用不同标记的乙型肝炎病毒DNA探针对肝组织内病毒基因进行原位核酸分子杂交

APPLICATION OF DIFFERENTLY LABELED HEPATITIS B VIRUS PROBES TO IN SITU HYBRIDIZATION ON LIVER TISSUE

本文报道采用国产[α-(55)~S]dATP及进口[3~H]dNTP与生物素标记HBV DNA全基因探针进行原位核酸分子杂交,均可获得阳性结果。其中[3~H]dNTP标记探针的敏感性与特异性最佳。对6例慢性乙型肝炎患者肝组织切片进行原位核酸分子杂交,结果与肝组织提取物经Southern吸印转移HBV DNA杂交的阳性率完全一致。原位核酸杂交显影后的银颗粒呈灶性分布于肝细胞内,提示病毒在肝内扩散为细胞——细胞间传播方式进行。对1例有复制型HBV DNA的肝癌组织进行原位核酸分子杂交,发现HBV DNA主要分布在光镜下癌细胞旁肝细胞胞浆中,而癌细胞中未测得HBV DNA,其意义有待更多病例数检测后才能阐明。

Three different chemicals,namely.[~3H]dNTP,[aS]dATP and biotinylated dUTP were used to label cloned HBV DNA as probes for in situ hybridization on liver tissue sections.It was found that[~3H]dNTP labeled probe was the best in sensitivity and specificity.In 6 chronic hepatitis B cascs,the positive rate of HBV DNA hybridization by means of DNA extraction,gel electrophoresis and Southern blotting was identical with that of in situ hybridization.The silver granules that developed after autoradiography in liver sections revealed focal distribution,which suggested that the virus was spread by intercclIular transmission.By means of in situ Hybridization on liver tissue sections of a case with liver cancer,it was found that the integration as well as replication of HBV DNA was present mainly in the plasma of morphologically normal hcpatocytes nearby the cancer.Howcvcr,HBV DNA was not detected in those hcpatocytes with malignant changes.