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中国循证儿科杂志

中国循证儿科杂志 ›› 2016, Vol. 11 ›› Issue (4): 290-294.

• 论著 • 上一篇    下一篇

自然杀伤细胞与丙型肝炎病毒感染的Huh7.5细胞体外共培养后的功能变化

胡姚1,张婷2,叶颖子1,王晓红1,俞蕙1   

  1. 1 复旦大学附属儿科医院感染科 上海，201102；2 上海交通大学附属儿童医院消化科 上海，200040
  • 收稿日期:2016-06-08 修回日期:2016-08-29 出版日期:2016-08-25 发布日期:2016-08-25
  • 通讯作者: 俞蕙

Functional change of natural killer cells cocultured with hepatitis C virus infected Huh7.5 cells in vitro

HU Yao1, ZHANG Ting2, YE Ying-zi1,WANG Xiao-hong1,YU Hui1   

  1. 1 Department of Infectious Diseases, The Children′s Hospital, Fudan University, Shanghai 201102; 2 Department of Gastroenterology, Shanghai Children′s Hospital, Shanghai Jiaotong University, Shanghai 200040, China
  • Received:2016-06-08 Revised:2016-08-29 Online:2016-08-25 Published:2016-08-25
  • Contact: YU Hui

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摘要：

目的 探讨体外丙型肝炎病毒（HCV）感染对NK细胞功能产生的影响及其可能的机制。方法 利用质粒JC1-Flag2体外转录得到的HCV感染性颗粒（HCVcc）以MOI4.8感染Huh7.5细胞（Huh7.5-HCVcc），与健康人外周血分离得到的NK细胞进行共培养。与Huh7.5-HCVcc细胞共培养前后，采用ELISA法和MTT比色法分别检测NK分泌细胞因子IFN-γ、TNF-α和IL-10的水平和细胞杀伤活性，以评估HCV感染对NK细胞功能的影响。结果 与MOI4.8的Huh7.5-HCVcc细胞共培养，NK细胞分泌IFN-γ、TNF-α和IL-10水平受到抑制，其中IFN-γ在共培养6 h（P<0.001）、9 h（P<0.001）、12 h（P<0.001）受到显著抑制，TNF-α在共培养6 h（P<0.001）、9 h（P<0.001）、12 h（P=0.001）受到显著抑制，IL-10在共培养6 h（P<0.001）、9 h（P=0.006）受到显著抑制；且NK细胞分泌细胞因子IFN-γ、TNF-α和IL-10的功能均在共培养6 h受到的抑制效应最大，平均抑制率分别为24.1%、20.7%和24.3%。NK细胞杀伤活性在共培养6 h（P=0.023）受到抑制，平均抑制率为16.6%。结论 在体外与MOI4.8的Huh7.5-HCVcc细胞共培养，NK细胞分泌细胞因子(IFN-γ、TNF-α和IL-10)和细胞杀伤功能均受到抑制，且在不同时间点受到的抑制程度不同，提示NK细胞在HCV感染的不同阶段可能起到不同的作用。

Abstract:

Abstract ObjectiveThe numbers, subsets and function of natural killer (NK) cells altered in chronic hepatitis C virus (HCV) infected patients which indicating its association with chronic HCV infection, but the exact mechanisms remain unclear. To explore the functional effect of HCV infection on NK cells in vitro and the possible mechanism behind it.Methods Human hepatoma Huh7.5 cells were transfected with genomic RNA from cell culture adapted JC1 genotype 2a HCV strain (Huh7.5-HCVcc) and co-cultured with NK cells isolated from whole peripheral blood of concentrated healthy donors. The functional effect of HCV infection on NK cells was assessed by testing cytokine-secreting and cytotoxicity before and after co-culture.Results When co-cultrued with Huh7.5-HCV of MOI 4.8, the levels of IFN-γ, TNF-α and IL-10 secreted by NK cells were inhibited. The inhibition of secreting IFN-γ was specially significant at 6(P<0.001), 9(P<0.001) and 12(P=0.001) hours co-cultured with Huh7.5-HCV. The inhibition of secreting TNF-α was specially significant 6 h(P<0.001), 9 h(P<0.001) and 12 h(P=0.001) after co-cultured with Huh7.5-HCVcc. And the significant inhibition of secreting IL-10 appeared at 6 h(P<0.001) and 9 h(P=0.006) after co-cultured with Huh7.5-HCVcc. The strongest cytokine-secreting inhibition appeared 6 h after co-cultured with Huh7.5-HCVcc, the inhibition rate was 24.1%, 20.7% and 24.3%, respectively. The cytotoxicity decreased significantly 6 h after co-cultured with Huh7.5-HCVcc(P=0.023), and the average inhibition rate was 16.6%.Conclusion When in vitro cocultrued with Huh7.5-HCVcc of MOI 4.8, the NK cells functions of IFN-γ, TNF-α and IL-10 cytokine-secreting and cytotoxicity were inhibited. The degree of inhibition was different at different time points, which indicated that NK cells may act differently during HCV infection.