【作者】 陈文林；

【导师】 杨大国；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2014， 博士

【摘要】 慢性丙型肝炎是一个严重危害人类健康的公共卫生问题。据WHO统计全球丙型肝炎病毒（HCV）感染率3%,约1.7亿人，每年新发丙肝3.5万例。根据2010年9月国家卫生部公布的最新流行病学调查结果显示，我国抗HCV阳性率为0.43％，大概全国HCV感染者达500多万。HCV自然感染史长，其慢性化率为50%～85%。肝硬化和肝癌是慢性丙型肝炎患者的主要死因，在感染的5～0年后30%～40%将发展为肝硬化，10～20年后5%～7%发展为肝癌。成为现今严重危害人民健康的传染病之一。尽管现代医学在慢性丙型肝炎的治疗上取得一些突破性进展，其三联疗法成为基因1b型等一些难治性丙肝的最新方案。但对于耐药、抗病毒无效者及在治疗中出现药物毒副作用、停药后复发、病毒学突破、部分应答、无效应答或无应答、基因1型或4型HCV感染、高病毒载量(HCV RNA≥1.5×106IU/mL)、合并其他病毒感染,或同时合并其他如糖尿病、甲状腺功能障碍、血友病、器官移植、肾脏疾病及血液透析等自身免疫功能障碍有关的疾病及肝硬化失代偿期等问题的存在，使得丙肝治疗依然是临床难题，即所谓的难治性慢性丙型肝炎。目前由HCV基因1型感染所致的慢性丙型肝炎在我国约占78%,高病毒载量的慢性丙型肝炎约占69%,所以可以说在我国大多数的慢性丙型肝炎属于难治性丙型肝炎。RHC形成复杂，因而其临床表现也多种多样。通过“十一五”对CHC中医症候辨证分型规范研究显示：正虚邪恋、肝郁脾虚、肝肾阴虚三型占所有证型的73%，故虚实夹杂的临床表现是CHC的主要特征，若不实行临床干预，任其进一步发展，必将形成肝硬化等难治性肝病，其预后不佳，对患者生命将形成极大的威胁，对治疗造成巨大的挑战。在RHC治疗方面，并无特别有效的方法。聚乙二醇化a干扰素(PEG-IFN-a)联合利巴韦林进行优化治疗或标准方案联合蛋白酶抑制剂对基因1型的治疗仍然是当前的首要选择。但是由于干扰素、利巴韦林及蛋白酶抑制剂具有难以适应的毒副作用，使RHC治疗更为困惑。因此，进一步探求难治性丙型肝炎免疫发病机制及寻找其治疗的有效方案是医学和临床的迫切需求，中医药以其独特优势，在防治慢性丙型病毒性肝炎方面积累了丰富的经验。从传统的辨证论治、专病专方专药、针灸、单药、民间验方，发展到现在的中西医结合治疗方案，都有中医药作出的贡献。多年的研究也证实，中医药在慢性丙型肝炎的防治中不仅可以缓解患者症状，减轻肝脏炎症，而且能抑制或抗病毒，逆转肝脏早期肝纤维化，减轻西药的毒副作用，延缓其慢性化，增强机体免疫，提高患者生活质量，以达到延长生存期，预防肝硬化、肝癌等终末期肝病，意义重大。目的：本研究在“十一五”慢性丙型肝炎证候规律及中西医结合治疗方案研究的基础上，依托国家“十二五”难治性丙型肝炎的中医药治疗方案，以中药辨证方（益气解毒方、疏肝健脾方、补肾柔肝方）治疗难治性丙型肝炎，观察其临床疗效、转氨酶和HCV RNA转阴率及下降率（HCV RNA下降2log），并对其免疫功能与转氨酶、病毒载量以及中医证型与T淋巴细胞亚群、HCV RNA等相关性进行研究，运用计算机统计学软件进行数据处理和综合分析，以探讨中药辨证方对难治性丙型肝炎的临床疗效以及RHC的病毒载量、肝脏炎症、免疫调节机制与中医证型之间的相关性，以便进一步提高难治性丙型病毒性肝炎的临床治疗水平。方法：实验分三个部分进行第一部分：中医药辨证方治疗难治性丙型肝炎的疗效观察将54例难治性丙型肝炎患者随机、对照分为试验组28例和对照组26例。试验组给予中药辨证方：益气解毒方、疏肝健脾方、补肾柔肝方治疗，对照组给予中药安慰剂治疗。治疗12，24，36，48周后观察并比较2组血HCV RNA阴转率和下降率、肝功能变化及2组中医症状积分变化。第二部分：难治性丙型肝炎患者外周血T细胞亚群分析与病毒载量相关性研究对153例难治性丙型肝炎感染者按基因1b、抗病毒治疗阴转后复发及肝硬化分成3组，分别采用流式细胞术和荧光定量PCR技术分别检测外周血中T淋巴细胞亚群(CD3+T、CD4+T、CD8+T、CD4+T/CD8+T)和HCV RNA载量。另选同期健康体检者150人外周血中T淋巴细胞亚群作为对照组。第三部分：难治性丙型肝炎中医证型与T淋巴细胞亚群及HCV RNA载量等相关性研究对153例RHC患者进行中医证型研究,用流式细胞仪检测其患者和150例正常健康对照者外周血T淋巴细胞亚群变化，荧光定量PCR检测患者RHCRNA含量、基因分型和肝功能检测，观察肝功能、血清RHC RNA载量及T淋巴细胞亚群指标与中医证型之间的关系。结果：第一部分：中医药辨证方治疗难治性丙型肝炎的疗效观察与对照组比较中药辨证方（益气解毒方、疏肝健脾方、补肾柔肝方）组能明显降低转氨酶水平、提高HCV RNA阴转率和下降率（P＜0.05)。第二部分：难治性丙型肝炎患者外周血T细胞亚群分析与病毒载量相关性研究1与对照组相比RHC患者组CD3+T、CD4+T下降有统计学意义（p＜0.05），而CD8+T、CD4+T/CD8+T无统计学意义（p＞0.05）。2高病毒载量患者组与对照组相比较CD3+T、CD4+T下降有统计学意义（P＜0.05），而CD8+T、CD4+T/CD8+T变化无显著差异（P＞0.05），低病毒载量患者组与对照组比较CD8+T下降有统计学意义（P＜0.05）。RHC高病毒载量患者组与低病毒载量患者T淋巴细胞亚群比较，差异无统计学意义（P＞0.05）。3抗病毒治疗阴转后复发组与对照组比较CD3+T、CD4+T、CD8+T下降明显，差异有统计学意义（P＜0.05）。4肝硬化组与对照组比较CD3+T、CD8+T下降明显，而CD4+T/CD8+T升高明显，差异有统计学意义（P＜0.05）。5基因1b组与对照组相比较CD4+T、CD8+T下降明显，差异有统计学意义（P＜0.05）。6治疗阴转复发组与肝硬化组无统计学意义，与基因1b型比较CD3+T、CD8+T下降明显，差异有统计学意义（P＜0.05）。7肝硬化组与基因1b型组比较CD3+T、CD8+T下降明显、而CD4+T/CD8+T升高明显，差异有统计学意义（P＜0.05）。第三部分：难治性丙型肝炎中医证型与T淋巴细胞亚群及HCV RNA载量等相关性研究1难治性丙型肝炎常见的中医证型依次为正虚邪恋、肝郁脾虚、肝胆湿热、瘀血阻络、肝肾阴虚及脾肾阳虚等六型。2ALT水平与中医证型的关系：即ALT从高到低在各证型中的排列顺序为：湿热中阻型、脾肾阳虚型、肝肾阴虚型、瘀血内阻型、肝郁脾虚型、正虚邪恋型，其湿热中阻型与其他证型相比较ALT明显升高，差异有统计学意义(P＜0.05)； AST水平与中医证型的关系：即AST从高到低在各证型中的排列顺序为：脾肾阳虚型、湿热中阻型、瘀血内阻型、肝肾阴虚型、肝郁脾虚型、正虚邪恋型，其中脾肾阳虚型与与其他类型相比较明显升高，差异有统计学意义(P＜0.05)；ALB与中医证型的关系为：脾肾阳虚型ALB与其他中医类型明显降低，差异有统计学意义(P＜0.05)。3RHC RNA水平与中医证型有一定规律性，即湿热中阻型＞正虚邪恋型＞肝肾阴虚型＞瘀血内阻型＞肝郁脾虚型＞脾肾阳虚型；湿热中阻型与瘀血内阻型、脾肾阳虚型相比较，RHC RNA明显升高，差异有统计学意义(P＜0.05)。4T淋巴细胞亚群指标(CD3+T、CD4+T、CD4+T/CD8+T)与中医辨证分型有一定相关性，与正常对照组相比较湿热中阻型CD8+T明显升高（P＜0.01），CD4+T/CD8+T明显下降（P＜0.05）；脾肾阳虚型和肝郁脾虚型与正常对照组相比较CD3+T、CD4+T明显下降（p＜0.01），CD8+T明显下降（P＜0.05）；正虚邪恋型、瘀血内阻型、肝肾阴虚型与正常对照组相比较(CD3+T、CD4+T、CD4+T/CD8+T)差异无统计学意义(P＞0.05)。5在各证型相比较中脾肾阳虚型T淋巴细胞计数明显下降，与正虚邪恋型、湿热中阻型、瘀血内阻型、肝肾阴虚型差异有统计学意义（P＜0.05）；肝郁脾虚型T淋巴细胞计数明显下降，与湿热中阻型、肝肾阴虚型差异有统计学意义（P＜0.05）；肝胆湿热型T淋巴细胞计数明显升高，与瘀血内阻型、脾肾阳虚型比较差异有统计学意义（P＜0.05）；CD4+T/CD8+T明显下降，与瘀血内阻型比较差异有统计学意义（P＜0.05）。结论：1、中药辨证方能改善难治性丙型病毒性肝炎肝脏炎症，且具有一定的抗病毒作用。2、长期慢性HCV感染会引起机体细胞免疫功能低下和免疫功能紊乱，可能是基因1b型、抗病毒治疗阴转后复发及肝硬化形成难治性的主要原因之一， RHC患者病毒载量高，病毒复制活跃，肝脏炎症亢进，免疫功能低下及免疫紊乱程度随HCV载量增高而逐渐加重。3、RHC中医证型依次为正虚邪恋型、肝郁脾虚型、湿热中阻型、瘀血内阻型、肝肾阴虚型及脾肾阳虚型等六型;4、RHC转氨酶、病毒载量及T淋巴细胞亚群与中医证型有一定相关性。更多还原

