【作者】 田辉；

【导师】 王占英；

【作者基本信息】 中国医科大学 ， 内科学， 2002， 硕士

【摘要】 前言 丙型肝炎病毒(HCV)感染呈世界性分布，其特征是80％以上的患者转为慢性肝炎，进而发展为肝硬化，甚至原发性肝癌。目前研究认为HCV感染慢性化与病毒因素和宿主因素均有关。 HCV为一单股正链RNA病毒，整个基因组具有高度变异性。在感染者体内HCV可同时存在多种变异株，以高度多样化的病毒群体即准种(quasispecies)的形式存在。近年来的研究提示不仅HCV准种特性与HCV感染慢性化相关，而且急性期HCV准种的动态变化规律可以预测感染的结局。不过，就目前的研究资料来看同时对急性、慢性感染HCV准种比较研究较少，病程长短与HCV准种变化的研究更少。为此，本研究采用聚合酶链反应-单链构象多态性分析(PCR-SSCP)法对30例丙肝患者进行了HCV准种的检测，探讨HCV准种与丙型肝炎慢性化的关系。 材料与方法 以30例丙型肝炎患者为研究对象。其中急性丙型肝炎8例，慢性丙型肝炎15例，肝硬化7例。所有病例诊断均符合2000年9月(西安)全国传染病与寄生虫病学术会议修订标准。所有病人血清抗-HCV和HCVRNA(5NC区PCR)均阳性，其它肝炎病毒感染标志均阴性，而且均未应用过干扰素及免疫抑制剂或免疫增强剂。取上述30例病人静脉血各3ml，分离血清。首先通过RTPCR检测HCVRNA{高变区1(HVR1)}，然后采用SSCP分析进行准种检测，比较分析了临床类型、感染途径及年龄、性别、ALT水平、病程与SSCP条带数的关系。 结 果 本文30例HCVRNA（HVRI）阳性标本经SSCP分析均可见清晰的SSCP条带，条带数为2－9条，平均4．7条。其中急性丙型肝炎8例，SSCP条带数为2－5条，平均3．13条；慢性丙型肝炎15例，SSCP条带数为3－7条，平均5．07条；肝硬化7例，SSCP条带数为3－9条，平均5．57条。慢性肝炎组与急性肝炎组SSCP条带数比较有非常显著差异f值为0．004。肝硬化组与急性肝炎组SSCP条带数比较有显著差异J值为0．015。慢性肝炎组与肝硬化组SSCP条带数比较未见明显差异，P值为0．639。 本文中通过输血或使用血制品感染HCV者共17例，SSCP条带数为 3－9条，平均 5．3条。散发感染组共 11例，SSCP条带数为2－7条，平均3．9条，与输血感染组比较有显著差异J值为0．040。通过吸毒感染HCV者2例，SSCP条带数为3－4条，平均3．5条，与散发感染组比较未见有显著差异J值为0．839。 本文中22例慢性肝炎和肝硬化患者SSCP条带数与性别、年龄人LT水平均无相关性。而SSCP条带数与病程显著相关，相关系数分别为0．572（Pearson、相关），0．556（SPe－an、相关）。 讨 论 在本文中，8例急性丙肝，15例慢性丙肝和7例HCVRNA阳性的肝硬化患者SSCP条带数分别为3．13。1．07，5．07。1．48，5．57。2．15，慢性丙肝和肝硬化的HCV准种数量显著高于急性丙肝，由此表明HCV准种特性与丙型肝炎慢性化密切相关，与国内外的一些研究结果一致。实际上，准种呈现出一个快速移动的靶 ·2·位，宿主免疫系统似乎对之不能完全控制。也就是说HCV准种是HCV在体内得到保护，逃脱宿主兔疫攻击，造成感染持续的重要策略之一。近年来陆续有研究证实急性感染期HCV准种的演变规律可以预测感染的结局。丙肝慢性化的过程就是HCV准种产生速度大于宿主兔疫清除能力的结果。 本文结果表明输血或输血制品感染者HCV准种数量显著高于散发感染者，说明输血或输血制品可增加 HCV感染次数及不同准种重叠感染的机会。本文吸毒感染组HCV准种数量与散发感染组比较未见显著差异月能与吸毒感染组病例少有关。 本文显示持续感染时间越长，HCV准种数量越高，HCV准种数量与病程正相关。可见HCV准种数量与丙型肝炎的疾病进展可能有关。理论上准种内的变异株反映的是在特定环境内以它们的复制能力为基础的这些突变株间竞争性选择的结果，即复制效力最强的HCV准种将成为优势株。但由于病毒RNA聚合酶的错配复制及宿主免疫选择压力的作用，随感染时间延长，不断有新的准种出现。这也从另一个角度说明由于HCV准种的存在，造成感染持续，使病情逐步加重。 本文表明慢性丙肝病人HCV准种数量与血清ALT水平元关，提示肝病活动度与准种数量多少并无相关性。但目前就HCV准种与肝病活动度的关系还末达成共识，争议颇多。造成这种研究结论差异的原因，可能与方法学上的差异，如引物选择不同造成PCR扩增产物片段大小不一，导致SSCP分析时出现某些结论的不一致；病例选择上的偏倚，研究样本大小及检测时间不一致等因素有关。对此应进一步深人研究。 本文采用PCR－SSCP分析法进行了准种检测，结果令人满意。较传统的克隆测序法相对省时省力，花费少，简单易行，提示PCR．SSCP法可作为检测HCV准种的常规手段而广泛开展应用。 ·3· 结 论 1．慢性丙型肝炎HCV准种数量高于急性丙型肝炎，表明HCV准种数量与丙肝慢性化密更多还原

