Molecular Biological Studies on Pathogenesis of Biliary Atresia Using PCR method

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Between November 1989 and March 1991 we have performed surgical liver biopsy on 14 cases with neonatal hepatitis (NH), 3 with choledochal cyst (CDC) and 8 with no hepatobiliary disorders. Among the 14 patients with BA, specimens from 11 were taken during the radical operation, 5 at the conversion or closure operation of stoma and 2 at the re-radical operation. Liver specimens were investigated in all 14 patients and in 3 of the 14 samples from BA, extrahepatic bile duct or its remnants and lymphnodes of hepatoduodenal ligament were also examined.When the purified RNA of reovirus 3 was used as the template, the PCR yielded a single amplified fragment of the expected size of 385 bp. The identities of the amplified DNA fragment were confirmed by cleavage with ClaI or EcoRV, which yielded the predicted bands of 97 and 288 or 87 and 298 bp.To evaluate the sensitivity of the PCR assay, amplification was performed on 10-fold serial dilutions of purified reovirus 3 RNA ranging from ng to 1 pg.It was possible to visually detect 10 ng of the amplified product in the first 30 cycles after polyacryleamide gel electrophoresis (PAGE). When an additional 30 cycles of the same primers was examined, the detection level of RNA was as little as 100 pg. Accordingly an increased sensitivity was achieved by using the second PCR amplification.No specific band were detected in 11 liver specimens from BA at the time of radical operation. In 3 of these cases, in addition to liver specimens, extrahepatic bile duct or its remnants (BD) and lymphnodes of hepatoduodenal ligament (LN) were examined. These tissue specimens showed also no PCR products. In 7 liver specimens from BA obtained at the conversion or re-radical operation, reovirus 3 was not detected by the second PCR amplification. PCR analysis performed on liver specimens with CDC, NH and those without hepatobiliary disorders did not show any PCR product uner our experimental conditions.