本文選題：BTLA　切入點(diǎn)：抑制　出處：《蘇州大學(xué)》2012年碩士論文

【摘要】：BTLA是2003年發(fā)現(xiàn)的Ig家族成員，是與CTLA-4及PD-1類似的抑制性受體。人的BTLA基因定位于染色體3q13.2，與小鼠具有48%的同源性。BTLA mRNA在脾臟及淋巴結(jié)組織中高表達(dá)，在腎臟、肝臟、肺、腦、心臟及肌肉等組織中表達(dá)量都很低，甚至不表達(dá)。BTLA廣泛表達(dá)于T細(xì)胞、B細(xì)胞、巨噬細(xì)胞、樹突狀細(xì)胞和NK細(xì)胞表面。 BTLA含有一個(gè)信號(hào)序列、一個(gè)免疫球蛋白可變區(qū)（IgV）樣的胞外段、跨膜區(qū)和大約100個(gè)氨基酸的胞漿區(qū)。BTLA胞漿區(qū)存在3個(gè)重要的含酪氨酸殘基的基序:潛在的Grb2結(jié)合位點(diǎn)，免疫受體酪氨酸抑制基序和免疫受體酪氨酸轉(zhuǎn)換基序。研究表明，，BTLA與其配體HVEM結(jié)合可以抑制T細(xì)胞的增殖和細(xì)胞因子的分泌，推遲細(xì)胞周期的進(jìn)程，從而抑制或終止免疫應(yīng)答。在持續(xù)的抗原刺激下，BTLA高度選擇性表達(dá)在無(wú)能CD4+T細(xì)胞上，表明BTLA信號(hào)在誘導(dǎo)與維持T細(xì)胞無(wú)能狀態(tài)的過(guò)程中有重要作用。另外，BTLA不僅在在維持外周免疫耐受中具有重要作用，還可以預(yù)防自身免疫疾病的發(fā)生，終止炎癥反應(yīng)，抑制移植免疫應(yīng)答。 BTLA在初始和活化T細(xì)胞上都有表達(dá)，因此，Murphy提出BTLA可能在T細(xì)胞活化的各個(gè)階段尤其是早期階段發(fā)揮作用。本研究首先分析了BTLA-HVEM順式復(fù)合物的存在對(duì)細(xì)胞表面BTLA分子表達(dá)水平檢測(cè)的影響，進(jìn)而研究了BTLA在T細(xì)胞活化起始和早期階段的調(diào)節(jié)作用。 一、BTLA-HVEM順式復(fù)合物對(duì)細(xì)胞表面BTLA分子檢測(cè)的影響 【目的】T細(xì)胞和U937細(xì)胞表面存在BTLA和HVEM共表達(dá)的現(xiàn)象。探討B(tài)TLA-HVEM順式復(fù)合物存在對(duì)細(xì)胞表面BTLA分子檢測(cè)的影響。 【方法】分離人外周血單個(gè)核細(xì)胞，經(jīng)陰性選擇磁珠分離純化獲得T淋巴細(xì)胞。采用流式細(xì)胞術(shù)的方法，用抗不同BTLA位點(diǎn)的商品化單抗MIH26和本室研制的單抗8H9分別檢測(cè)U937和T細(xì)胞等細(xì)胞表面BTLA分子的表達(dá)水平。293T/BTLA基因轉(zhuǎn)染細(xì)胞上BTLA分子的檢測(cè)采用8H9單抗。 【結(jié)果】商品化單抗MIH26(0.5μg)檢測(cè)T細(xì)胞和U937細(xì)胞上BTLA分子的表達(dá)量分別為90%以上，而8H9單抗（1μg）檢測(cè)到二者的表達(dá)水平分別為42.2%、30.8%。采用0.02μg和0.05μg的8H9單抗檢測(cè)293T/BTLA轉(zhuǎn)基因細(xì)胞株，顯示BTLA分子的表達(dá)量分別為80%和91.4%。 【結(jié)論】T細(xì)胞和U937細(xì)胞上共表達(dá)的BTLA和HVEM形成順式復(fù)合物，影響8H9對(duì)BTLA的檢測(cè)，而不影響MIH26的檢測(cè)。 二、BTLA對(duì)T細(xì)胞活化啟動(dòng)和早期階段的調(diào)節(jié)作用 【目的】觀察BTLA在T細(xì)胞活化過(guò)程中的表達(dá)變化，探討其在T細(xì)胞活化各個(gè)階段尤其是早期階段的抑制作用。 【方法】分離人外周血單個(gè)核細(xì)胞，經(jīng)磁珠分選獲得純化的T細(xì)胞。用激發(fā)型CD3抗體和CD28抗體刺激T細(xì)胞活化，采用流式細(xì)胞術(shù)，檢測(cè)BTLA、CTLA-4和PD-1在T細(xì)胞活化過(guò)程中的動(dòng)態(tài)表達(dá)。激發(fā)型CD3抗體和CD28抗體在加入BTLA單抗8H9的情況下刺激T細(xì)胞活化，流式細(xì)胞術(shù)檢測(cè)T細(xì)胞活化早期標(biāo)志分子CD25和CD69的表達(dá)。激發(fā)型CD3抗體和CD28抗體聯(lián)合HVEM-Fc或者BTLA單抗8H9，觀察BTLA信號(hào)在活化后的不同時(shí)間對(duì)T細(xì)胞增殖的作用。同時(shí)，采用ELISA方法檢測(cè)8H9對(duì)CD3單抗聯(lián)合CD28單抗刺激的T細(xì)胞分泌IL-2和IFN-γ的影響。預(yù)先用CD3和CD28抗體刺激T細(xì)胞活化后，在24h，48h，72h分別加入8H9抗體并交聯(lián)，觀察缺失BTLA信號(hào)對(duì)T細(xì)胞活化抑制的減弱程度。 【結(jié)果】BTLA在T細(xì)胞上組成性高表達(dá)，活化后表達(dá)有所降低，然后迅速回升；CTLA-4在靜止T細(xì)胞上不表達(dá)，PD-1的表達(dá)水平很低，二者都是活化后被誘導(dǎo)表達(dá)。激發(fā)型CD3抗體和CD28抗體聯(lián)合8H9刺激T細(xì)胞后，CD25和CD69的表達(dá)均受到抑制。激發(fā)型CD3抗體和CD28抗體聯(lián)合8H9或者HVEM-Fc刺激后，在刺激后的1d，2d，3d，4d檢測(cè)，均對(duì)T細(xì)胞有抑制作用。同時(shí)，8H9對(duì)CD3單抗聯(lián)合CD28單抗刺激的T細(xì)胞分泌的IL-2和IFN-γ具有抑制效應(yīng)。CD3抗體和CD28抗體預(yù)先活化T細(xì)胞24h，48h，72h后，再加入8H9抗體仍然具有抑制功能，但不如在T細(xì)胞活化之初加入8H9的效應(yīng)。 【結(jié)論】BTLA可以在T細(xì)胞活化的啟動(dòng)和早期階段發(fā)揮重要的負(fù)性調(diào)節(jié)作用，從而抬高T細(xì)胞活化的閾值。