【Abstract】 Chronic hepatitis c(HCV) is a public health problem whichcause serious damage to the human health. According to the WHO global HCV infection rate was3%, about170million people, thereare35000new hepatitis c patients every year. According to thelatest published by the state ministry of health in September，2010,the results of epidemiological investigation anti HCV positive ratewas0.43%in our country, probably the HCV infected more than5million.Nature of HCV infection history is long, a rate ofchronic at50%to85%. Liver cirrhosis and liver cancer is theleading cause of death among patients with chronic hepatitis c, theinfection of30%to40%will develop cirrhosis after5-10years,5%to7%after10-20years for the development of liver cancer. Oneof the most infectious diseases become the serious harm people’shealth. Although modern medicine in the treatment of chronichepatitis c made some breakthrough in the triple therapy as genotype1b，etc.some lastest scheme of refractory hepatitis c. But someunable to antiviral and virological breakthrough in the treatment,partial response, invalid response or no response, the side effectsof drugs, relapse after the drug was stopped, genotype1or4typeof HCV infection,a high viral load,merging other virus infection,orwith other such as diabetes, thyroid dysfunction Hemophilia organtransplant kidney disease and hemodialysis related diseases suchas autoimmune dysfunction,and the existence of the problems suchas cirrhosis of the liver decompensation period, makes thehepatitis c treatment is still a clinical problem, namely so-calledrefractory chronic hepatitis c. At present,caused by infection ofHCV genotype1chronic hepatitis c in China accounts for about78%,high viral load of chronic hepatitis c accounts for about69%,sothe vast majority of chronic hepatitis c in China belong to the refractory hepatitisc.refractory hepatitis c be formed complex,Andso its clinical manifestations are diverse. Through the11thfive-year plan for CHC symptoms of TCM syndrome classification’study shows：zheng xu xie lian、liver depression and spleendeficiency、kidney Yin deficiency type three accounts for more than73%of all kind of this syndrome，So the factors of the clinicalmanifestations of intermingled deficiency and excess are the maincharacteristics of CHC， If people do not implement clinicalintervention, let it to further development,it will be formed theintractable liver disease such as cirrhosis，The poor prognosis，which would be a great threat to The life of the patients, and poseda great challenge to treat. There is no special effective methodin terms of RHC therapies. Polyethylene glycol (peg) a interferon(P E G a IF N a) plus ribavirin optimized treatment or standardsolutions combined with protease inhibitors for the treatment ofgenotype1is still the first choice. But because of interferon、ribavirin and protease inhibitors are difficult to adapt to the sideeffects of the refractory hepatitis c treatment more confused.Therefore, further to seek immune pathogenesis of refractoryhepatitis c and further