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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 investigate the role of polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) in the in vitro infection of tree shrew hepatocytes with HCV. Methods Two-step perfusion method was used to isolate hepatocytes from tree shrews and cultured in L15 medium. After 3 days of inoculation of serum, HCV-RNA positive sera were inoculated with 40 g · L -1 PEG and 15 g · L -1 HCV-RNA positive sera were incubated for 6h to 8h, and cells and supernatants of different phases were collected for detection of HCV positive and negative strand RNA by reverse transcription-polymerase chain reaction (PT-PCR). Results When PEG and DMSO were not added, the first detection of positive and negative intracellular RNA was detected on d5 and d10 after infection, and the positive detection of culture supernatant was the first positive after d3 and d10 infection. DMSO, the first detection of negative intracellular chain is detected after d3, to d17 can still be detected intermittently, the first detection of positive intracellular chain is infected d3, d15 can still be detected intermittently, the culture supernatant positive strand RNA for the first time After the infection d3, to d15 also showed positive. Conclusions The positive and negative RNAs of PEG plus DMSO cells were detected for the first time with higher detection rate and longer duration.