细胞因子在登革病毒感染人皮肤成纤维细胞中的作用

The roles of chemokines in the interaction between dengue virus and human skin fibroblast

目的探讨登革病毒(denguevirus,DV)对人皮肤成纤维细胞(HSF)的感染性和细胞因子在DV感染HSF中的作用。方法采用微量病毒空斑法检测登革Ⅱ型病毒(denguetype-2virus,DV2)感染HSF后病毒繁殖动态变化,间接免疫荧光法检测HSF内DV抗原。透射电镜观察病毒感染细胞的超微结构改变。用不同浓度的IL-6、TNF-α、GM-CSF分别作用于DV2感染HSF的不同环节(病毒吸附时和病毒吸附后),于感染后48h收集感染上清,测病毒滴度;用DV2感染HSF后,于不同时间收集感染上清,用ELISA法定量测定IL-6、TNF-α的含量。结果病毒感染后24h即可在培养上清中测出病毒,病毒滴度在48h达到高峰,以后逐渐下降。用间接免疫荧光法证明感染的HSF胞浆及胞膜上携带DV抗原。在光镜和电镜下,感染细胞均未见明显的形态和结构改变。在病毒吸附时10ng/ml浓度的IL-6能显著提高病毒产量;在病毒吸附时和吸附后100ng/ml浓度的TNF-α能抑制病毒的产量。GM-CSF对DV感染HSF无明显影响。DV感染能促进HSF分泌IL-6;对TNF-α的分泌无明显影响。结论HSF是DV的允许性细胞。HSF可能是蚊叮咬后在原位组织中首先支持DV感染的细胞之一;细胞因子在DV感染HSF的致病和免疫过程中起重要作用。

Objective To study dengue virus（DV） infection to human skin fibroblasts（HSF） by exploring the effect of cytokines on replication of DV 2 in HSF and cytokine production by DV 2 infected HSF . Methods To establish culture of HSF in vitro from skin samples. The recovered cells were identified as fibroblasts by their shape characteristics in light microscope and by immuno-histochemistry. DV2 infected HSF in vitro, culture super- natants were collected at different time after infection, the titer of virus was measured by microplaque forming assay. DV antigen in HSF was demonstrated by an indirect immunofluorescent assay. The infected HSF were examined by electron microscopy. II,-6, TNF-α, GM-CSF of high, medium and low concentration were incubated with HSF during and after DV2 absorption. Cultured supernatants were collected at 48 h after infection and virus titers were determined. The production of IL-6, TNF-α secreted by DV2 infected HSF was checked by ELISA. Results Titers of infectious DV2 were found as early as 24 h after infection and reached the peak by 48 h. In addition, no evident cytopathogenic effect was seen in DV2 infected HSF. IL-6 at a concentration of 10 ng/ml can increase DV2 replication in HSF when the cytokine was incubated with HSF during DV2 absorption. TNF-α at a concentration of 100 ng/ml can protect uninfected HSF from DV2 infection when cytokine was incubated with HSF during and after DV2 absorption, but GM-CSF has no effect on the DV2 replication in HSF. Level of IL-6 was greatly increased in the culture supernatants of HSF after DV2 infection and there was no change in the level of TNF-a secreted by DV2 infected HSF. Conclusion HSF is the target of dengue virus infection. They may be one of the initial target cells supporting DV2 infection at the site of entry; cytokines play an important role in the immuno-pathogenesis of DV2 infection in HSF.