to find effective scheme for the treatmentof refractory hepatitis c is the urgent need of medical and clinicaltraditional Chinese medicine for its unique advantages, in theprevention and treatment of chronic viral hepatitis c hasaccumulated rich experience. From the traditional treatment basedon syndrome differentiation, ZhuanBing specially designed medicine,acupuncture, medicine, folk prescription, development to combinetraditional Chinese and western medicine treatment, has thecontribution of Chinese medicine. Years of research also confirmed that the traditional Chinese medicine in the prevention andtreatment of chronic hepatitis c is liver enzyme and antiviraleffect, to turn the early suppression of liver fibrosis has beenrecognized, to reduce the rate of chronic, chemical medicine sideeffects to improve compliance, improve immunity, improvingsymptoms and quality of life, prolong the survival period, reducethe mortality and the incidence of major events is more obviousadvantages.Purpose：This study on the basis of the "11th five-year plan" Thestudy of Chronic hepatitis c syndrome regularity and the treatmentprescription with the method of intergrated traditional andwestern medicine, relying on the national “12th five-year”refractory hepatitis c treatment of traditional Chinese medicine,in traditional Chinese medicine (TCM) syndrome (yiqi jiedu party,liver, spleen, kidney and liver) to treat refractory hepatitis c,and observe its clinical curative effect, transaminase and HCV RNAturn rate and falling rate drops2log (HCV RNA), and the immunefunction and transaminase, viral load and TCM syndrome type and Tlymphocyte subsets, HCV RNA and other correlation is studied, usingthe computer statistical software for data processing andcomprehensive analysis, to explore the clinical curative effect ofChinese medicine syndrome differentiation on refractory hepatitisc and RCHC viral load, liver inflammation, immune regulationmechanism and the correlation between TCM syndrome types, in orderto further improve the level of clinical treatment of refractorychronic viral hepatitis c.Methods: the experiment divided into three partsThe first part: the syndrome differentiation of traditional Chinese medicine clinical observation on treatment of refractorychronic hepatitis c54patients with refractory chronic hepatitis c were randomized,compared,28cases were divided into test group and control groupin26cases. Experimental group were given Chinese medicinesyndrome differentiation: yiqi jiedu, liver spleen, kidney andliver treatment, control group given a placebo treatment.,24,36,4812weeks after treatment to observe and compare the two groups ofblood HCV RNA Yin turn rate and falling rate, liver function andtwo groups of traditional Chinese medicine symptom integralchanges.The second part: the peripheral blood T cell subsets in patientswith refractory chronic hepatitis c correlation research and viralload153cases of intractable chronic HCV infection in1b gene,recurrence and liver cirrhosis after antiviral treatment Yin turninto three groups, respectively, by using flow cytometry andfluorescence quantitative PCR detection respectively in theperipheral blood T lymphocyte subsets (CD3+T，CD4+T，CD8+T，CD4+T/CD8+T) and HCV RNA loads. Alternate year healthy physicalexamination,150people in the peripheral blood T lymphocytesubgroup as control group.The third part: refractory of TCM syndrome types of