聚乙二醇与二甲基亚砜促进HCV体外感染树鼩肝细胞的作用

A promotion of PEG and DMSO in HCV infection of adult normal Tupaia hepatocytes in primary culture in vitro

目的观察聚乙二醇(PEG)与二甲基亚砜(DMSO)在HCV体外感染树鼩肝细胞中的作用。方法我们采用两步灌流法分离树鼩肝细胞,并用L15培养基予以培养,培养3d接种HCV-RNA阳性血清,在接种感染血清前90min 加40g·L^1PEG与15g·L^1,然后再接种HCV-RNA阳性血清温育6h～8h,并收集不同时相的细胞和上清以逆转录-聚合酶链反应(PT-PCR)法检测其中HCV正负链RNA。结果未加PEG与DMSO时,细胞内正负链RNA首次检出是感染后d5,至d10仍可间断检出,培养上清正链首次检出是感染后d3,d10亦呈阳性;加PEG与DMSO时,细胞内负链首次检出是感染后d3,至d17仍可间断检出,细胞内正链首次检出是感染后d3,至d15仍可间断检出,培养上清正链RNA首次检出感染后d3,至d15亦呈阳性。结论加PEG与DMSO细胞内正负链RNA首次检出时间较早,检出率较高,持续时间较长。

OBJECTIVE To explore the effect of polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) on infection of adult normal Tupaia hepatocytes in primary culture by hepatitis C virus in vitro. METHODS When hepatocytes of adult Tupaia were isolated by a two-stage perfusion method and cultured in a suitable medium for 3 days, culturing hepatocytes were inoculated with the human sera positive for HCV RNA. Before inoculating the human sera positive for HCV RNA about 90min, 40g·L^1 PEG and 15g·L^1 DMSO were added into culturing hepetocytes. After incubation with sera positive for HCV RNA for 6h 8h, the cells and supernatant were harvested at various phases during the culturing periods. Both the plus and minus strands of HCV RNA in infected cells or supernatant were examined by reverse transcription polymerase chain reaction (RT-PCR). RESULTS When PEG and DMSO were absent, the intracellular plus or minus strands of HCV RNA were first detected on day 5 post-incubation and then intermittently detected on day 10 post-incubation. When PEG and DMSO were added, the intracellular plus or minus strands of HCV RNA were first detected on day 3 post-incubation and then could be intermittently detected on day 17 postincubation. The supernatant could be intermittently detected on day 3 15 post-incubation. CONCLUSION PEG and DMSO are benifitial to promote HCV infection of adult normal Tupaia hepatocytes in primary culture in vitro. They can prolong the period of detection of viral RNA in cells.