【作者】 潘焕峰；

【导师】 李东复；

【作者基本信息】 吉林大学 ， 内科学， 2009， 博士

【摘要】 肝细胞癌(HCC)是导致人类死亡的第三大肿瘤相关性病因,在我国居恶性肿瘤病死率的第二位,其发病与乙型肝炎病毒(HBV)感染有关,并呈明显HBV流行性地理分布。研究证实HLA-Ⅱ类分子的某些等位基因与HBV慢性持续性感染有关,进而明确HLA-Ⅱ类抗原基因多态性与肝癌发生发展关系具有重要的临床意义。本研究应用序列特异性引物PCR扩增技术(PCR-SSP)检测了吉林地区汉族人群慢性乙型肝炎(CHB)、肝炎肝硬化(HC)、HCC不同疾病组患者及正常健康对照组HLA-DRB1、DQB1等位基因分布情况;应用酶免疫法检测了肝癌患者外周血AFP水平;应用免疫组化技术检测了与HBV感染相关肝癌癌组织、癌旁组织及正常健康对照肝脏组织的HLA-DR、DQ分子表达,并跟踪随访了HCC患者,旨在从基因层面探索乙型肝炎病毒慢性持续感染相关肝癌发生与HLA-DRB1、HLA-DQB1多态性相关关系;阐明HLA-DRB1和/或DQB1等位基因与HCC患者血清AFP表达水平及HLA-DR、DQ抗原在肝癌组织中的表达与意义,进而阐明抗原表达与HCC病理分级、临床分期及患者生存期等相关性。结果表明,慢性乙型肝炎组DRB1*1201-3、DRB1*0701等位基因表达频率均高于正常对照组(P<0.05,RR=2.391;P<0.01,RR=6.35);乙型肝炎肝硬化组DRB1*0701等位基因表达频率明显高于正常对照组(P<0.01,RR=7.00);肝癌组DRB1*0701等位基因表达频率明显高于正常对照组(P<0.01,RR=6.236),HLA-DRB1*1501-5等位基因表达频率高于正常对照组(P<0.05,RR=2.303)、慢性乙型肝炎组(P<0.05,RR=2.550)及肝硬化组(P<0.05,RR=2.635)。具有HLA-DRB1*0701及/或HLA-DRB1*1501-5等位基因型组HCC患者血清AFP表达水平高于无HLA-DRB1*0701及/或HLA-DRB1*1501-5等位基因型组(P<0.05)。39.5 %肝癌细胞膜和/或浆中表达HLA-DR抗原(P<0.05),病理分化较好组癌组织DR抗原表达率高于分化较差组(P<0.05)。结论:吉林地区汉族人群慢性乙型肝炎患病与HLA-DRB1*1201-3、HLA-DRB1*0701等位基因型相关联,乙型肝炎肝硬化患病与HLA-DRB1*0701等位基因型相关联; HBV慢性持续感染相关肝癌患病与HLA-DRB1*0701、HLA-DRB1*1501-5等位基因型相关联;具有HLA-DRB1*0701及/或HLA-DRB1*1501-5等位基因型的HCC患者血清AFP高表达;肝癌组织HLA-DR抗原的表达量与肝癌患病及分化程度相关。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most important causes of cancer death worldwide. It has great regional differences in the epidemiology and has extremely poor prognosis due to failure in diagnosing it early. Persistent infection of hepatitis B virus (HBV) is the main etiological factor of HCC. China is one of the most epidemic areas of HBV infection in the world. Up to today many pathopoiesis component elements of HCC have not been illuminated because of complicated nosogenesis of HCC. Reproduction of HBV and immune clearance response of organism to HBV are the two basic pathopoiesis factors of HBV infection-related diseases. Especially, different immune functional status of organism result in different clinical manifestations and different prognosises after HBV infection. T cell-mediated immune response acts most importantly in virus clerance and nosogenesis. Human leucocyte antigens (HLA) possibly participate genetic control of immune response. More and more studys have showed that HLA-Ⅱallels especially HLA-DR, DQ allels polymorphism are associated with chronic hepatitis B and posthepatitis B hepatic cirrhosis. Moreover, the population characteristics of association of HLA-Ⅱallels polymorphism with HBV persistent infection are differrent because of various races and various areas in the world. Consequently HLA-Ⅱallels polymorphism would be associated with HBV infection-related hepatocellular carcinoma. It is key for clinical therapy prospect of HCC how to build some new methods including early diagonosis, prognosis evaluation and effective therapy in gene level.Objective: To investigate the association of human leucocyte antigen-DRB1, DQB1 alleles and expression of HLA-DR, DQ antigen with HBV infection-related hepatocellular carcinoma in Han population of Jilin area. Some new methods would be built about early diagonosis, prognosis evaluation of HCC and some evidences would be provided for effective therapy in the future.Methods: 1. Alleles of HLA-DRB1, DQB1 in 61 patients with chronic hepatitis B, 44 patients with posthepatitis B hepatic cirrhosis, and 50 healthy controls were detected by using polymerase chain reaction/sequence specific primer (PCR/SSP) technique. The results were analyzed by gel imaging analytical system after agarose gel electrophoresis to explore the association of HLA-DRB1, DQB1 alleles with chronic hepatitis B, posthepatitis B hepatic cirrhosis. 