本文選題：登革病毒 + 人樹突狀細(xì)胞��； 參考：《暨南大學(xué)》2005年碩士論文

【摘要】：目的:以人髓系樹突狀細(xì)胞為靶細(xì)胞,研究登革病毒對(duì)人樹突狀細(xì)胞的感染性及產(chǎn)生細(xì)胞因子的變化和這些細(xì)胞因子對(duì)登革病毒感染人樹突狀細(xì)胞的影響,探討樹突狀細(xì)胞在登革病毒感染中的作用機(jī)制,為闡明登革病毒感染的發(fā)病機(jī)制并指導(dǎo)臨床治療提供科學(xué)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(gè)核細(xì)胞,以細(xì)胞因子GM-CSF、IL-4進(jìn)行誘導(dǎo)培養(yǎng)成樹突狀細(xì)胞并通過形態(tài)學(xué)特征、細(xì)胞表型和淋巴細(xì)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(xì)胞,甲基纖維素微量空斑試驗(yàn)測定病毒滴度,間接免疫熒光法檢測細(xì)胞上病毒抗原表達(dá),透射電鏡觀察病毒顆粒及病毒感染后細(xì)胞超微結(jié)構(gòu)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(xì)胞因子TNF-α、IL-6、IFN-γ水平的動(dòng)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(shí)MTT法測定感染后48hDCs的數(shù)量變化。 結(jié)果:(1)人外周血單核細(xì)胞經(jīng)細(xì)胞因子GM-CSF和IL-4體外誘導(dǎo)培養(yǎng)1周成樹突狀細(xì)胞,形態(tài)學(xué)觀察可見細(xì)胞形態(tài)不規(guī)則,細(xì)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(dá)率達(dá)80%～95%,樹突狀細(xì)胞刺激同種淋巴細(xì)胞增殖反應(yīng)表明本法獲得的樹突狀細(xì)胞具有強(qiáng)大的促進(jìn)淋巴細(xì)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(dá)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(nèi)可見大量病毒顆粒。(3)登革病毒感染促進(jìn)樹突狀細(xì)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(xì)胞因子對(duì)病毒感染的樹突狀細(xì)胞數(shù)量無明顯影響。 結(jié)論:樹突狀細(xì)胞是登革病毒感染的靶細(xì)胞之一,病毒可在細(xì)胞內(nèi)復(fù)制并促進(jìn)細(xì)胞分泌細(xì)胞因子如TNF-α、IL-6等,這些增高的細(xì)胞因子又可通過增強(qiáng)或抑制病毒在樹突狀細(xì)胞內(nèi)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(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 趙衛(wèi),曹虹,張文炳,何小艷;登革病毒甲基纖維素微量蝕斑技術(shù)的建立[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2001年S1期

2 王平忠,白雪帆;細(xì)胞因子/趨化因子在漢坦病毒感染發(fā)病機(jī)制中的作用[J];國外醫(yī)學(xué).流行病學(xué)傳染病學(xué)分冊;2004年03期

3 王明軍;樹突狀細(xì)胞的形成、功能及可能的應(yīng)用[J];國外醫(yī)學(xué)(免疫學(xué)分冊);2001年04期

4 潘宇;Th細(xì)胞及其細(xì)胞因子與登革病毒致病機(jī)制關(guān)系的研究進(jìn)展[J];國外醫(yī)學(xué)(免疫學(xué)分冊);2003年03期

5 李雪蓮,武峰,王瑩;登革病毒組織培養(yǎng)技術(shù)[J];河南醫(yī)學(xué)研究;2002年03期

6 曹艷英,郭輝玉;TNFα在登革病毒感染發(fā)病機(jī)制中作用的實(shí)驗(yàn)研究[J];暨南大學(xué)學(xué)報(bào)(自然科學(xué)與醫(yī)學(xué)版);1997年05期

7 許化溪,王勝軍,嚴(yán)俊,潘志超,夏圣,劉恭植;人外周血樹突狀細(xì)胞的分離與鑒定[J];免疫學(xué)雜志;1997年03期

8 張靜,李偉華,陳順英,郭秀英,李黎;用蝕斑法進(jìn)行麻疹疫苗的病毒滴定[J];微生物學(xué)免疫學(xué)進(jìn)展;1999年01期

9 江振友,曹艷英,林晨;TNFα和IL-10在巨細(xì)胞病毒感染人胚肺成纖維細(xì)胞中的作用[J];中國病理生理雜志;2002年03期

10 曹艷英,郭輝玉;IL-6與登革病毒感染相互關(guān)系的實(shí)驗(yàn)研究[J];中國病理生理雜志;1999年02期

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