Department of Hepatobiliary Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, Jiangsu Province, China.
World J Gastroenterol. 2011 Jun 14;17(22):2774-80. doi: 10.3748/wjg.v17.i22.2774.
To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes.
Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assay kits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells.
The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high 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 cell was infected by the supernatant.
Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.
研究壳聚糖纳米纤维支架对猪原代肝细胞表达的猪内源性逆转录病毒（PERV）产生和感染性的影响。
新鲜分离的猪原代肝细胞分别在有无壳聚糖纳米纤维支架（定义为纳米组和肝组）的情况下培养 7 天。每天收集培养液，用 RT 活性测定试剂盒和逆转录-聚合酶链反应（PCR）以及 PERV 特异性引物的实时 PCR 检测逆转录酶（RT）活性和 PERV RNA。用每日回收细胞的裂解物进行 Western 印迹，以确定 PERV 蛋白 gag p30。此外，通过孵育人胚肾 293（HEK293）细胞来测试上清液的体外感染性。
实时 PCR、RT 活性测定和 Western 印迹均观察到两组之间相似的变化趋势。发现 PERV 表达有两个高峰，分别在 10 小时和第 2 天，随后呈规律下降。两组之间没有发现显著差异，除了纳米组在第 6 天的 PERV RNA 和第 5 天的 PERV 蛋白水平显著高于肝组。在体外感染实验中，上清液未感染 HEK293 细胞。
壳聚糖纳米纤维支架可能延长猪肝细胞中 PERV 的分泌时间，但不会明显影响其产生量和感染性，因此可应用于生物人工肝，而不会增加病毒传播的风险。
