【作者】 张达坤；

【导师】 邝卫红；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2011， 博士

【摘要】 研究背景肝内胆汁淤积是导致肝硬化进程加快的一个重要危险因素,胆汁淤积时血清生化指标和肝脏组织结构有明显改变。分子生物技术发现,肝细胞和胆小管细胞膜上胆盐输出泵BSEP、膜转运体NTCP是调控胆汁分泌和排泄的重要分子通路,其调控的异常直接导致了肝内的胆汁淤积。肝内胆汁淤积属于中医“黄疸”范畴,中医药独特的疗效对本病的治疗有明显的特色和优势,在实际临床工作中经常运用利胆退黄法来治疗胆汁淤积,疗效明确。虽有临床研究探讨利胆退黄法的治疗效果,但从文献报道来看,回顾性研究多,前瞻性研究少,且诊断、疗效判定标准不规范统一。绝大多数前瞻性研究只是简单的临床观察,而缺乏严格的科研设计,无对照组,资料重复性差,统计不完全,非多中心研究,循证医学证据力度较小。目前针对利胆退黄法作用机制的实验研究少,且选用指标单调,未能结合目前分子生物学的发展。研究目的采用复合因素“肥甘饮食+湿热环境+致病因子”建立肝内胆汁淤积湿热证大鼠模型,模拟病证结合的动物模型,研究利胆退黄法中药复方、单味及活性成分对该模型大鼠胆盐输出泵及膜转运体表达的影响,并同时观察其血清生化指标、肝脏组织病理的变化,为利胆退黄法治疗肝内胆汁淤积症提供理论基础,以期从分子水平进一步探讨利胆退黄法的作用机理及靶点。并结合临床经验,在严谨的科研设计下进行临床随机对照研究,进一步证明利胆退黄法的临床疗效。研究内容与方法实验部分：1造模：选择长夏季节造模,将实验动物放于湿热造模箱内,并喂饲葡萄糖和猪油脂混合液,形成湿热证；再采用α-异硫氰酸萘酯(ANIT)造成肝损害形成肝内胆汁淤积湿热证大鼠模型。2分组及干预方法：三月龄雄性SD大鼠(SPF级动物)75只,随机分为5组,每组15只。五组分别为：正常对照组、模型组、茵陈蒿汤组、单味茵陈组、苦参碱组复方茵陈蒿汤组：造模第8天开始每天下午给100%茵陈蒿汤2mL/100g灌胃,一直到造模结束后28天,共用药42天。单味中药茵陈蒿组：造模第8天开始每天下午给100%茵陈蒿2mL/100g灌胃,一直到造模结束后28天,共用药42天。苦参碱：造模第8天开始每天下午按每100 g大鼠15 mg苦参碱灌胃,一直到造模结束后28天,共用药42天。正常对照组和模型组都予0.9%生理盐水2mL/100g灌胃。3检测指标及方法：3.1采用全自动生化分析仪检测各组大鼠的血清的ALT、AST、ALP、TB、DB。3.2各组大鼠肝脏组织行病理切片常规HE染色检查,显微镜观察肝脏组织结构变化。3.3采用免疫组化染色,检测各组大鼠肝组织NTCP和BSEP。3.4采用荧光实时定量逆转录聚合酶链式反应(Real-time RT-PCR)检测各组大鼠肝组织NTCP多肽和胆盐输出泵BSEP。临床部分：利胆退黄法临床疗效评价研究：本课题依据诊断标准共收集病例60例,病例来自于广州中医药大学第一附属医院脾胃科、广州市第八人民医院的住院患者。采用随机、阳性对照的方法分为两组,对照组予口服熊去氧胆酸250mg/次,2次/d。治疗组予口服加味茵陈蒿汤(基本方由茵陈、栀子、大黄、赤芍、郁金、金钱草、虎杖等组成)。主要观察临床症状、体征、舌象、脉象、肝功能(TB、DB、ALT、GGT)旨标的变化以及临床综合疗效、中医证候疗效。从而对利胆退黄法治疗肝内胆汁淤积的临床疗效进行评价研究,为其临床推广提供数据支持。研究结果1模型建立后利胆退黄法各组较模型组均能有效改善肝内胆汁淤积湿热证的症状、体征以及降低胆红素、总胆汁酸、血清肝酶的水平并减轻病理损害。2针对利胆退黄法调控BSEP及NTCP的表达、促进胆盐代谢的初步研究,通过免疫组织化学法和RT-PCR法分析了各组大鼠肝组织中NTCP和BSEP表达变化的规律,研究显示模型组肝组织中BSEP和NTCP表达均明显下调,通过干预后利胆退黄法各组大鼠虽未恢复至正常组水平,但其BSEP和NTCP的表达有所上调,经统计学比较,与模型组有显著性差异。3临床研究发现利胆退黄方组与西药对照组比较,两组在临床疾病疗效改善方面相似,在改善患者血清胆红素和肝酶方面均有显著疗效,两者差异无统计学意义,两组均未出现明显的毒副作用。但利胆退黄方组在改善中医证候积分、证候疗效方面优于后者。研究结论1从病因、症状、体征、实验指标及药物反证合参等方面综合评价得出本研究建立的动物模型符合肝内胆汁淤积湿热证,是比较满意的中西医结合的病证结合模型。通过利胆退黄法干预该模型后证实利胆退黄法能有效改善肝内胆汁淤积湿热证大鼠的相关症状、体征和胆红素、总胆汁酸及肝功能量化指标以及减轻病理损害。2针对利胆退黄法调控胆盐输出泵及膜转运体表达、促进胆盐代谢进行了初步研究,采用免疫组织化学、RT-PCR等技术分析了肝内胆汁淤积湿热证大鼠肝组织中NTCP和BSEP表达变化的规律。发现肝内胆汁淤积时BSEP和NTCP表达均明显下调,利胆退黄法能通过上调BSEP和NTCP的表达,增加肝细胞对胆盐的摄取和排泌,特别是促进胆盐转运系统功能的恢复。3以利胆退黄法组成的加味茵陈蒿汤治疗肝内胆汁淤积湿热证患者在本次临床研究中取得了满意的临床疗效。与西药对照组比较,在改善中医证候积分、疗效方面优于后者。为利胆退黄法的临床应用提供了可靠的证据支持。更多还原

【Abstract】 BackgroundIntrahepatic cholestasis is an important risk factor of the speeding up of cirrhosis of the liver. There are significant changes in serum biochemical parameters and in organizational structure of liver. It was found by molecular biology technology that the bile salt export pump(BSEP) in liver cells and bile duct cell membrane, and the membrane transporter NTCP, are important molecular pathways for regulation of bile secretion and excretion. The abnormal regulation of BSEP and NTCP led to the intrahepatic cholestasis directly. Cholestatic jaundice are equal to the jaundice in Traditional Chinese Medicine (TCM). Treatments in TCM have showed unique effect and advantages. In clinical work normalizing the gallbladder function to treat jaundice is an effective way. But the mechanism of normalizing the gallbladder function was not illuminated in reported researches. Most