Detection of PERV by polymerase chain reaction and its safety in bioartificial liver support system

AIM： To establish a method detecting porcine endogenous retrovirus （PERV） in China experimental minipigs and to evaluate the safety of PERV in three individuals treated with bioartificial liver support systems based on porcine hepatocytes. METHODS： Porcine hepatocytes were isolated with two-stage perfusion method, then cultured in the bioreactor, which is separated by a semipermeable membrane （0.2μm） from the lumen through which the patients＇ blood plasma was circulated. After posthemoperfusion, patients＇ blood was obtained for screening. Additionally, samples of medium collected from both intraluminal and extraluminal compartments of the laboratory bioreactor and culture supernate in vitro was analyzed. The presence of viral sequences was estimated by polymerase chain reaction （PCR） and reverse transcriptase-polymerase chain reaction （RTPCR）. Finally, the infection of virus in the supernate of common culture was ascertained by exposure to the fetal liver cells. RESULTS： PERV-specific gag sequences were found in the porcine hepatocytes using RT-PCR. and were detected in all samples from the intraluminal, extraluminal samples and culture supernate. However, culture supernatant from primary porcine hepatocytes （cleared of cellular debris） failed to infect human fetal liver cells. Finally, RT-PCR detected no PERV infection was found in the blood samples obtained from three patients at various times post-hemoperfusion. CONCLUSION： The assays used are specific and sensitive, identified by second PCR. PERVs could be released from hepatocytes cultured in bioreactor without the stimulation of mitogen and could not be prevented by the hollow fiber semipermeable membrane, indicating the existence of PERV safety in extracorporeal bioartificial liver support system （EBLSS）.

瞄准：为了建立在中国试验性的微型猪检测猪的内长的制动火箭病毒(PERV ) 的一个方法并且在三评估 PERV 的安全，个人们基于猪的 hepatocytes 与简历 artificial 肝支持系统对待。方法：猪的 hepatocytes 与二阶段的灌注方法被孤立，在生物反应器然后有教养，它被半渗透的膜(0.2 microm ) 与病人的血浆通过被传播的腔分开。在 post-hemoperfusion 以后，病人的血为屏蔽被获得。另外，媒介的样品从实验室生物反应器的管腔内、额外的钠分隔空间收集了，文化上层清液在试管内被分析。病毒的序列的存在被聚合酶链反应(PCR ) 和反向的 transcriptase 聚合酶链反应(RT-PCR ) 估计。最后，在普通文化的上层清液的病毒的感染被暴露查明到胎儿的肝细胞。结果：PERV 特定作呕序列用 RT-PCR 在猪的 hepatocytes 被发现。并且从管腔内的、额外的钠样品和文化上层清液在所有样品被检测。然而，从主要猪的 hepatocytes 的文化上层清液(细胞的碎片变清) 没能感染人的胎儿的肝细胞。最后， RT-PCR 没检测感染在在各种各样的时间 post-hemoperfusion 从三个病人获得的血样品被发现的 PERV。结论：使用的试金特定、敏感，由第二 PCR 识别了。没有 mitogen 的刺激， PERV 能被免除在生物反应器有教养的 hepatocytes 并且不能被空纤维阻止半渗透的膜，显示在身体外的简历 artificial 肝的 PERV 安全的存在支持系统(EBLSS ) 。