CAJ | 학술논문

目的病毒安全性是猪源性生物人工肝(BAL)不可忽略的安全问题.本研究旨在探讨影响BAL中猪内源性逆转录病毒(PERV)透过的因素.方法构建双循环的新型猪源性BAL.两循环间采用膜孔径为10 nm、20 nm、30 nm和35 nm的血浆成分分离柱以及500 nm膜孔径的血浆过滤柱分隔两循环.反应器内填充共培养的猪肝细胞-骨髓间充质肝细胞或原代猪肝细胞并连续循环72 h.每12 h抽取内、外循环内培养液检测PERV RNA、逆转录酶(RT)活性以及其体外感染性.结果血浆过滤柱组中,内外循环内PERV检测结果完全相同,提示500 nm血浆滤过柱对PERV并无阻隔作用.在血浆成分交换柱各组中,各时间点内循环中均能检出RNA和RT活性,但是外循环中并未检出RT活性,而且随着膜孔径减小,RNA的检出时间逐渐延迟,且内循环RNA水平逐渐增加.各培养液均未发现能够体外感染HEK293细胞.结论膜孔径、循环时间以及内循环病毒浓度是影响BAL中PERV透过的主要原因.
목적 병독안전성시저원성생물인공간(BAL)불가홀략적안전문제.본연구지재탐토영향BAL중저내원성역전록병독(PERV)투과적인소.방법 구건쌍순배적신형저원성BAL.량순배간채용막공경위10 nm、20 nm、30 nm화35 nm적혈장성분분리주이급500 nm막공경적혈장과려주분격량순배.반응기내전충공배양적저간세포-골수간충질간세포혹원대저간세포병련속순배72 h.매12 h추취내、외순배내배양액검측PERV RNA、역전록매(RT)활성이급기체외감염성.결과 혈장과려주조중,내외순배내PERV검측결과완전상동,제시500 nm혈장려과주대PERV병무조격작용.재혈장성분교환주각조중,각시간점내순배중균능검출RNA화RT활성,단시외순배중병미검출RT활성,이차수착막공경감소,RNA적검출시간축점연지,차내순배RNA수평축점증가.각배양액균미발현능구체외감염HEK293세포.결론 막공경、순배시간이급내순배병독농도시영향BAL중PERV투과적주요원인.
Objective To identify the factors influencing the transfer of porcine endogenous retroviruses across the membrane of a new bioartificial liver (BAL),which is a necessary caution to consider for BALs carrying porcine hepatocytes.Methods A novel porcine BAL composed of two circuits was constructed using a plasma filter with membrane pore size of 500 nm and a plasma component separator with membrane pore sizes 10 nm,20 nm,30 nm,and 35 nm.Co-cultured cells of porcine hepatocytes and mesenchymal stem cells or single porcine hepatocytes were incubated in the bioreactors.The BAL was continuously worked for 72 hours during which the supernatant was collected from the internal and external circuits every 12 hours.PERV RNA,reverse transcriptase (RT) activity,and in vitro infectivity from the supernatant were detected.Results In the plasma filter group,the PERV RNAlevels were the same in both circuits,suggesting little to no effect of the plasma filter on the PERV RNA's crossing.With plasma component separators,PERV RNA was found in the external circuits at different times without positive RT activity and HEK293 cell infection,but its level was reduced significantly.The larger the membrane pore size was,the earlier and the more RNA was detected.Conclusions The membrane pore size,the treatment time and the viral level in the internal circuit are contributing factors influencing the transfer of PERV RNA across the membrane in a BAL.