[Abstract]:BTLA is found in 2003 the members of the Ig family, is similar to the CTLA-4 and PD-1 inhibitory receptors. The BTLA gene is located on chromosome 3q13.2 with 48% homology to.BTLA mRNA high expression in spleen and lymph node tissues in the kidney, liver, lung, brain and heart in mice, expression and muscle tissue volume is very low, or no expression of.BTLA was widely expressed in T cells, B cells, macrophages, dendritic cells and NK cell surface.
BTLA contains a signal sequence, a variable region of immunoglobulin (IgV) extracellular sample, there are 3 important motifs containing tyrosine residues in transmembrane and about 100 amino acid cytoplasmic.BTLA cytoplasmic region: potential Grb2 binding sites, immunoreceptor tyrosine inhibitory motifs and the immune receptor tyrosine conversion motif. The study shows that the combination of BTLA and its ligand HVEM can inhibit the proliferation and cytokine secretion of T cells, delay the progression of the cell cycle and inhibit or terminate the immune response. In the continuous stimulation of antigen, expression of BTLA highly selective in anergic CD4+T cells, suggesting that BTLA signal is an important role in the process of induction and maintenance of T cell anergy. In addition, BTLA not only play an important role in sustaining peripheral tolerance, but also can prevent the occurrence of autoimmune diseases, termination of inflammatory reaction, inhibition of graft immune response.
In the initial BTLA and activated T cells have the expression, therefore, Murphy put forward the various stages of the BTLA activation in T cells especially the early stage play a role. This study first analyzes the existing BTLA-HVEM CIS complex expression level detection of cell surface BTLA molecules, and study the regulation of initiation and activation of BTLA in the early stage of T cells.
The effect of BTLA-HVEM CIS complex on the detection of BTLA molecules on the surface of cells
[Objective] the co expression of BTLA and HVEM on the surface of T cells and U937 cells was investigated. The effect of BTLA-HVEM CIS complex on the detection of BTLA molecules on the cell surface was investigated.
[method] isolated from human peripheral blood mononuclear cells were isolated and purified by negative selection using T cells. By using the method of flow cytometry, developed by different anti BTLA site commercial monoclonal antibody MIH26 and monoclonal antibody 8H9 were detected in the laboratory detection of BTLA molecular level.293T/BTLA gene transfected cells on the expression of U937 using 8H9 monoclonal antibody and T cell BTLA cell surface molecules.
[results] the commercialization of monoclonal antibody MIH26 (0.5 g) to detect the expression of BTLA molecule on T cells and U937 cells were more than 90%, while the 8H9 monoclonal antibody (1 g) to detect the expression level of two were 42.2%, 30.8%. by 0.02 g and 0.05 g 8H9 monoclonal antibody 293T/BTLA transgenic fine cell lines showed that the expression level of BTLA molecules were 80% and 91.4%.