【Abstract】 Hepatitis C virus (HCV) is an important public health problem because it is a major cause of chronic hepatitis, cirrhosis, and hepato-cellular carcinoma worldwide. The most striking feature of this virus is its ability to induce chronic infection in at least 80% of infected individuals. The recent studies have shown that the chronicity of HCV infection relates to both viral factors and host factors.HCV is a single - stranded positive sense RNA virus, the whole genome displaying a higher degree of genetic variability. HCV exists as a highly heterogenous population of closely related variants in vivo, which is called quasispecies. In recent years, several studies have shown that not only HCV quasispecies nature is strongly correlated with chronic infection, but also the evolutionary dynamics of the viral quasispecies during the acute phase of hepatitis C can predicts whether the infection will resolve or become chronic. However, the evidence is still preliminary. Comparative study of HCV quasispecies of the patients with acute and chronic infection is rare, the investigations of the relationship between the duration of HCV infection and HCV quasispecies are fewer. Thus, to explore the relationship between HCV quasispecies and HCV persistent infection , HCV quasispecies of 30 patients with hepatitis C was detected by single - strand conformation polymor-phism (SSCP) analysis.Materials and methodsThe serum samples of 30 patients with hepatitis C were used in this study. The study group consisted of 8 patients with acute hepatitis, 15 patients with chronic hepatitis and 7 patients with liver cirrhosis. The diagnosis was based on the diagnosis criteria of communicable and parasitic disease branch of China Medical Association. The serum of all patients were HCVRNA(5’NC) positive and the markers of other hepatitis virus were negative, and none of the patients had received any immunosuppressive drug and interferon before the study. HCVR-NA(HVR1) was tested by RT - PCR, and the number of HCV qua-sispecies was detected by SSCP analysis. To study the relationship between HCV quasispecies and chronic infection of HCV, the number of bands in SSCP analysis was compared among clinical types, transmission routes and age, sex, ALT levels, duration of HCV infection.ResultsThe average number of SSCP bands per sample was 4. 7 with a range from 2 to 9. In the present study , the average number of HCV quasispecies in the patients with acute hepatitis, chronic hepatitis and liver cirrhosis was 3. 13, 5. 07, 5. 57, respectively. The number of viral quasispecies in the patients with chronic hepatitis and liver cirrhosis was significantly higher than that in the patients with acute hepatitis C (P < 0.05). However, the number of viral quasispecies in the patients with chronic hepatitis was not significantly higher than thatin the patients with liver cirrhosis (P >0. 1 ).In the present study, the average number of SSCP bands in 17 patients whose infection was caused by blood transfusion or blood products was 5.3 (range 3 -9) , the average number of SSCP bands in 11 patients whose infection was acquired by sporadic infection was 3.9 (range 2 -7) , the difference between the posttransfusion and sporadic hepatitis groups was significant ( P <0. 05) . However, in 2 patients whose infection was acquired by I. V. drug use, the average number of SSCP bands was 3.5 ( range 3-4 ) , no significant difference was found between the I. V. drug use and sporadic hepatitis groups.In the present study, statistical analysis of the results revealed that age, sex, ALT levels were not correlated with the number of bands in SSCP analysis. The number of SSCP bands in the 22 patients with chronic hepatitis and cirrhosis was significant correlation with the duration of HCV infection.DiscussionIn the present study,3. 13 ±1.07,5.07 ±1.48 and 5. 57 ±2.15 SSCP bands were detected in 8 patients with acute hepatitis, 15 patients with chronic hepatitis and 7 patients with liver cirrhosis, respectively. The number of viral quasispecie更多还原