猪肝细胞和培养上清液中猪内源性逆转录病毒的检测

建立了猪肝细胞及其培养上清液中猪内源性逆转录病毒(PERV)的检测方法，探讨了其在猪肝细胞生物人工肝应用中的意义。以PERV gag基因为靶序列，选用特定的引物，PCR检测中国实验用小型猪肝细胞PERV前病毒DNA；RT-PCR检测猪、犬、大鼠以及HBV阳性病人血清和猪肝细胞培养6h、24h时的上清液PERV RNA，同时检测猪肝细胞猪线粒体DNA(mtDNA)。研究结果表明：检测5份中国实验用小型猪血清、肝细胞及培养猪肝细胞24h时的上清液PERV均为阳性，而5份培养猪肝细胞6h时的上清液、5份犬血清、5份大鼠血清和5份HBV阳性病人血清PERV检测结果均为阴性，猪肝细胞中均可检测到猪mtDNA。因此，中国实验用小型猪肝细胞携带PERV；PERV可释放到血清中；猪肝细胞培养24h后该病毒颗粒已释放到培养液中；PCR和RT-PCR方法检测PERV具有特异性强、简便的特点。

Detection of Porcine Endogenous Retrovirus in Porcine Hepatocytes and the Supernatant of Cultured Porcine Hepatocytes

In this study, we constructed the determination methods of porcine endogenous retrovirus (PERV) in porcine hepatocytes and the supernatant and studied the significance in the application of bioartificial liver based on porcine hepatocytes. Proviral PERV gag sequences in Chinese experimental miniature pig hepatocytes were detected by PCR using specific primers. PERV RNA in the sera from pigs, canines, rats and HBV patients and the supernatant of cultured porcine hepatocytes at 6 h, 24 h was detected by RT-PCR using specific primers. In the same time, porcine mitochondrial DNA (mtDNA) was detected in porcine hepatocytes. The results showed that proviral PERV gag sequences and porcine mtDNA were detectable in porcine hepatocytes from 5 Chinese experimental miniature pigs. PERV RNA was detectable in the porcine serum and the supernatant of cultured porcine hepatocytes at 24 h. Negative results were showed in the supernatant at 6 h and sera from 5 pigs, 5 canines, 5 rats and 5 HBV patients. Therefore, Chinese experimental miniature pig hepatocytes contain PERV and PERV particles can be released into the serum. PERV particles have been released into the supernatant of cultured porcine hepatocytes at 24 h. PCR and RT-PCR methods we used are of high specificity and convenience.