Molecular Biological Studies on Pathogenesis of Biliary Atresia Using PCR method

从1989年11月到1991年3月，我们对14例新生儿肝炎（NH）， 3例胆总管囊肿（CDC）和8例无肝胆疾病的患者进行了手术肝活检。14例BA患者中，11例在根治术中采集标本，5例在造口转换或闭合术中采集标本，2例在再根治术中采集标本。所有14例患者均行肝脏标本检查，其中3例患者均行肝外胆管或其残余物及肝十二指肠韧带淋巴结检查。以呼肠孤病毒3的纯化RNA为模板，PCR扩增得到预期大小为385 bp的单个扩增片段。用ClaI或EcoRV对扩增的DNA片段进行剪切，得到的预测条带分别为97和288 bp或87和298 bp。为了评估PCR检测的敏感性，对纯化的呼肠孤病毒3 RNA进行10倍连续稀释，范围从ng到1 pg，在聚丙烯酰胺凝胶电泳（PAGE）后的前30个循环中可以目测到10 ng的扩增产物。当同一引物的另外30个循环被检测时，RNA的检测水平低至100 pg。因此，通过使用第二次PCR扩增实现了灵敏度的提高。11例BA肝标本根治时未检出特异性条带。其中3例除肝脏标本外，还检查了肝外胆管或其残余物（BD）和肝十二指肠韧带（LN）淋巴结。这些组织标本也未显示PCR产物。转化或再根治手术获得的7例BA肝脏标本中，第二次PCR扩增未检出呼肠孤病毒3。在我们的实验条件下，对CDC、NH和无肝胆疾病的肝脏标本进行PCR分析均未发现PCR产物。

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.