chronichepatitis c and T lymphocyte subgroup and HCV RNA loads such ascorrelation studiesTCM syndrome types was studied for the153patients, using flowcytometry to test which patients and150normal healthy controlsperipheral blood T lymphocyte subsets changes, fluorescence quantitative PCR detection RCHC RNA content, genotyping, andpatients with liver function test, to observe the liver function,serum RCHC RNA loads and T lymphocyte subgroup indicators and therelationship between the TCM syndrome type.Results:the first part: the syndrome differentiation of traditionalChinese medicine clinical observation on treatment of refractorychronic hepatitis cTraditional Chinese medicine (TCM) syndrome compared with controlsparty (yiqi jiedu, liver, kidney and liver, spleen and party party)group can obviously reduce transaminase level, improve the HCV RNAYin turn rate and falling rate (P＜0.05) in the second part:peripheral blood T cell subsets in patients with refractory chronichepatitis c correlation research and viral load.1RHC patients compared with the control group of CD3+T，CD4+Tdecline was statistically significant (P＜0.05), while CD8+T, CD4+T/CD8+T no statistical significance (P>0.05).2A group of patients with high viral load compared with thecontrol group CD3+T, CD4+T decline was statistically significant (P<0.05), and CD8+T, CD4+T/CD8+T change there was no significantdifference (P>0.05), a group of patients with low viral load CD8+Tdecline was statistically significant compared with controls (P <0.05). RCHC group of patients with high viral load and low viralload in patients with T lymphocyte subsets, there was nostatistically significant difference (P>0.05).3After antiviral treatment turn Yin relapse group and controlgroup comparison of CD3+T, CD4+T, CD8+T decreased significantly, thedifference was statistically significant (P <0.05). 4liver cirrhosis group and control group comparison of CD3+T,CD8+T decreased significantly, and CD4+T/CD8+T increasesignificantly， the difference was statistically significant (P <0.05).5Gene1b group compared with control group, CD4+T, CD8+Tdecreased significantly, the difference was statisticallysignificant (P <0.05).6Treatment Yin turn recurrence, no statistical significancebetween the groups with and cirrhosis of the liver, compared withgenotype1b CD3+T, CD8+T decreased significantly, the differencewas statistically significant (P <0.05).7Cirrhosis group compared with genotype1b group of CD3+T,CD8+T decreased significantly, and CD4+T/CD8+T increasesignificantly，the difference was statistically significant (P <0.05).The third part: refractory of TCM syndrome types of chronichepatitis c and T lymphocyte subgroup and HCV RNA loads such ascorrelation studies.1Refractory common TCM syndrome types of chronic hepatitisc in the order is empty and, to change, liver and gallbladder dampand hot, blood stasis resistance winding, liver and kidney Yindeficiency and spleen kidney Yang deficiency and so on six type.2ALT level and based on the relationship between TCM syndrometype：which is the ALT from high to low in sequence for each cardtype：humid heat to hinder type，spleen and kidney Yang deficiencytype，liver-kidney Yin deficiency type，blood stasis resistancetype， change the type is empty and type, including humid heat tohinder type compared with other syndrome types ALT increased significantly, the difference was statistically significant(P<0.05); AST