【作者】 蒋业贵；

【导师】 王宇明；

【作者基本信息】 第三军医大学 ， 内科学， 2002， 博士

【摘要】 为了探讨HLA-ⅡI类和Ⅲ类基因与乙型病毒性肝炎之间的相关性，我们作了如下研究： 1．采用免疫组化法和Western-blot法对36例慢性乙型肝炎和20例正常肝组织中HLA-DR和HLA-DQ的表达进行了检测，采用免疫组化双染色技术对HLA-DR／HBcAg和HLA-DQ／HBcAg的表达进行了检测，分析HLA-DR、HLA-DQ在慢性乙型肝炎肝组织中的表达及其意义。 2．采用免疫组化法对36例慢性乙型肝炎和20例正常肝组织中HSP27、HSPT0和HSP90α的表达进行了检测，采用免疫组化双染色技术对HSP70／HBcAg和HSP90α／HBcAg的表达进行了检测，采用原位杂交法对HSP70 mRNA的表达进行了检测，分析HSP27、HSP70和HSP90α在慢性乙型肝炎肝组织中的表达及其意义。 3．应用PCR／SSP法对106例正常健康人、52例慢性乙型肝炎、30例急性乙型肝炎和32例慢性重型乙型肝炎患者HLA-DRB1、HLA-DQA1、HLA-DQB1等位基园多态性进行了检测，分析HLA-DRB1、HLA-DQA1和HLA-DQB1等位基因多态性与乙型病毒性肝炎之间的相关性。 4．采用MTT法检测52例慢性乙型肝炎和30例急性乙型肝炎患者的外周血淋巴细胞对重组乙型肝炎核心抗原(rHBcAg)的增殖反应，分析淋巴细胞增殖指数与HLA-DRB1、HLA-DQA1、HLA-DQB1等位基因多态性的关系。 5．应用EBV体外感染52例慢性乙型肝炎、30例急性乙型肝炎和32例慢性重型乙型肝炎患者外周血淋巴细胞建立永生化的淋巴母细胞系(LCL)；应用结晶紫染色法检测LCL分泌TNF能力，分析TNF活性水平 与乙型病毒性肝炎临床表型和HLA－DRB、HLA－DQA、HLA－DQB等位 基因多态性的关系。 主要结果和结论如下： 1．正常肝组织中肝细胞未见 HLA－DR、HLA－DQ表达。慢性乙型肝炎 肝细胞 HLA－DR、HLA－Do表达阳性率分别为 22．22O、19．44O。其中，中 度和重度肝炎HLA－DR、HLA－DQ阳性率O7．50、37．50）明显高于轻度肝 炎（10％、5％），两者相比差异显著（ X；‘＝＝3．89，X＊＊曰5．99，P＜0．05）口 HLA＊R 和 HLAoQ过度表达，可能与病毒活动和肝细胞坏死有关，在慢性乙型肝 炎发病机制中起重要作用。HLA－DR（HLA－DQ）和 HBCAg同时阳性的肝细 胞变性、坏死明显，在慢性乙型肝炎的免疫损伤机制中 HLA－DR（HLA－DQ） 和HBCAg可能具有协同作用。 2．慢性乙型肝炎和正常肝组织中肝细胞HSP27表达阳性率分别为 25O和 20O，两者相比差异无显著性（X乙0．18，P＞0．05）。慢性乙型肝炎中 HSP70和 HSP90 a阳性率（44．44％、38．89％）明显高于正常肝组织（15％、 15％X 两者相比差异显著（X；’－4．97，X。’叫．97，P叼刀5卜中度和重度肝炎 HSP70、HSP90 a阳性率（68．75％、62．5％）明显高于轻度肝炎（25％、20％）， 两者相比差异显著（X广＝6．89，X／－6．76，P＜0＊5）。HSP70、HSP90 Q表达阳 性的肝细胞大多数无HBCAg表达。HBV在肝细胞内感染可诱导HSP70和 HSP90 a表达增加，而对 HSP27的诱导作用则不明显，HSP70和 HSP90 a 可作为慢性乙型肝炎时肝细胞损害的一种标志。HSP70、HSP90 a可能干 扰了HBV蛋白的产生或装配。 3．共检出HLA－DRB等位基因14个、HLA－DoAl等位基因10个、 HLA－DQ田等位基因 13个，正常健康人中检出率较高的 HLA0RB等位 基因有HLA－DRBI＊1201／1202、”1501／1502、“0901和”0401／04if，等位基 因频率分别为 16刀4％、16刀4％、15．09％和 11．32％。HLA－DQAI等位基因 有 HLA－DQAI“0301、“0102、”0501和“0601，等位基因频率分别为 26．89％， ZI．23％，门＊8％和 10．85％。HLA－DQBI等位基因有 HLA－DQBI＊0301、 “0303、”0201、“0502和”0601，等位基因频率分别为18．87％、16．sl％、 ·IX· ＼10．85o、9．430和9．430。符合我国南方汉族人群的遗传学特点。 4．慢性乙型肝炎组的HLA－DRB”0301、HLA－DQAI‘0501 和HLA－DQBI’0301等位基因频率（17．310，25．960，35．580）明显高于正常对照组（5．67％，13石8％，18．87％），两者相比差异显著（X；’吕12．3068，PCI＝0．0074，MI＝＝4．15．X。’＝9．2002，PCZ＝0＊157，肌＝2．87．X。’＝＝15．5938，PC3＝0＊075，RR3＝4刀刀。慢性乙型肝炎组的HLA－DRB门 八 02和HLA－DQAI“0301等位基因频率（．96o，14．42o）明显低于急性乙型肝炎组（13．330，30o），两者相比差异显著（X广s11．9206，PCI＝0．0145，RR1＝18．55．X。‘＝7＊781，PC。＝0．0388，RR＝3．70）。慢性重型乙型肝炎组的HLA－DRBI“1001的等位基因频率＊刀9％更多还原

