Molecular Biological Studies on Pathogenesis of Biliary Atresia Using PCR method

1989年11月至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例无肝胆疾病的新生儿进行了手术肝活检。14例BA患者中11例在根治术时取材，5例在造口转闭术时取材，2例在再次根治术时取材。以呼肠孤病毒3型的纯化RNA为模板，PCR扩增出385 bp的特异性片段，其中3例BA肝外胆管或其残端及肝十二指肠韧带淋巴结的DNA片段为阳性。扩增的DNA片段的同一性通过用ClaI或EcoRV切割来确认，其产生97和288或87和298 bp的预测条带。扩增在10-纯化的呼肠孤病毒3 RNA的10倍系列稀释，范围从ng到1 pg。聚丙烯酰胺凝胶电泳（PAGE）后循环。用同样的引物重复检测30个循环后，RNA的检测量仅为100 pg，因此，采用第二次PCR扩增提高了灵敏度，在11例BA患者根治术时的肝组织中均未检测到特异性条带。其中3例除肝脏标本外，还检查了肝外胆管或其残余（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。在转基因或再根治术时获得的7份BA肝标本中，第二次PCR扩增未检出呼肠孤病毒3型。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分析未显示任何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.