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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 investigate the effect of dengue virus (DV) on human dermal fibroblast (HSF) infectivity and cytokines in DV infected HSF. Methods Virulence of HSV infected with denguetype-2 virus (DV2) was detected by cytomegalovirus plaque assay. DV antigen in HSF was detected by indirect immunofluorescence assay. Transmission electron microscopy was used to observe the ultrastructural changes of virus-infected cells. The different concentrations of IL-6, TNF-α and GM-CSF were used to treat the different sections of HSV infected by DV2 (after virus adsorption and virus adsorption), the supernatant was collected 48h after infection and the virus titer was measured. After infection with HSF, the supernatant was collected at different times, and the levels of IL-6 and TNF-α were measured by ELISA. Results The virus was detected in the culture supernatant 24h after virus infection. The virus titer peaked at 48h and then decreased gradually. Indirect immunofluorescence was used to confirm that DV antigen was carried in the cytosol and plasma membrane of infected HSF. Under light microscope and electron microscope, no obvious morphological and structural changes were observed in infected cells. IL-6 at a concentration of 10 ng / ml significantly increased viral production at virus adsorption; TNF-a at a concentration of 100 ng / ml after virus adsorption and after adsorption inhibited virus production. GM-CSF had no significant effect on DV infected HSF. DV infection can promote HSF secretion of IL-6; TNF-α secretion had no significant effect. Conclusion HSF is a permissive cell for DV. HSF may be one of the first cells that support DV infection in situ after mosquito bites; cytokines play an important role in the pathogenicity and immunity of DV-infected HSF.