基因1型戊型肝炎病毒 ORF3对人外周血来源的树突状细胞成熟和活化功能的影响

The effect of ORF3 of genotype 1 hepatitis E virus on the maturation and activation of dendritic cells derived from human peripheral blood

目的:探讨基因1型戊型肝炎病毒(HEV)开放读码框3(ORF3)对人外周血来源的树突状细胞(DC)成熟和活化功能的影响。方法:抽取健康志愿者的外周血分离单个核细胞,经体外培养、诱导为DC。然后分别用含有ORF3的慢病毒载体以及空载载体感染DC 48h,流式细胞仪检测ORF3组、空载组和空白组细胞表面CD_(80)、CD_(83)、CD_(86)的表达,ELISA法检测DC上清中IFN-β、IL-4、IL-10和IL-12的分泌。之后再加入TNF-α进一步刺激DC成熟,流式细胞仪检测各组细胞CD_(80)、CD_(83)、CD_(86)以及IFN-β、IL-4、IL-10、IL-12的表达。最后将DC与T淋巴细胞共培养,CCK8法检测T淋巴细胞增殖。结果:含有ORF3组的DC与空载组相比,加入TNF前后,其细胞表面CD_(80)、CD_(83)、CD_(86)以及细胞上清中IFN-β、IL-4、IL-10和IL-12的表达都明显较低(P<0.05)。ORF3组与空载组相比其刺激T细胞增殖的能力也较弱(P<0.05)。结论:基因1型HEV ORF3可以抑制人外周血来源DC的成熟,抑制其分泌IFN-β、IL-4、IL-10和IL-12等细胞因子,抑制DC对T淋巴细胞的增殖刺激作用,从而削弱机体对HEV的免疫反应,实现免疫逃逸,可能是导致戊型肝炎慢性化的原因之一。

Objective:To study the effect of gene type 1 hepatitis E virus(HEV)open reading frame 3(ORF3)on the maturation and activa-tion of dendritic cells(DCs) derived from human peripheral blood. Methods:Peripheral blood from healthy volunteers was cultured into dendritic cells in vitro. Then,DC was infected with the ORF3-containing lentiviral vector and the empty vector for 48h,and the expression of CD80,CD83, and CD86 of DCs were detected by flow cytometry. IFN-β,IL-4,IL-10 and IL-12 in DC supernatant were detected by ELISA. After that adding TNF-α to further stimulate DC maturation. Then the expression of CD80,CD83,CD86 and IFN-β,IL-4,IL-10 and IL-12 in each group of cells were examined again. Finally,each group of DCs was co-cultured with T lymphocytes. The CCK8 method was used to detect the proliferation of T lymphocytes. Results:The expression of IFN-β,IL-4,IL-10 and IL-12,CD80,CD83,CD86 of DCs containing ORF3 group compared with that of the empty vector group are significantly lower before and after TNF addition. The ability of ORF3 group to stimulate T cell proliferation was also weaker than that of the empty vector group. Conclusion:HEV ORF3 of genotype 1 can inhibit the maturity of human peripheral blood derived DC,inhibit the secretion of IFN-beta,IL 4,IL-10 and IL-12,inhibits the proliferation and stimulation of DC on T lymphocytes,thereby weakens the body’s immune response to HEV and achieving immune escape,maybe one reason leading to chronic hepatitis.