【作者】 邱冰；

【导师】 李东复；

【作者基本信息】 吉林大学 ， 内科学， 2010， 博士

【摘要】 HBV持续感染所致的疾病是全球性分布的慢性病毒感染性疾病,HBV的持续感染是引起慢性乙型肝炎、肝硬化和原发性肝癌(hepatocellular carcinoma,HCC)等不同临床结局的重要原因。HBV感染慢性化机理颇为复杂,除了与病毒、环境因素有关外,宿主本身,尤其是免疫遗传因素发挥了相当重要的作用。目前人们更多地关注细胞因子和抗原提呈系统及其介导的免疫细胞间的相互作用,正是这些免疫分子与细胞参与了HBV持续感染的免疫应答,不同个体免疫遗传状态的差异决定了HBV持续感染的结局。其中人体重要的免疫调节因子肿瘤坏死因子-α(tumour necrosis factor–α,TNF-α),及MHC-Ⅰ类分子限制的抗原递呈途径中的重要分子抗原提呈相关转运体(transporter associated with antigen processing,TAP)等位基因多态性对慢性HBV感染结局的影响, TNF-α、TAP1表达与HBV相关肝细胞癌的关系一直是人们研究的热点,因为HBV持续感染是肝细胞癌发生发展的重要因素。目前国内外关于TNF-α、TAP多态性与慢性HBV感染及表达与肝癌患病关系研究尚少,报道也不一致。为此本课题选取我国东北地区(吉林、黑龙江)汉族人群慢性HBV感染患者,包括慢性乙型肝炎(Chronic hepatitis B, CHB)、乙型肝炎肝硬化(HB cirrhosis, HC)和HBV相关肝癌(Hepatocellular carcinoma, HCC)患者,并以HBV感染自然恢复者(Spontaneous recovery,SR)作为对照,应用聚合酶链反应-限制性片段长度多态性(Polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)技术检测不同疾病组患者和对照组HBV感染自然恢复者的TNF-α、TAP等位基因分布情况,并构建TAP等位基因的单体型;应用免疫组化技术EnVision方法检测HBV相关肝癌及正常肝组织肝细胞中TNF-α及TAP1分子的表达。旨在从免疫遗传学角度探讨慢性HBV感染与TNF-α、TAP基因多态性相关关系,探讨TNF-α、TAP1分子在HBV相关肝癌组织中的表达及意义,进而阐明TNF-α/TAP1与HBV相关肝癌病理分级的关系。结果表明:1.慢性乙型肝炎和乙型肝炎肝硬化组患者TNF-α-857C等位基因频率显著高于对照组HBV感染自然恢复者(OR=1.52,95%CI 1.08-2.13,P=0.01;OR= 1.47,95%CI 1.06-2.04,P=0.02);2.慢性乙型肝炎、乙型肝炎肝硬化和HBV相关肝癌组患者TNF-α-863A等位基因频率均显著高于对照组HBV感染自然恢复者(OR=1.72,95%CI 1.19-2.50,P= 0.004;OR=1.81,95%CI 1.26-2.61,P=0.001;OR=1.90,95%CI 1.33-2.73,P<0.001);3.慢性乙型肝炎和乙型肝炎肝硬化组患者TAP1-637-Gly等位基因频率显著高于对照组HBV感染自然恢复者(OR=1.53 ,95%CI1.05-2.23,P=0.026;OR=1.73,95%CI1.20-2.48,P=0.003);4. HBV相关肝癌组患者TAP2-651-Cys等位基因频率显著高于对照组HBV感染自然恢复者(OR=2.24,95%CI1.45-3.48,P<0.001);5.慢性乙型肝炎组患者TAP2-687-Gln等位基因频率显著高于对照组HBV感染自然恢复者(OR=1.36,95%CI1.02-1.82,P=0.039);6.慢性乙型肝炎、乙型肝炎肝硬化、HBV相关肝癌组患者TAP单体型[-687C;-651T; -637G; -333A]频率均显著高于对照组HBV感染自然恢复者(P均<0.05);7.正常肝组织肝细胞均未见TNF-α及TAP1表达,TNF-α在HBV相关肝癌组织肝细胞中表达阳性率为60%,与正常肝组织表达情况相比较,有显著性差异(P<0.05),并且TNF-α在病理分化较好的癌组织阳性表达率高于分化较差者(P<0.05),TAP1在HBV相关肝癌组织肝细胞中阳性表达率为91.4%,与正常肝细胞比较有非常显著差异(P<0.01)。结论:在东北汉族人群中,TNF-α-857C等位基因可能增加慢性乙型肝炎及乙型肝炎肝硬化患病的易感性;TNF-α-863A等位基因可能增加慢性乙型肝炎、乙型肝炎肝硬化和HBV相关肝癌患病的易感性。TAP1-637-Gly等位基因可能增加慢性乙型肝炎和乙型肝炎肝硬化患病的易感性;TAP2-651-Cys等位基因可能增加HBV相关肝癌患病的易感性;TAP2-687-Gln等位基因可能增加慢性乙型肝炎患病的易感性;TAP单体型[-687C;-651T;-637G;-333A]可能增加慢性乙型肝炎、乙型肝炎肝硬化及HBV相关肝癌患病的易感性。TNF-α、TAP基因多态性与东北地区汉族人群慢性HBV感染不同临床结局相关。HBV相关肝癌组织中TNF-α表达量与HCC患病及组织分化程度相关,HBV相关肝癌组织中TAP1分子异常表达。更多还原

【Abstract】 Chronic infection of HBV is an important factor leading to different clinical outcomes including chronic hepatitis B, hepatitis B cirrhosis and hepatocellular carcinoma (HCC). Each year, about 1.2 million people worldwide die from chronic HBV infection-related liver disease. It is high-endemic area of hepatitis B virus infection in China,where there is up to 9.09% of chronic HBV infection among crowd accounting for over 1/3 of the global total.Each year it occurs in patients with hepatitis B cirrhosis and hepatocellular carcinoma (HCC) accounting for 2% and 1% among patients with chronic hepatitis B,respectively. Chronic HBV infection is threatening to people,s health more and more. Therefore, the study about the chronic mechanism of HBV infection is still an important topic in the world. The chronic mechanism of HBV infection is quitely complex, involving the combined effects of a host, viruses, environment and other factors, the combination of host immune and genetic factors may play an important role in the occurrence and progression of chronic HBV infection.lt has been payed more attention to antigen-presenting system and cytokines, because they are involved in immune response to HBV infection, and may be associated with the susceptibility to HBV infection and the clinical outcomes of infection. In which the effects of polymorphisms of TNF-α(tumour necrosis factor-α)as an important