[Conclusion] the co expression of BTLA and HVEM on T and U937 cells forms CIS complex, which affects the detection of BTLA by 8H9, but does not affect the detection of MIH26.
Two, BTLA regulates the activation and early stages of T cell activation
[Objective] to observe the expression of BTLA in the activation of T cells, and to explore the inhibition of the activation of T cells in various stages, especially in the early stage.
[method] isolated from human peripheral blood mononuclear cells, purified T cells by Macs. Agonist CD3 and CD28 antibody stimulated the activation of T cells, flow cytometry was used to detect BTLA, CTLA-4 and PD-1 in T cell activation in the dynamic process. The expression of agonist CD3 and CD28 antibody in join the BTLA mAb 8H9 under the condition of stimulated T cell activation, activation of T cells was detected by flow cytometry the expression of early marker CD25 and CD69. Agonist CD3 and CD28 antibody combined with HVEM-Fc or BTLA mAb 8H9, to observe the effect of BTLA signal on T cell proliferation in different time after activation. At the same time, the influence of ELISA method for detecting 8H9 of CD3 monoclonal antibody combined with CD28 mAb stimulated T cells secreting IL-2 and IFN- gamma. Advance with CD3 and CD28 antibodies to stimulate T cell activation, in 24h, 48h, 8H9 and 72h were added to the antibody cross-linking, observe the deletion of BTLA signal to T cells The degree of attenuation of activation inhibition.
[results] BTLA of high expression in T cells after activation, expression decreased, and then quickly rebounded; CTLA-4 was not expressed in resting T cells, the expression level of PD-1 is very low, the two are activated are induced. Combined with 8H9 agonist CD3 and CD28 antibody stimulated T cells. The expression of CD25 and CD69 were suppressed. Agonistic CD3 antibody and CD28 antibody combined with 8H9 or HVEM-Fc stimulation, after stimulation of 1D, 2D, 3D, 4D detection, all have inhibitory effect on T cells. At the same time, the secretion of 8H9 CD3 monoclonal antibody and CD28 monoclonal antibody of T cells stimulated by IL-2 and IFN- with gamma the inhibitory effect of.CD3 antibody and CD28 antibody pre activated T cells 24h, 48h, 72h after adding 8H9 antibody has inhibiting function, but not in T cell activation at the beginning of accession to the 8H9 effect.
[Conclusion] BTLA can play an important negative regulatory role in the initiation and early stages of T cell activation, thus raising the threshold of activation of T cells.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 嚴(yán)俊,姚X,周瑤璽;T細(xì)胞活化信號(hào)傳遞的分子基礎(chǔ)[J];國(guó)際免疫學(xué)雜志;1993年04期