2. Alleles of HLA-DRB1, DQB1 in 62 patients with HCC were detected by PCR/SSP technique and were compared among subjects of four groups in order to illuminate the association of HLA-DRB1 and DQB1 alleles with HBV infection-related hepatocellular carcinoma in gene level. 3. Serum AFP levels were detected by enzyme immunoassay technique and compared in HCC patients with associated allels with in those without associated allels in order to explore the relationship of HLA-Ⅱallels with serum AFP levles of HCC. 4. The expressions of HLA-DR and DQ antigens in 38 of 62 liver carcinoma tissues, tissues adjacent to HCC and 10 nomal liver tissues obtained through exairesis were detected by using immunohistochemical technique in order to explore the expression situation and its significance of HLA-DR, DQ antigen in HBV infection-related HCC. All the HCC patients were followed up postoperatively, and the relationship were analyzed among pathological typing, clinical staging (TNM staging), the patient survival parameters and HLA-Ⅱantigen expression.Results:1. The frequency of HLA-DRB1*1201-3 allele in chronic hepatitis B group was higher than that in healthy control group(17.21 % vs. 8.00 %,P=0.0427, RR=2.391). The frequency of HLA-DRB1*0701 allele in chronic hepatitis B group, hepatic cirrhosis group was markedly higher than that in healthy control group(11.48 % vs. 2.00 %, P=0.0066, RR=6.35; 12.50 % vs. 2.00 %, P=0.0046, RR=7.00 respectively). This study indicated that alleles of HLA-DRB1*1201-3 and HLA-DRB1*0701 were close associated with chronic hepatitis B. HLA-DRB1*0701 was also close associated with hepatic cirrhosis. Alleles of HLA-DRB1* 0701 and HLA-DRB1*1201-3 were probably the predisposing genes and/or linked genes of chronic hepatitis B. Allele of HLA-DRB1* 0701 was probably the predisposing genes and/or linked genes of hepetic cirrhosis. 2. The frequency of HLA-DRB1*0701 allele in HCC group was markedly higher than that in healthy control group (11.29 % vs. 2.00 %, P=0.0073,RR=6.236). The frequency of HLA-DRB1*1501-5 allele in HCC group was higher than that in healthy control group (18.55 % vs. 9.00 %, P=0.0423, RR=2.303), in chronic hepatitis B group (11.29 % vs. 8.20 %, P=0.0172,RR=2.550)and in hepatic cirrhosis group (11.29 % vs. 7.95 %, P=0.0292,RR=2.635). Alleles of HLA-DRB1*1501-5, HLA-DRB1*0701 were probably the predisposing genes and/or linked genes of HBV infection-related hepatocellular carcinoma. 3. The serum level of AFP was higher in HCC group with HLA-DRB1*1501-5 and/or HLA-DRB1*0701 allels than that in HCC group without HLA-DRB1*1501-5 and/or HLA-DRB1*0701 allels(1000±1537 vs. 268.55±933.10), and the statistical difference was significant(Z= 2.57,P= 0.010). 4. The expression of DR antigen was negative in hepatocytes of nomal liver tissues except on antrum endotheliocytes and Kupffer cells. Several chronic hepatitis tissues adjacent to HCC expressed DR antigen on antrum endotheliocytes, Kupffer cells and inflammatory reaction areas. Sinus hepaticus and fibra spacing expressed DR antigen strongly in several hepatic cirrhosis tissues adjacent to HCC. The expressions of DR antigen were negative on cytomembranes and/or in cytoplasms of cirrhosis haptic cells.There was no statistical difference between nomal liver tissues and tissues adjacent to HCC about HLA-DR expression( P=1.000), while 39.5 % of HCC tissues expressed DR antigen which distributed on the cytomembranes and/or in cytoplasms intensively