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目的建立外周血淋巴细胞HLA-A mRNA的检测方法,探讨其在慢性乙型肝炎、肝炎后肝硬化、肝癌患者中的应用价值。方法改良法提取人外周血淋巴细胞RNA,应用逆转录-聚合酶链反应(RT-PCR)一步法扩增HLA-A mRNA,琼脂糖凝胶电泳结果采用Bandscan软件分析相对灰度值,半定量HLA-A的基因表达。结果半定量HLA-A所得平均相对灰度值在慢性乙型肝炎组、肝硬化组、肝癌组、正常对照组分别为0．54±0．21、0．34±0．27、0．22±0．16、0．95±0．19。慢性乙型肝炎组、肝硬化组、肝癌组均明显低于正常对照组(P<0．05),慢性乙型肝炎组与肝癌组,肝硬化组与肝癌组相比差异有统计学意义(P<0．05),慢性乙型肝炎组与肝硬化组有下降趋势。结论成功建立检测HLA-A基因的RT-PCR一步法,从基因转录水平上证实慢性乙型肝炎、肝炎后肝硬化、肝癌患者外周血淋巴细胞HLA-A下调,且与相应患者细胞免疫功能减弱密切相关。
Objective To establish a method for the detection of HLA-A mRNA in peripheral blood lymphocytes and to explore its value in chronic hepatitis B, post-hepatitis cirrhosis and liver cancer. Method Amelioration method was used to extract human peripheral blood lymphocyte RNA. HLA-A mRNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) in one step. The results of agarose gel electrophoresis were analyzed by Bandscan software. HLA-A gene expression. Results The average relative gray value of semi-quantitative HLA-A was 0.54 ± 0.21, 0.34 ± 0.27 and 0.22 respectively in chronic hepatitis B group, cirrhosis group, liver cancer group and normal control group ± 0.16, 0.95 ± 0.19. Chronic hepatitis B group, cirrhosis group and hepatocellular carcinoma group were significantly lower than those in normal control group (P <0.05), there was significant difference between chronic hepatitis B group and hepatocellular carcinoma group, cirrhosis group and hepatocellular carcinoma group (P < P <0.05), chronic hepatitis B group and cirrhosis group have a downward trend. Conclusion One-step reverse transcription-polymerase chain reaction (RT-PCR) for detection of HLA-A gene was successfully established. HLA-A levels in peripheral blood lymphocytes of patients with chronic hepatitis B, posthepatitis cirrhosis and hepatocellular carcinoma were confirmed by gene transcription. closely related.