of these researches were simple clinical observation. The number of experimental reports and the reported indicators were relatively little. Most of the clinical observations showed a lack of rigorous design, and of control group. The data and statistics method were relatively poor. And most of them were non-multi-center studies. The criteria of diagnosis and efficacy was not standardized.PurposeTo make the intrahepatic cholestasis rat model with dampness-heat syndrome by the composite factors as hot and damp environment and fat dietary and etiopathogenisis. By the combination of disease and syndrome animal model, the effect of herbal compound, a single herb and the active ingredients of herb could be studied by the expressions of BSEP(bile salt export pump) and NTCP. The serum biochemical parameters and structural changes in the liver were observed at the same time. This could provide a theoretical basis for the treatments of jaundice by normalizing the gallbladder function, and so to discuss the targets.Subjects and MethodsExperiment on rats:1 modeling:kept the rats in hot and humid boxes in the long summer festival, and fed them with the mixture of glucose and pig fat to form damp-heat syndrome. Then usedα-naphthyl ester isothiocyanate (ANIT) to form the intrahepatic cholestasis rat model.2 grouping and intervention:150 three-month-old male SD rats were randomly divided into 5 groups, each group had 15 rats. Here were the five groups:normal control group, model group, YCHT group, single-flavor capillaris group, and matrine groupCompound YCHT Group:Lavaged the rats with 100% Yin Chen Hao Decotion 2mL /100g everyday from the 8th afternoon of modeling to the 28th days after modeling.Single herb Artemisia Group:Lavaged the rats with 100% Artemisia 2mL/ 100g everyday from the 8th afternoon of modeling to the 28th days after modeling.Matrine:Lavaged the rats with 100% Matrine 2mL/100g everyday from the 8th afternoon of modeling to the 28th days after modeling.Normal control group and model group were lavaged with the 0.9% saline 2mL/100g.3 indicatrix and methods:3.1 Analyzed the ALT, AST, ALP, TB, DB in rats serum by automatic biochemical analyzer.3.2 Got the liver tissue to do routine pathological examination in HE staining, to observe the structural changes in liver tissue by microscopy.3.3 detected the NTCP and BSEP in the liver tissue by the immunohistochemical staining.3.4 detected the BSEP and NTCP in liver tissue by fluorescent real-time quantitative reverse transcription polymerase chain reaction (Real-time RT-PCR). Clinical research:Clinical research on efficacy of normalizing the gallbladder function to treat jaundice:Collected 60 patients who met relevant standards from Digestion Department in First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou Eighth People’s Hospital. This research used a randomized, positive control method. All patients were divided into two groups. The control group were treated with conventional symptomatic treatment, intravenous think Mattel and glutathione 1.0g 1.2g; and took ursodeoxycholic acid 250mg/time,2 times/d orally. The herb group