level and based on the relationship between TCM syndrometypes: which is the AST from high to low in sequence for each cardtype：spleen kidney Yang deficiency type, humid heat to hinder type,blood stasis resistance type, liver-kidney Yin deficiency type,change the type is empty and type, the spleen and kidney Yangdeficiency type and compared with other types increasedsignificantly, the difference was statistically significant(P<0.05); Propagated and based on the relationship between TCMsyndrome types: spleen kidney Yang deficiency type A significantlylower with other types of traditional Chinese medicine, thedifference was statistically significant(P <0.05).3RHC RNA levels and TCM syndrome types have a certain regularity,namely courage humid heat to hinder type> is empty and>liver-kidney Yin deficiency type> blood stasis resistance type>change the type> spleen kidney Yang deficiency type.The damp andhot resistance type and blood stasis resistance type compared thespleen kidney Yang deficiency type,RHC RNA increasedsignificantly,the difference was statistically significant(P<0.05).4Index of T lymphocyte subsets (CD3+T, CD4+T, CD4+T/CD8+T) oftraditional Chinese medicine and have a certain correlation,compared with normal control group humid heat to hinder type CD8+T significantly increased (P<0.01), CD4+T/CD8+T decreasedobviously (P <0.05); Spleen and kidney Yang deficiency type andchange the type compared with normal control group T CD3+T, CD4+TT cells decreased obviously (P <0.01), and CD8+T significantlydecreased (P<0.05); Is virtual love evil, blood stasis resistance compared with the normal control group, liver and kidney Yindeficiency type (CD3+T, CD4+T, CD4+T/CD8+T) has no statisticalsignificance(P＞0.05).5In each type of spleen kidney Yang deficiency syndromecompared T lymphocyte count declined obviously, and is empty andtype, humid heat to hinder type, blood stasis resistance type，liver and kidney Yin deficiency syndrome, type difference wasstatistically significant(P <0.05); Change the type of Tlymphocyte count drops obviously, and humid heat to hinder type,kidney Yin deficiency type difference was statisticallysignificant(P<0.05); Liver syndrome T lymphocyte count increasedsignificantly, compared with the blood stasis resistance type,spleen and kidney Yang deficiency type difference was statisticallysignificant(P <0.05)； CD4+T/CD8+T significantly decreased,compared with blood stasis type resistance difference wasstatistically significant(P <0.05).Conclusion:1Chinese medicine syndrome differentiation can improverefractory chronic viral hepatitis c liver inflammation, and hascertain antiviral effect.2long-term chronic HCV infection can cause low organismcellular immunity function and immune dysfunction, may be thegenotype1b and antiviral treatment after turn Yin recurrence andliver cirrhosis formed one of the leading causes of refractory, RCHCpatients with high viral load, active viral replication, liverinflammation disease, weakened immune system and the immune levelof turbulence gradually aggravated with HCV higher loads.3TCM syndrome type of RHC in turn is empty and type, change the type, liver and gallbladder damp and hot resistance type, bloodstasis resistance type, liver and kidney Yin deficiency type andspleen kidney Yang deficiency type and so on six type.4RHC transaminase, viral load and T lymphocyte subsets andTCM syndrome types have certain relevance.更多还原