本文選題：登革病毒 + 人樹突狀細胞　； 參考：《暨南大學》2005年碩士論文

【摘要】：目的:以人髓系樹突狀細胞為靶細胞,研究登革病毒對人樹突狀細胞的感染性及產(chǎn)生細胞因子的變化和這些細胞因子對登革病毒感染人樹突狀細胞的影響,探討樹突狀細胞在登革病毒感染中的作用機制,為闡明登革病毒感染的發(fā)病機制并指導臨床治療提供科學依據(jù)。 方法:課題分4部分,第一部分人外周血常規(guī)分離單個核細胞,以細胞因子GM-CSF、IL-4進行誘導培養(yǎng)成樹突狀細胞并通過形態(tài)學特征、細胞表型和淋巴細胞刺激能力鑒定。第二部分DV_2感染DCs,于感染后6h、24h、48h、72h、96h分別收集上清液和細胞,甲基纖維素微量空斑試驗測定病毒滴度,間接免疫熒光法檢測細胞上病毒抗原表達,透射電鏡觀察病毒顆粒及病毒感染后細胞超微結構的變化。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法檢測細胞因子TNF-α、IL-6、IFN-γ水平的動態(tài)變化。第四部分用高、中、低濃度的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,測病毒滴度,同時MTT法測定感染后48hDCs的數(shù)量變化。 結果:(1)人外周血單核細胞經(jīng)細胞因子GM-CSF和IL-4體外誘導培養(yǎng)1周成樹突狀細胞,形態(tài)學觀察可見細胞形態(tài)不規(guī)則,細胞表面大量樹枝狀突起,免疫組化及間接免疫熒光CD1a陽性表達率達80%～95%,樹突狀細胞刺激同種淋巴細胞增殖反應表明本法獲得的樹突狀細胞具有強大的促進淋巴細胞增殖能力。(2)病毒感染后6h即可在培養(yǎng)上清中測出病毒,病毒滴度在48h達到高峰,以后逐漸下降。間接免疫熒光法證明感染的DCs胞漿及胞膜上攜帶病毒抗原。透射電鏡下在病毒感染48h后DCs胞漿內可見大量病毒顆粒。(3)登革病毒感染促進樹突狀細胞分泌TNF-α、IL-6,但分泌IFN-γ水平無明顯改變(4)中、低濃度的IL-6能顯著提高病毒產(chǎn)量,以中濃度的IL-6為主;高、中濃度的TNF-α則抑制病毒產(chǎn)量,以高濃度為主。細胞因子對病毒感染的樹突狀細胞數(shù)量無明顯影響。 結論:樹突狀細胞是登革病毒感染的靶細胞之一,病毒可在細胞內復制并促進細胞分泌細胞因子如TNF-α、IL-6等,這些增高的細胞因子又可通過增強或抑制病毒在樹突狀細胞內的增殖從而影響疾病的發(fā)生發(fā)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- 偽 and IL-6IFN- 緯 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- 偽 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- 偽 and IL-6, but the level of IFN- 緯 did not change significantly. Low concentration of IL-6 could significantly increase the virus yield. TNF- 偽 of high and middle concentration inhibited the yield of virus, and high concentration of TNF- 偽 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- 偽 and IL-6. These cytokines may influence the development of the disease by increasing or inhibiting the proliferation of virus in dendritic cells.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392

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