壳聚糖纳米纤维支架对猪肝细胞分泌猪内源性逆转录病毒的影响

Porcine Endogenous Retroviral Expression and Infectivity by Porcine Hepatocytes Cultured on Chitosan Nanofiber Scaffold

探讨壳聚糖纳米纤维支架对肝细胞的PERV分泌量以及其感染性的影响。将新鲜分离的猪肝细胞接种于壳聚糖纳米纤维支架或普通条件下连续培养7 d,分为实验组(Nano组)和对照组(Hep组)。每天收集培养液检测逆转录酶(RT)活性,并通过RT-PCR和实时定量PCR测定PERV RNA水平。通过western blot检测细胞裂解液中PERV gag 30蛋白含量。细胞培养液体外孵育HEK293细胞以测定其感染性。结果表明:实时定量PCR、RT活性以及western blot具有相似的变化趋势。在体外培养10 h和2 d出现PERV分泌高峰然后逐渐下降。除第6 dPERV RNA水平和第5 d蛋白水平实验组明显高于对照组外,其余均无明显差异。体外感染实验无HEK293细胞感染。壳聚糖纳米纤维支架会延长猪肝细胞的PERV分泌时间但对分泌量以及体外感染性并无明显影响,可安全用于生物人工肝。

This study is aimed to investigate the production and infectivity of PERV expressed by porcine hepatocytes cultured with chitosan nanofiber scaffold.Primary porcine hepatocytes were cultured with or without chitosan nanofiber scaffold（defined as Nano group and Hep group） for 7 days.Reverse transcriptase（RT） activity and PERV RNA in daily collection of culture medium was detected with RT activity assay kits,RT-PCR and real time PCR with the PERV specific primers.And PERV protein gag p30 was determined by western blot the lysates of daily retrieved cells.Besides,the HEK 293 cells were incubated with the supernatants to test in vitro infectivity.Results obtained from real time PCR,RT activity assay and western blot showed similar changing trends between the two groups.Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline.No significant difference was found in the two groups except the significantly higher level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group.And in the in vitro infection experiment,no HEK293 cells were infected by the supernatants.Chitosan nanofiber scaffold prolonged PERV secreting time of hepatocytes but no obvious influence on the productive amount and infectivity of the PERV expressed by porcine hepatocytes.It could be applied safely in the bioartificial liver.