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利用体外细胞感染模型,分别检测HIV-1感染的细胞系中转录和翻译水平ISG15的表达及细胞上清中p24蛋白水平,探讨HIV-1感染对ISG15表达的影响及后者对前者的抑制效应。6种HIV-1易感细胞系中,以IFN-α 2b刺激作为阳性对照,利用HIV-1感染性克隆pNL4-3转染293T、TZM-bl和HeLa细胞;HIV-1假病毒感染Jurkat、MT-4和THP-1细胞,24h收细胞提取RNA,利用荧光定量PCR(qPCR)的方法检测转录水平ISG15的表达情况。48h后收细胞提取总蛋白,Western blot检测蛋白水平ISG15的表达情况。HIV-1感染或转染后明显上调ISG15转录水平,且THP-1和TZM-bl细胞中上调尤为显著。但除了TZM-bl细胞,其他5种细胞系中没有表现出ISG15蛋白水平的上调。ISG15真核表达质粒与HIV-1感染性克隆pNL4-3共转染结果显示,293T细胞中ISG15能直接抑制HIV-1子代病毒颗粒的产生和成熟,并且这种抑制作用具有时间剂量依赖性;TZM-bl中虽然也有显著的抑制作用,但是趋势不同。HIV-1感染明显上调ISG15转录但却无ISG15蛋白产生,预示该病毒能对抗固有免疫细胞的识别活化及其效应功能,具体机制还有待进一步阐明。
Using in vitro cell infection model, the transcription and translation level of ISG15 in HIV-1 infected cell lines and the level of p24 protein in cell supernatant were detected respectively to investigate the effect of HIV-1 infection on ISG15 expression and the inhibitory effect of the latter on the former . HIV-1 susceptible cell lines were transfected with 293T, TZM-bl and HeLa cells using HIV-1 infectious clone pNL4-3 as a positive control with IFN-α 2b stimulation; HIV-1 pseudovirus infected Jurkat, MT-4 and THP-1 cells were harvested for 24 h, RNA was extracted from the cells for 24 h, and the expression of ISG15 at the transcription level was detected by qPCR. After 48 hours, cells were harvested to extract total protein, Western blot was used to detect the expression of protein ISG15. The transcription of ISG15 was significantly up-regulated after HIV-1 infection or transfection, especially in THP-1 and TZM-bl cells. However, except for TZM-bl cells, none of the other five cell lines showed upregulation of ISG15 protein levels. ISG15 eukaryotic expression plasmid and HIV-1 infectious clone pNL4-3 cotransfection results showed that ISG15 293T cells can directly inhibit HIV-1 progeny virus particle generation and maturation, and this inhibitory effect of a dose-dependent manner Although TZM-bl also has a significant inhibitory effect, but the trend is different. HIV-1 infection significantly increased ISG15 transcription but no ISG15 protein production, suggesting that the virus can fight innate immune cells recognition and activation of its effector function, the specific mechanism remains to be further elucidated.