用双探针原位杂交法检测常见肝病组织中输液传染性病毒DNA

Assay of transfusion transmitted virus DNA in the tissue of the frequent liver diseases with in situ hybridization of double-chain probe

目的 :建立肝组织输液传染性病毒DNA(TTVDNA)的原位杂交检测技术 ,探讨TTVDNA在肝组织中的分布。方法 :采用PCR扩增法 ,分别合成地高辛标记的G1a、G2b两种亚型的双链TTVDNA探针。同时应用两型探针对 2 2例血清学检查无甲 -庚型肝炎病毒感染的肝病患者肝组织TTVDNA进行原位杂交检测 ,并将检测结果与巢式PCR法检测配对血清TTVDNA的结果进行比较。结果 :原位杂交阳性信号主要定位于肝细胞核 ,少数位于胞浆。 2 2例肝病患者肝组织标本中TTVDNA原位杂交阳性率 6 8 2 % (15 / 2 2 ) ,其中慢性肝炎、肝硬化、肝细胞癌患者中原位杂交阳性率83 3% (15 / 18) ,4例肝破裂、肝良性肿瘤患者TTVDNA原位杂交检测皆为阴性。配对血清TTVDNA阳性率 6 3 6 % (14/ 2 2 )。血清与肝组织TTVDNA检测符合率 86 4 % (19/ 2 2 )。结论 :双探针原位杂交检测具有较高的特异性和灵敏性 ,有助于肝病病因的分析。TTVDNA在慢性肝炎、肝硬化、肝细胞癌的肝组织中检出率较高 。

Aim: To establish a technique of in situ hybridization (ISH) for transfusion transmitted virus DNA (TTVDNA) in liver tissue. Methods: Two subtypes of double-chain probe of TTVDNA genotype 1a and 2b, labelled with digoxigenin was, respectively, synthesized by means of polymerase chain reaction (PCR). Hepatic tissues of 22 patients with liver disease, whose serum without the infection of A-G hepatitis virus, were tested for TTVDNA with two subtypes of probe at the same time. The results of ISH assay were compared with those of nested-PCR assay for TTVDNA in paired serum. Results: TTVDNA was found mainly in the nucleus of hepatocytes and hepatic carcinoma cells ,and rarely in the cytoplasm of them. The positive rate of TTVDNA was 68.2% (15/22) in the liver tissue of 22 patients, 83.3% in those patients with chronic hepatitis, hepatocirrhosis or hepatocellular carcinoma . The results were all negative in liver tissue in 4 patients with hepatorrhexis or benign tumor. The positive rate of TTVDNA was 63.6% (14/22) in paired serum of 22 patients . The results of ISH in liver tissue tally with those of PCR in paired serum in 86.4% (19/22) patients. Conclusion: The assay of ISH using two subtypes of probe of TTVDNA at the same time holds high sensitivity and specificity, and will be helpful to the study of etiology of hepatic diseases. The positive rate of TTVDNA in liver tissue of chronic hepatitis, hepatocirrhosis and hepatocellular carcinoma is higher than that of others, which suggests that TTV may be related to these hepatic diseases.