【Abstract】 To investigate the association between HLA class II, III genes and viral hepatitis B, We performed the following investigations:1. HLA-DR and HLA-DQ were determined in specimens of 36 cases of chronic hepatitis B and 20 cases of normal liver tissues by immunohistochemistry and Western-blot, and HLA-DR/HBcAg and HLA-DQ/HBcAg were determined by immunohistochemical double-labelling staining to analyse the expression and significance of HLA-DR and HLA-DQ in chronic hepatitis B.2. HSP27, HSP70 and HSP90 a were determined in specimens of 36 cases of chronic hepatitis B and 20 cases of normal liver tissues by immunohistochemistry. HSP70/HBcAg and HSP90 a /HBcAg were determined by immunohistochemical double-labelling staining and HSP70 mRNA was determined by in situ hybridization technique to analyse the expression and significance of HSP27, HSP70 and HSP90 a in chronic hepatitis B.3. HLA-DRB1, HLA -DQA1 and HLA-DQB1 alleles in 54 patients with chronic hepatitis B, 30 patients with acute hepatitis B, 32 patients with chronic severe hepatitis B and 106 normal control subjects were determined by the polymerase chain reaction/sequence specific primer (PCR/SSP) to analyse the association between the polymorphism of HLA-DRB1, HLA-DQA1, HLA-DQB1 alleles and viral hepatitis B.4. Perpheral blood lymphocyte response to recombinant hepatitis B core antigen (rHBcAg) was determined by MTT in 54 patients with chronic hepatitis B and 30 patients with acute hepatitis B to analyse the associationinbetween the lymphocyte proliferation index and the polymorphism of HLA-DRB1, HLA-DQA1 and HLA-DQB1 alleles.5. Immortalized lymphoblastoid cell lines (LCL) were created by EBV infecting peripheral blood lymphocytes in 54 patients with chronic hepatitis B, 30 patients with acute hepatitis B and 32 patients with chronic severe hepatitis B in vitro, and TNF production in LCL was determined by crystal violet staining to analyse the association between the TNF producton in LCL and clinical phenotype of viral hepatitis B and the polymorphism of HLA-DRB1, HLA-DQA1 and HLA-DQB1 alleles .The main results and conclusions were as follows:1. HLA-DR and HLA-DQ were not detected in hepatocytes of normal liver tissues. The positive rates of HLA-DR and HLA-DQ were 22.22% and 19.44% in chronic hepatitis B, respectively. The positive rates of HLA-DR and HLA-DQ in moderate and severe hepatitis ( 37.5%, 37.5% ) were higher than in mild hepatitis ( 10%, 5% ), with significant correlation between them (x 12=3.89, x 22=5.99, PO.05). These findings suggest that overexpression of HLA-DR and HLA-DQ are closely associated with HBV activity and hepatocyte necrosis, and play an important role in pathogenesis of chronic hepatitis B. HLA-DR (HLA-DQ) and HBcAg simultaneously positive hepatocytes obviously denaturalized and necrosed. These findings suggest that both HLA-DR (HLA-DQ) and HBcAg may play a cooperative role in immune-induced damage of chronic hepatitis B.2. The positive rates of HSP27 were 25% and 20% in chronic hepatitis B and normal liver tissues, respectively. There was no significant correlation between them (x 2=0.18, P>0.05). The positive rates of HSP70 and HSP90 a in chronic hepatitis (44.44%, 38.89%) were higher than in normal liver tissues (15%, 15%), with significant correlation between them ( x ,2=4.97, x 22=4.97, P<0.05). The positive rates of HSP70 and HSP90 a in moderate and severehepatitis (68.75%, 62.5%) were higher than in mild hepatitis (25%, 20%), with significant correlation between them ( x 12=6.89, x 22=6.76, P<0.05). HSP70 and HSP90 a mainly expressed in HBcAg negative hepatocytes. These findings suggest that increation of HSP70 and HSP90 a expression may be induced by HBV infection in hepatocytes, but HSP27 not. HSP70 and HSP90 a may be a marker of hepatocyte damage in chronic hepatitis B, and may interfere with production or assemblation of HBV protein.3. Fourteen HLA-DRB1 alleles, ten HLA-DQA1 alleles and thirteen HLA-DQB1 alleles更多还原