Localization And Distribution Of Ttv Dna In Liver Tissue In The Patients With Ttv

Part I In Situ Hybridization in the assay of TTVDNA in liver tissue: Establishment of the technique and assessment of the sensitivity and specificity Objective This study was designed to establish a technique of In Situ Hybridization (1511) for transfusion transmitted virus (TTV) DNA in liver tissue. Methods A pair of primers were designed according to the high conserved region of TTV nucleotides sequences reported by Okamoto et al. Two subtypes of double-chain probe of TTVDNA, genotype la and 2b, labelled with digoxigenin was, respectively, synthesized by means of polymerase chain reaction (PCR). Liver tissues of patients with the disease of liver 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. Partial gene within QRF1 of a TTV isolate (599), random sampling in the positive cases of 1811 assay, was cloned and sequenced and the result was compared with the sequences of TTV isolates (N22, Gla) reported by Okamoto et al. Results In the patients with serum positive TTVDNA, 13 of 14 patients showed positive TTVDNA in liver tissue. TTVDNA in liver tissue was negative in 6 of 8 patients with negative TTVDNA in the 3 serum. The result sequenced for partial gene within ORF1 of 899 showed that the nucleotide homology was 97.79% between 899 and N22, and 98.16% between S99 and Gla. Conclusion The sensitivity of JSH assay may be improved by using two subtypes of probe of TTVDNA at the same time. The specificity of JSH assay has relationship to the length and the specificity of probe, the strictness of hybridization and the stringent condition for post-hybridization washing. JSH assay for TTVDNA in liver tissue is a value technique of molecular pathology for studing TTV infection. Part II The study of localization and distribution of TTYDNA in liver tissue Objective To observe the feature of localization and distribution of TTVDNA in liver tissue and to study the relationship between TTV infection and hepatic injury. Methods ISH was used to detect TTVDNA of liver tissue for 26 patients with chronic hepatitis, hepatocirrhosis or hepatocellular carcinoma, and 18 cases of them were detected for TTVDNA in paired serum with nested-PCR also. The relationship of TTVDNA in liver tissue and TTVDNA in serum was analyzed. The liver tissues of the cases infected TTV only were stained by hematoxylin and eosin (HE). Excluding the influence of those known hepatitis virus, to study the change of pathology of liver tissue after TTV infection. 4 Results TTVDNA was found mainly in the nucleus of hepatocytes and hepatic carcinoma cells , and rarely in the cytoplasm of them. TTVDNA positive cells were scattered in liver tissue of chronic hepatitis, hepatocirrhosis or hepatocellular carcinoma. The difference of density of TTVDNA positive cell between tumorous liver tissues and peritumor liver tissues is not significant. The pathologic features oceuring in liver tissue specimens of patients with chronic hepatitis infected with TTV only were similar to those found in other viral hepatitis. Conclusion It...