慢性乙型肝炎组织病理学指标及病毒X基因变异与患者预后的相关性研究

The relationship between HBV-associated histopathological indexes, X gene mutations and prognosis in patients with chronic hepatitis B virus infection

目的探讨乙型肝炎病毒(HBV)相关组织病理学指标及病毒X基因变异与慢性乙型肝炎患者预后转归的关系。方法对1986—1994年间慢性乙型肝炎肝穿刺活检病例进行预后随访,83例样本按患者有无肝细胞癌和肝硬化的发生分为3组:癌变组、硬化组和非硬化组。另取20例HBV相关性肝细胞癌作为对照研究。肝损伤活动程度用Scheuer评分表示;免疫组化二步法显示肝组织内HBsAg、HBcAg、HBeAg和HBx蛋白的表达;荧光实时定量PCR检测肝组织HBV DNA含量;PCR方法扩增HBV X基因nt1583～1793区段,目的条带经胶回收后直接双向测序。结果HBV 4种病毒蛋白在肝组织内均有不同程度的表达,以HBsAg最强,HBeAg最弱;核苷酸错义突变发生于nt1632～1636 (AA 87/88)、1719(AA 116)、1725～1730(AA 118/119)、1752(AA 127)、1762和1764(AA 130/131)位点。统计学分析结果显示,Scheuer评分的G分级(P=0.001)和S分期(P=0.000)硬化组显著高于非硬化组;肝组织内HBeAg的表达硬化组也比非硬化组高(P=0.008)。nt1762/1764双突变在癌变组所占比例明显高于非癌变组(P=0.011);nt1725～1730野生型(P=0.024)和nt1762/1764突变型(P=0.001)在肝癌组中所占比率均明显高于慢性肝炎组。结论Scheuer评分和肝组织内HBeAg的表达与慢性乙型肝炎患者肝硬化的发生有关;nt1762/1764突变能显著增加慢性乙型肝炎患者肝细胞癌的发生率。

Objective To identify the relationship between HBV-associated histopathologieal indexes, X gene mutations and prognosis in patients with chronic hepatitis B. Methods Patients with liver biopsy samples in 1986-1994 were followed up. All of the 83 cases were divided into three groups： Carcinogenesis, Cirrhosis, and Non-cirrhosis. The rest 20 HBV-related hepatocellular carcinoma cases were also studied. Two-step immtmohistochemical staining showed the expression of HBsAg, HBeAg, HBeAg and HBx protein in situ. HBV DNA levels in liver were determined by fluorescence quantitative real-time PCR. nt1583-1793 in HBV X gene was amplified by PCR. Products were purified from agarose for sequence analysis in double directions. Results Among the four HBV antigens, the expression of HBsAg was the highest, while HBeAg was the lowest. Missense mutations were found in nt1632-1636（AA 87/88）, 1719（AA 116）, 1725-1730（AA 118/119）, 1752（AA127）, 1762 and 1764 （AA 130/131）. Statistic analysis showed that grading （ P = 0.001） and staging （ P = 0.000） of Seheuer scores were higher in Cirrhosis than those in Non-cirrhesis, as well as the expression of HBeAg in liver （ P = 0. 008）. The percentage of nt1762/1764 mutation in Carcinogenesis was significantly higher than that in Non-careinogenesis （P = 0.011）. The percentage of wild type of nt1725-1730 was obviously higher in carcinoma cases than that in chronic hepatitis cases （ P = 0.024）, while the percentage of nt1762/1764 mutations did so （ P = 0. 001 ）. Conclusion Scheuer scores and the expression of HBeAg in liver closely correlate with cirrhosis in patients with chronic hepatitis B. The mutation at nt1762/1764 obviously increases the risk for hepatocareinogenesis.