本文選題：SEA　切入點(diǎn)：原子力顯微鏡　出處：《暨南大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的：了解超抗原金黃色葡萄球菌腸毒素A(SEA)對(duì)人T淋巴細(xì)胞增殖活性與表面超微結(jié)構(gòu)的影響；以及在SEA作用下CD3分子、ZAP-70、TCR(?)鏈在T細(xì)胞上的形態(tài)學(xué)、分子生物學(xué)水平變化特點(diǎn)。探討SEA活化T細(xì)胞在信號(hào)傳導(dǎo)通路上的特點(diǎn),為SEA的應(yīng)用提供相關(guān)支持。 方法：1,利用T細(xì)胞體外培養(yǎng)法分別于正常人外周血淋巴細(xì)胞、Jurkat細(xì)胞加入不同濃度SEA培養(yǎng)不同時(shí)間；2,利用CCK-8法檢測細(xì)胞增殖活性；3,采用原子力顯微鏡(AFM)觀察經(jīng)SEA處理后T細(xì)胞表面超微結(jié)構(gòu)變化,利用CD3抗體修飾的探針考察CD3分子在T細(xì)胞表面分布；4,應(yīng)用激光共聚焦顯微鏡(LSCM)觀察經(jīng)SEA刺激后T細(xì)胞CD69分子表達(dá)以及ZAP-70細(xì)胞膜內(nèi)的分布邊集；5,采用SYBR GreenⅠ熒光定量PCR和相對(duì)定量檢測TCRζ鏈、ZAP-70在不同濃度SEA作用下的T淋巴細(xì)胞中表達(dá)情況,以β2微球蛋白基因(β2M)作為內(nèi)參,根據(jù)相對(duì)定量公式：2-△△Ct分析TCRζ鏈、ZAP-70基因表達(dá)差異。 結(jié)果：1,SEA可使人外周血T細(xì)胞CD69表達(dá),且適宜濃度10～0.01mg/LSEA作用下T細(xì)胞增殖活性最大；2,SEA作用于T細(xì)胞不同時(shí)間6-48小時(shí),T細(xì)胞表面超微結(jié)構(gòu)發(fā)生改變,隨著時(shí)間推移細(xì)胞表面粗糙度(Ra)先升高后降低,細(xì)胞表面陸續(xù)出現(xiàn)斑塊狀、裂隙狀、大顆粒狀等結(jié)構(gòu)；3,CD3分子在經(jīng)SEA作用后T細(xì)胞上呈現(xiàn)聚集趨勢(shì)。4,SEA適宜濃度10～0.01mg／L能夠引起ZAP-70在T細(xì)胞內(nèi)熒光強(qiáng)度迅速變化,同一濃度SEA在1小時(shí)內(nèi),ZAP-70熒光強(qiáng)度先升高后降低。5,不同濃度SEA誘導(dǎo)T細(xì)胞TCRζ鏈、ZAP-70基因表達(dá)下降,2-△△Ct值均小于1,且不同濃度的SEA所誘導(dǎo)基因下降的程度不同,0.1mg/LSEA誘導(dǎo)下降程度最大。 結(jié)論：1,0.1～10mg／L超抗原SEA48小時(shí)體外引起Jurkat T細(xì)胞、正常人外周血T細(xì)胞顯著增殖；2,SEA作用Jurkat T細(xì)胞不同時(shí)間(48小時(shí)內(nèi)),細(xì)胞表面超微結(jié)構(gòu)發(fā)生特異性改變；3,SEA作用正常人外周T細(xì)胞12小時(shí),T細(xì)胞表達(dá)活化CD69分子；4,TCR信號(hào)轉(zhuǎn)導(dǎo)途徑上的重要分子ZAP-70在SEA作用T細(xì)胞過程中有一定貢獻(xiàn)；5,SEA引發(fā)的TCR信號(hào)轉(zhuǎn)導(dǎo)途徑上TCRζ鏈、ZAP-70作用與經(jīng)典TCR信號(hào)轉(zhuǎn)導(dǎo)途徑不同；6,SEA活化T細(xì)胞可通過旁路途徑活化T細(xì)胞。
[Abstract]:Objective: to investigate the effects of superantigen Staphylococcus aureus enterotoxin Agna on proliferation activity and surface ultrastructure of human T lymphocytes, and to investigate the effect of CD3 molecule ZAP-70 on the proliferation and ultrastructure of human T lymphocytes. To study the characteristics of SEA activated T cells in signal transduction pathway and to provide relevant support for the application of SEA. Methods in vitro T cell culture method was used to culture normal human peripheral blood lymphocyte Jurkat cells with different concentrations of SEA for different time. CCK-8 assay was used to detect cell proliferation activity and atomic force microscope (AFM) was used to observe SEA. After treatment, the ultrastructure of T cell surface was changed. CD3 antibody modified probe was used to investigate the distribution of CD3 molecule on T cell surface. The expression of CD69 molecule in T cell stimulated by SEA and the distribution edge of ZAP-70 cell membrane were observed by laser confocal microscopy. SYBR Green 鈪，

本文編號(hào)：1625581