human immune regulator and TAP (transporter associated with antigen processing) as an important molecular of MHC-Ⅰrestricted antigen processing pathway on the clinical outcomes of chronic HBV infection,and expression of TNF-α,TAP1 on hepatocellular carcinoma had been studied little.The study aimed to clarify the correlation between the gene polymorphisms of TNF-α、TAP and clinical outcomes of chronic HBV infection ,and the association between expression of TNF-α、TAP1 and hepatocellular carcinoma due to HBV in a Han population in northeasten China .Objective:The aim of this study was to explore the potential relationship between polymorphisms of TNF-α,TAP and different clinical outcomes of chronic HBV infection,and the association of expression of TNF-α,TAP1 with occurrence and development of hepatocellular carcinoma due to HBV in a Han population in northeastern China. It is expected that results of the studies will be theoretical importance in exploring chronic mechanism of HBV infection and helpful to providing new indicators for diagnosis and prognosis ,and theoretical basis for the immune and gene therapy of chronic HBV infection-related diseases.Methods:1.189 HBV spontaneously recovered subjects (SR), 571 HBV chronically infected subjects including 180 chronic HB patients (CHB), 196 HB cirrhosis patients (HC) and 195 hepatacellular carcinoma patients due to HB(HCC) were included in this study. TNF-α-857 C/T and-863 C/A , TAP1-333 Ile/Val and-637 Asp/Gly ,TAP2-651 Arg/Cys and -687 Stop/Gln were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), The results were analyzed with gel imaging analytical system after agarose gel electrophoresis, and clarified the association of TNF-αgenotypes and alleles at position 857、863 , TAP alleles and four locus haplotype composed of TAP1-333,-637 and TAP2-651, -687 with chronic hepatitis B, HB cirrhosis,and hepatacellular carcinoma duo to HB by the method of case-control study.2.The expression of TNF-αand TAP1 in 35 liver carcinoma tissues, and 10 nomal liver tissues obtained through operation were detected with EnVision method belonged to immunohistochemical technique and compared with each other in order to clarify the expression and their significance of TNF-α,TAP1 in HCC due to HBV. Furthermore, the association with pathological typing was analyzed.Results:1.TNF-α-857C allele frequency was significantly higher in chronically HBV-infected individuals (CHB and HC) compared with that of SR subjects (P=0.01, OR=1.52,95%CI=1.08-2.13;P=0.02,OR=1.47,95%CI=1.06-2.04,respectively);2.TNF-α-863A allele frequency was significantly more common in HBV-infected individuals with chronic HBV infection (CHB, HC and HCC) than that of SR controls(P=0.004, OR=1.72, 95%CI=1.19-2.50;P=0.001, OR=1.81, 95%CI=1.26-2.61 and P<0.001, OR=1.90, 95%CI= 1.33-2.73, respectively).3. TAP1-637-Gly allele frequency was significantly more common in HBV-infected individuals with chronic HBV infection (CHB and HC) than that of SR controls(OR = 1.53, 95%CI1.05-2.23, P=0.026; OR =1.73, 95%CI1.20-2.48,P = 0.003);4. a statistically significant difference in the distribution of TAP2-651-Cys allele between HCC cases and SR controls(OR=2.24,95%CI1.45-3.48,P<0.001) was observed; 5.TAP2-687-Gln allele frequencie in CHB group was higher than that in SR group (OR=1.36,95% CI1.02-1.82,P=0.039); 6.Haplotype analysis also revealed that TAP haplotype [-687C;-651T; -637G; -333A] was strongly associated with chronic HBV infection (CHB, HC and HCC) compared with SR(P<0.001, P<0.05, P<0.001,respectively);7.The expression of TNF-α,TAP1 was negative in hepatocytes of normal liver tissues. 