原位聚合酶链技术检测肝细胞癌组织内丙型肝炎病毒核酸及其意义

DETECTION OF HEPATITIS C VIRUS RNA IN CELLS WITH HEPATOCELLULAR CARCINOMA AND ITS PACTICAL SIGIFICANCE BY IN SITU POLYMERASE CHAIN REACTION

应用一步反转录。聚合酶链原位扩增法（ＯＲＴ－ＰＣＲＩＳ）检测肝细胞癌（ＨＣＣ）及癌周组织内丙型肝炎病毒核糖核酸（ＨＣＶＲＮＡ），同时以一步反转录－ＰＣＲ（ＯＲＴ－ＰＣＲ）法检测血清及肝组织匀浆内ＨＣＶＲＮＡ。发现癌和癌周组织经原位扩增后ＨＣＶＲＮＡ检出率为７７．８％（２／２７）及８１．５％（２２／２７），而血清及组织匀浆内仅为２９．６％及３７．Ｏ％，两者与癌及癌周组织相比差异均有显著性（Ｐ＜０．０１）。ＯＲＴ－ＰＣＲＩＳ示在癌组织中ＨＣＶＲＮＡ以核型为主（１２／２１），阳性细胞点样分布，细胞内信号强度多为＋（５７．１％）；而癌周组织以核浆型为主（１４／２２），阳性细胞弥漫分布，信号强度多为＋＋＋（５０．０％）。无论在癌还是在癌周组织均未发现ＨＣＶｃＤＮＡ存在。结果提示ＯＲＴ－ＰＣＲＩＳ优于ＲＴ－ＰＣＲ，ＨＣＶ对本地区ＨＣＣ发生、发展起着极为重要的作用。癌周ＨＣＶ复制较癌组织内活跃，癌和癌周细胞核阳性表明ＨＣＶＲＮＡ在核内整合或复制，这种整合或复制对肝细胞基因组ＤＮＡ产生的影响可能与乙型肝炎病毒（ＨＢＶ）相类似。

ersistent infection with hepatitis Cvirus (HCV)is associated with chronic hepatitis and cirrhosis which may eventually develop into hepatocellular carcinoma(HCC).As far,the pathogenesis mechanism of HCC is unclear and nothing is known of the distribution,frequency or type of infected cells in HCC.One-step reverse transcription in situ polymerase chain reaction(ORT-PCRIS)to detect HCV RNA in HCC samples was developed in our laboratory,Liver tissues from 27 patients with HCC were investigated by this technique for freezed or paraffin-embedded sections. Meanwhile,HCV RNA in sera and extracts of specimens with HCC were assayed by RT-PCR.The positive rate of HCV RNA by ORT-PCRIS waS 81.5%(22/27) in the peritumor liver tissues and 77.8%(21/27) in the tumor tissues, significantly higher than 29.6%(8/27)in sera and 37.0%(10/27 ) in the extreats of HCC by RP-PCR (P<0.01).HCV RNA positive signals were found mainly in the nuclei of tumor cell,and mainly both the nuclei and cyto- plasms in peritumor cells and mainly both the nuclei and cytoplasms in peritumor cells(P<0. 05).Positive granules of HCV RNA were much more in peritumor cells than in the tumor cells. Positive cells were scat-tered mainly on the point-type in the cancer tissues and on the diffussion-type in the peritumor tissues. Our findings suggest that ORT-PCRIS for detecting HCV RNA in the cell with HCC remarkbly prior to trad-tional RT-PCR, HCV infection plays a relatively impor-tant role in determination of HCC development and perhaps HCV replication and its genomic RNA integration with hepatocyte DNA are involved in the course of the maglinant transformation of hepatocytes.