Detection Of PERV And Its Security In Bioartificial Liver Based On Porcine Hepatocytes

Fulminant hepatic failure (FHF) remains a disease with high mortality. Bioartificial liver, which can support the regulatory functions of the liver and bridge to the transplantation, has been a new therapy for the treatment of FHF. Porcine hepatocytes, including transgenic pig, have become an important cell source due to the shortage of human cell lines. Recently, concerns have been raised about the safety of this potential therapy, especially the possibility of cross species transmission of porcine endogenous retroviruses (PERV) since vitro infection of human cell lines has been demonstrated.PERVs, described by Breese et al in 1970 for the first time, are integrated into the genome of all pigs and belong to the gammaretroviruses type C retroviruses. Expression of PERVs has been shown in several pig tissues at the level of mRNA production. The conformations of the virue particle are main core protein of 27kD, gag prosome protein of 60kD and 41kD and small gag protein about 9kD and 7kD. The genome structure includes 5', gag, pol, env, 3' and could be classified into three various types, PERV-A, PERV-B and PERV-C according to the discrepancy of env gene. At least two subtypes of PERV, PERV-A and PERV-B, infect human cells in vitro. After vitro infection of human cell lines has been demonstrated by Patience, more infection experiments show that PERVs infect not only human cell lines and primary cells, but also a variety of cell lines, such as, T Lymphocyte line C8166, WIL2.NS.6TG, THP-1 and PBMC. Infective virus separated from the infected human cells has a wide range of infection and viral particles have been shown to be released by mitogen stimulated pig peripheral blood mononuclear cells, by cultured aorta endothelial cells, the kidney cell line PK-15 and pancreatic islet cells, et al. Recently, animal model of PERV infection has been established, which expand and deepen the researches in this field. Some observations also suggest that xenotransplantation products may be associated with some risk of PERV infection, but it is not clear whether the titer of expressed virus will be high enough to cause infection in recipients. Though clinical trialsdid not show virus infection had taken place, researches in vitro made it necessary in the further study of virus security in EBLSS with porcine hepatocytes. In this study, on the base of porcine hepatocytes isolated and cultured with two-stage perfusion method, PCR and RT-PCR assays were used to detect proviral DNA sequences and PERV RNA sequences. PERV detection of cultured hepatocytes in different cultivated patterns, used commonly in bioartificial, was observated to reveal the rule of virus releasing. Retrospectional detection of 2 patients treated with EBLSS used on porcine hepatocytes was carried out and the infection of PERV was valued. The main research results are as follows:1. All these PCR assays gave negative results on tissue and serum samples from 2 HBV patients, as well as 2 HCV patients and experimental animals, while positive results on porcine hepatocytes and serum samples. We also found that the Mg++ concerntration of PCR reaction was of importance in showing the picture definition. The results proved to be sensitive and specific.2. Observation on the three different models of cultivation showed the persistent function of the cultured cells, which were used commonly in the bioartificial liver support system. PERV could be detected in all the culture supernate in different time point without the stimulation of mitogen by the established method and could last till cell death. The existence of PERV in extralumen and intralumen of the bioreactor demonstrated the semipermeable membrane couldn't prevent the virus passing through.3. Sera from 2 patients treated with bioartificial liver support system based on porcine hepatocytes were tested by the method. The infection of virus in the cell -free supernatants from cultured porcine hepatocytes was ascertained by exposure to the fetal liver cells in order to mimic the bioartificial liver sup...