60% of HCC tissues expressed TNF-αwhich distributed in cytoplasms dispersively, There was significant difference between HCC and the normal hepatic tissues in expression of TNF-α( P<0.05),and the positive expression rate of TNF-αin well-differenciated HCC tissues was significantly higher than that in poorly-differenciated ones (P<0.05),which showed that the overexpression of TNF-αwas associated with the occurrence and development of HCC due to HB.91.4% of HCC tissues expressed TAP1 which distributed in cytoplasms intensively or dispersively, There was statistical difference in expression of TAP1 between HCC tissues and the normal hepatic tissues (P<0.05),which showed that TAP1-related MHC-Ⅰrestricted antigen processing pathway was possibly normal in HCC related to HB.Conclusion:In a Han population in northeastern China. 1.TNF-α-857C allele was close associated with CHB and HC, TNF-α-863A allele was close associated with CHB,HC and HCC. TNF-αalleles may be close associated with chronicity of HBV infection 2.TAP1-637-Gly allele was associated with CHB and HC; TAP2-651-Cys allele was associated with HCC;TAP2-687-Gln allele was associated with CHB; 3.TAP haplotype [-687C;-651T; -637G; -333A] was strongly associated with chronic HBV infection (CHB, HC and HCC),respectively;4.The expression quantity of TNF-αwas associated with occurrence and development of HCC due to HB;4.TAP1-related MHC-Ⅰrestricted antigen processing pathway is possibly normal in HCC related to HB.Compared with similar studies in China and worldwide,our research deserves to be studied and explored further which provided laboratory evidence for immune genetic mechanism of chronic HBV infection from gene and molecular level. The innovations of this research were as follows: It was the first study in Han population of northeastern China that:①TNF-αallele polymorphism was associated with chronic HBV infection including chronic hepatitis B, posthepatitis B hepatic cirrhosis and posthepatitis B hepatocellular carcinoma;②TAP allele and haplotype polymorphisms were associated with chronic HBV infection including chronic hepatitis B, posthepatitis B hepatic cirrhosis and posthepatitis B hepatocellular carcinoma;③The expression quantity of TNF-αwas associated with the occurrence and degree of pathological differentiation of HCC duo to HBV;④The strongly positive expression of TAP1 suggested that TAP1-related MHC-Ⅰrestricted antigen processing pathway is possibly normal in HCC related to HB.In summary,this study has illuminated the relationship between TNF-α、TAP alleles and TAP haplotype and different clinical outcomes of chronic HBV infection including chronic hepatitis B,posthepatitis B hepatic cirrhosis and posthepatitis B hepatocellular carcinoma in Han population in northeastern China for the first time.The correlation between gene polymorphisms of TNF-α、TAP and chronicity of HBV infection was established .we also clarified the association between the expression quantity of TNF-αand the occurrence and development of HCC related to HB,further found TAP1-related MHC-Ⅰrestricted antigen processing pathway was possibly normal in HCC related to HB by detecting the expression of TNF-αand TAP1 of liver carcinoma tissues through operation using immunohistochemical technique.our study has elucidated the mechanism of the chronicity of HBV infection.and found genetic markers of the new specific forecast for incidence of chronic HBV infection in different parts of our country, provided a theoretical basis for the immune and gene therapy of diseases from chronic HBV infection and new indicators for early disease diagnosis and prognosis of chronic HBV infection.更多还原