model HSC-T6 increases of Bcl-2 proteins were observed , while decrease of Bcl-2 protein levels was obtained . Compared with model, HSC-T6 functed medicated serums under the same conditions, decrease of Bcl-2 proteins were observed , while increases of Bcl-2 protein levels was obtained .Conclusions: alcoholic liver cirrhosis regularly occurs among the drinkers of male, 50 years old,officer or businessmen, when the drinkers consumed the equivalent of 160g perday .with a mean duration of 20 years, which was approximate to a total alcoholic consumption of 1500kg.The characteristics of TCM syndrome of alcoholic liver cirrhosis mainly manifested deficiency and excess syndrome; The visceral position was mainly in liver,gallbladder,kidney and spleen; The result suggested that stagnation of qi, blood stasis, damp heat, with deficiency of yin genuine of liver and kidney and with deficiency of yang genuine of spleen and kidney were the interpretation of the causes of alcoholic liver cirrhosis. Through cells cultivation, we studied the inhibiting proliferation effects and the apoptosis-promoting effects of dog serum after being fed with Radix Paeoniae Rubra Distilled Substances on HSC-T6 builded model by acetaldehyde in vitro.We find the function of Radix Paeoniae Rubra Distilled Substances might be one of the mechanisms to counteract against alcoholic liver fibrosis.更多还原