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AIM:To establish a method detecting porcineendogenous retrovirus(PERV)in China experimentalminipigs and to evaluate the safety of PERV in threeindividuals treated with bioartificial liver support systemsbased on porcine hepatocytes.METHODS:Porcine hepatocytes were isolated withtwo-stage perfusion method,then cultured in thebioreactor,which is separated by a semipermeablemembrane(0.2μm)from the lumen through whichthe patients’blood plasma was circulated.After post-hemoperfusion,patients’blood was obtained forscreening.Additionally,samples of medium collectedfrom both intraluminal and extraluminal compartmentsof the laboratory bioreactor and culture supernate invitro was analyzed.The presence of viral sequenceswas estimated by polymerase chain reaction(PCR)andreverse transcriptase-polymerase chain reaction(RT-PCR).Finally,the infection of virus in the supernate ofcommon culture was ascertained by exposure to thefetal liver cells.RESULTS:PERV-specific gag sequences werefound in the porcine hepatocytes using RT-PCR.andwere 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 fetalliver cells.Finally,RT-PCR detected no PERV infectionwas found in the blood samples obtained from threepatients at various times post-hemoperfusion.CONCLUSION:The assays used are specific and sensitive,identified by second PCR.PERVs could bereleased from hepatocytes cultured in bioreactor withoutthe stimulation of mitogen and could not be preventedby the hollow fiber semipermeable membrane,indicatingthe existence of PERV safety in extracorporeal bioartificialliver support system(EBLSS).
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 withtwo-stage perfusion method, then cultured in the bioreactor , which is separated by a semipermeable metambrane (0.2 μm) from the lumen through which the patients ’blood plasma was circulated. After the post-hemoperfusion, patients’ blood was obtained forscreening. Additionally, samples of medium collected from both intraluminal and extraluminal compartments of the laboratory bioreactor and culture supernate invitro was analyzed. The presence of viral sequences was estimated by polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) .Finally, the infection of virus in the supernate ofcommon culture was ascertained by exposure to thefetal liver cells .RESULTS: PERV-specific gag sequences werefound in the porci ne hepatocytes using RT-PCR. and were detected in all samples from the intraluminal, extraluminal samples and culture supernate. Despite, culture supernatant from primary porcine hepatocytes (cleared of cellular debris) failed to infect human fetalliver cells. Infin, RT-PCR detected no PERV infectionwas found in the blood samples obtained from threepatients at various times post-hemoperfusion. CONCLUSION: The assays used are specific and sensitive, identified by second PCR. PERVs could be bereleased from hepatocytes cultured in bioreactor without the stimulation of mitogen and could not be preventedby the hollow fiber semipermeable membrane, indicatingthe existence of PERV safety in extracorporeal bioartificialliver support system (EBLSS).