树突状细胞在登革病毒感染中的作用研究

发布时间：2018-05-12 12:25

本文选题：登革病毒 + 人树突状细胞　；参考：《暨南大学》2005年硕士论文

【摘要】：目的:以人髓系树突状细胞为靶细胞,研究登革病毒对人树突状细胞的感染性及产生细胞因子的变化和这些细胞因子对登革病毒感染人树突状细胞的影响,探讨树突状细胞在登革病毒感染中的作用机制,为阐明登革病毒感染的发病机制并指导临床治疗提供科学依据。方法:课题分4部分,第一部分人外周血常规分离单个核细胞,以细胞因子GM-CSF、IL-4进行诱导培养成树突状细胞并通过形态学特征、细胞表型和淋巴细胞刺激能力鉴定。第二部分DV_2感染DCs,于感染后6h、24h、48h、72h、96h分别收集上清液和细胞,甲基纤维素微量空斑试验测定病毒滴度,间接免疫荧光法检测细胞上病毒抗原表达,透射电镜观察病毒颗粒及病毒感染后细胞超微结构的变化。第三部分DV_2感染DCs,于感染后6h、24h、48h、72h、96h收集感染上清,ELISA法检测细胞因子TNF-α、IL-6、IFN-γ水平的动态变化。第四部分用高、中、低浓度的IL-6、TNF-α作用于DV_2感染的DCs,于感染后6h、24h、48h、72h、96h收集上清,测病毒滴度,同时MTT法测定感染后48hDCs的数量变化。结果:(1)人外周血单核细胞经细胞因子GM-CSF和IL-4体外诱导培养1周成树突状细胞,形态学观察可见细胞形态不规则,细胞表面大量树枝状突起,免疫组化及间接免疫荧光CD1a阳性表达率达80%～95%,树突状细胞刺激同种淋巴细胞增殖反应表明本法获得的树突状细胞具有强大的促进淋巴细胞增殖能力。(2)病毒感染后6h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。间接免疫荧光法证明感染的DCs胞浆及胞膜上携带病毒抗原。透射电镜下在病毒感染48h后DCs胞浆内可见大量病毒颗粒。(3)登革病毒感染促进树突状细胞分泌TNF-α、IL-6,但分泌IFN-γ水平无明显改变(4)中、低浓度的IL-6能显著提高病毒产量,以中浓度的IL-6为主;高、中浓度的TNF-α则抑制病毒产量,以高浓度为主。细胞因子对病毒感染的树突状细胞数量无明显影响。结论:树突状细胞是登革病毒感染的靶细胞之一,病毒可在细胞内复制并促进细胞分泌细胞因子如TNF-α、IL-6等,这些增高的细胞因子又可通过增强或抑制病毒在树突状细胞内的增殖从而影响疾病的发生发展。
[Abstract]:Objective: to study the infection of dengue virus on human dendritic cells and the effect of these cytokines on the infection of human dendritic cells with human myeloid dendritic cells. To explore the role of dendritic cells in dengue virus infection and provide scientific basis for clarifying the pathogenesis of dengue virus infection and guiding clinical treatment. Methods: in the first part, mononuclear cells were routinely isolated from human peripheral blood. Dendritic cells were induced by GM-CSF- IL-4 and identified by morphological characteristics, cell phenotypes and lymphocyte stimulation ability. The second part of DV_2 was infected with DCS. Supernatants and cells were collected at 6 h, 24 h, 48 h, 72 h and 96 h after infection. The titer of virus was determined by methylcellulose microplaque test, and the expression of virus antigen was detected by indirect immunofluorescence assay. The ultrastructural changes of virus particles and cells after virus infection were observed by transmission electron microscope. The third part was DV_2 infected with DCS. The dynamic changes of cytokine TNF- 伪 and IL-6IFN- 纬 levels were detected by Elisa in the supernatant of DV_2 collected at 24 h or 48 h or 72 h / 96 h after infection. In the fourth part, high, medium and low concentrations of IL-6 TNF- 伪 were used to treat DV_2 infected DCS. The supernatants were collected at 6 h, 24 h, 48 h, 72 h and 96 h after infection. The titer of 48hDCs was measured by MTT method. Results Human peripheral blood mononuclear cells were induced by cytokines GM-CSF and IL-4 for 1 week to form dendritic cells. Morphological observation showed that the morphology of the cells was irregular and a large number of dendritic processes appeared on the surface of the cells. The positive expression rate of immunocytochemistry and indirect immunofluorescence CD1a was 80% and 95%. The dendritic cells stimulated the proliferation of allogeneic lymphocytes showed that the dendritic cells obtained by this method had a strong ability to promote lymphocyte proliferation. The virus can be detected in the culture supernatant. The titer of the virus reached its peak at 48 h, and then decreased gradually. Indirect immunofluorescence assay showed that the infected DCs cytoplasm and membrane carried viral antigen. Transmission electron microscope (TEM) showed that a large number of viral particles. 3) Dengue virus infection promoted dendritic cells to secrete TNF- 伪 and IL-6, but the level of IFN- 纬 did not change significantly. Low concentration of IL-6 could significantly increase the virus yield. TNF- 伪 of high and middle concentration inhibited the yield of virus, and high concentration of TNF- 伪 mainly inhibited the yield of virus. Cytokines had no significant effect on the number of dendritic cells infected by virus. Conclusion: dendritic cells are one of the target cells infected with dengue virus. The virus can replicate in the cells and promote the secretion of cytokines such as TNF- 伪 and IL-6. These cytokines may influence the development of the disease by increasing or inhibiting the proliferation of virus in dendritic cells.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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