took Yin Chen Hao Decotion Jia Wei. (The basic formula is capillaris, gardenia, rhubarb, red peony root, turmeric, money grass, Polygonum etc) orallyChanges in clinical symptoms and signs, tongue, pulse, liver function (TB, DB, ALT, GGT), clinical comprehensive efficacy, and TCM syndromes were observed. Evaluated the clinical efficacy of normalizing the gallbladder function to treat jaundice on intrahepatic cholestasis patients and provided clinical data.Result1 After modeling, the symptoms and signs of cholestasis and damp-heat syndrome, the bilirubin, total bile acid, serum liver enzymes and the pathological changes in the tissue could be improved in the herb groups.2 Research on the changes of BSEP and NTCP in normalizing the gallbladder function to treat jaundice showed that:expression of BSEP and NTCP in model group decreased significantly,while the ones’expression in herb groups increased but still not recover to the nomal level. There were significant differences between the herb groups and the model group by statistics.3 The result of clinical research showed that the improvements of clinical efficacy in the herb group were similar to the control group. The serum bilirubin and liver enzymes in both groups improved and there was no difference between them. None of them showed side effects In the improvements of syndromes’score and syndromes’efficacy, the herb group showed better efficacy.Conclusion1 According to the causes of disease, symptoms, signs, laboratory indicators and treatment efficacy, the rat model in this reseach fit both syndrome in Chinese Medicine and intrahepatic cholestasis. It was more satisfying integrative medicine model of disease and syndrome. It was confirmed that intervention by normalizing the gallbladder function to treat jaundice could effectively improve symptoms, signs and bilirubin, total bile acid and liver function of the rats model, and reduce the pathological damage of liver tissue.2 In this research, the regulation of NTCP and BSEP’s expression in rats’ liver tissue was explored by immunohistochemistry, RT-PCR analysis and other techniques. It was found that expression of BSEP and NTCP was significantly reduced in intrahepatic cholestasis rat model. The intervention of normalizing the gallbladder function to treat jaundice in Chinese Medicine could help increase the expression of BSEP and NTCP, increase bile salt uptake and excretion in liver cell, and in particular, promote functional recovery of bile salt transport system. The result provided the clinical theory basis for treatment of normalizing the gallbladder function to treat jaundice in intrahepatic cholestasis patients.3 The representative decotion of normalizing the gallbladder function to treat jaundice was Jia Wei Yin Chen Hao Decotion. The clinical efficacy of Jia Wei Tin Chen Hao Decotion to intrahepatic cholestasis patients was satisfactory in this research. Compared with western medicine control group, the scores of syndrome and efficacy were improved better in herb group. This provided reliable evidence for application of normalizing the gallbladder function to treat jaundice更多还原