besides on antrum endotheliocytes, Kupffer cells. There was statistical difference between HCC tissues and the other two kinds of hepatic tissues in expression of HLA-DR antigen( P<0.05). This result indicated that the expression quantity of HLA-DR antigen was associated with occurrence of HCC. The expression of DQ antigen was negative in hepatocytes of nomal liver tissues, while 8 cases of tissuses adjacent to HCC and 6 cases of HCC tissues expressed DQ antigen on the cytomembranes and/or in cytoplasms. There were no statistic differences among tissues of nomal liver, adjacent to HCC and HCC in expression of HLA-DQ antigen (P>0.05). There was no statistical difference in TNM staging between HCC patients with HLA-DR antigen expression and HCC patients without HLA-DR antigen expression (P>0.05). The rate of HLA-DR antigen expression in well-differenciated cases was higher than that in poorly-differenciated ones (P<0.05), which showed that overexpression of HLA-DR antigen in HCC tissues was associated with the occurrence and development of HCC. There was no statistical difference in the survival between HCC patients with HLA-DR antigen expression and HCC patients without HLA-DR antigen expression (χ~2= 0.3042, P=0.5813). There were statistical differences in the survival between HCC patients well-differentiated and HCC patients poorly–differenciated (χ~2=16.2542, P<0.0001); as well as between HCC patients withⅠ,Ⅱstage of TNM staging and patients withⅢ,Ⅳstage of TNM staging(χ~2=12.2002,P=0.0005).Conclusions:1. Alleles of HLA-DRB1*1201-3 and HLA-DRB1*0701 were close associated with CHB, allele of HLA-DRB1*0701 was also close associated with posthepatitis B HC. HLA-DRB1 allel polymorphism was close associated with chronicity of HBV infection in Han population of Jilin area. 2. Alleles of HLA-DRB1*1501-5, HLA-DRB1*0701 were close associated with HBV infection-related HCC in Han population of Jilin area. 3. The serum expression level of AFP was high in HCC patients with HLA-DRB1*1501-5 and/or HLA-DRB1*0701 allels. 4. The expression quantity of HLA-DR antigen was associated with the occurrence and differentiated degree of HBV infection-related HCC in Han population of Jilin area. The expression of DR antigen was not associated with TNM staging and the survival of HCC patients.The experience and results of this study deserved to be expolored and researched further which provided laboratory evidences for immunological genetics mechanism of HCC occurrence in gene level. The innovative points of this study were as follow: It was illuminated for the first time in Han population of Jinlin area that①. HLA-DRB1 allel polymorphism was associated with chronic hepatitis B, posthepatitis B hepatic cirrhosis and HBV infection-related hepatocellular carcinoma.②. The serum level of AFP expression was high in HCC patients with associated HLA-DRB1 allels.○3 The expression quantity of HLA-DR antigen was associated with the occurrence and differentiated degree of HCC.This study has illuminated the relationship between HLA-DRB1, DQB1 allels polymorphism and HCC in Han population of Jinlin area for the first time. It will be helpful for high risk population of HCC to be early screened and diagnosed by detecting HLA-DRB1 allel combined with serum AFP. Detection of HLA-DR antigen expression in HCC tissues combined with pathological typing, clinical staging will help to evaluate prognosis of HCC patients. This study will also provide theory evidences for immune gene therapy of HBV infection-related HCC in the future.更多还原