2 高靜;與T細(xì)胞有關(guān)的惡性增生[J];國(guó)際免疫學(xué)雜志;1991年04期

3 徐紅梅;TGF-β通過(guò)下調(diào)MyD88表達(dá)抑制LPS誘導(dǎo)的TLR4活化[J];中國(guó)腫瘤生物治療雜志;2005年03期

4 楊琳;郭明秋;;成熟和衰老T細(xì)胞活化后鈣信號(hào)的研究進(jìn)展[J];中國(guó)免疫學(xué)雜志;2006年02期

5 筱雅;;五味子對(duì)人上皮細(xì)胞中嗜酸細(xì)胞活化趨化因子分泌和嗜酸粒細(xì)胞遷移的抑制作用[J];現(xiàn)代藥物與臨床;2010年01期

6 吳丹丹;汪慧英;;CD69在細(xì)胞活化、凋亡中的雙向免疫調(diào)節(jié)作用[J];中國(guó)病理生理雜志;2010年09期

7 ;免疫細(xì)胞生物學(xué)[J];國(guó)外科技資料目錄(醫(yī)藥衛(wèi)生);1997年12期

8 Frijns C.J.M.;Kasius K.M.;Algra A.;張家墅;;蛛網(wǎng)膜下腔出血患者遲發(fā)性腦缺血與內(nèi)皮細(xì)胞活化標(biāo)記物的關(guān)系[J];世界核心醫(yī)學(xué)期刊文摘(神經(jīng)病學(xué)分冊(cè));2006年12期

9 張東葵 ,趙德容;人殺傷性T細(xì)胞活化因子[J];國(guó)際免疫學(xué)雜志;1989年01期

10 王鶴濱;;細(xì)胞活化專家維生素B_3[J];科學(xué)養(yǎng)生;2010年04期

相關(guān)會(huì)議論文 前10條

1 孫大康;李柏青;;PKCθ在結(jié)核桿菌抗原誘導(dǎo)人γδT細(xì)胞NF-κB活化中的作用[A];中國(guó)免疫學(xué)會(huì)第五屆全國(guó)代表大會(huì)暨學(xué)術(shù)會(huì)議論文摘要[C];2006年

2 薛興奎;孫文佶;胡潔;姚航平;蔡真;;單純皰疹病毒胸苷激酶基因轉(zhuǎn)染對(duì)T細(xì)胞活化和亞群分布的影響[A];2006年浙江省血液病學(xué)學(xué)術(shù)年會(huì)論文匯編[C];2006年

3 王兆豐;邵平揚(yáng);陳松勁;;單核細(xì)胞-血小板黏附、單核細(xì)胞活化功能與圍術(shù)期炎性反應(yīng)的關(guān)系[A];2007年浙江省醫(yī)學(xué)檢驗(yàn)學(xué)學(xué)術(shù)年會(huì)論文匯編[C];2007年

4 陽(yáng)芳;陳翔;粟娟;匡葉紅;常靜;;CD147在對(duì)親環(huán)素A誘導(dǎo)Jurkat細(xì)胞活化、增殖、趨化和基質(zhì)粘附過(guò)程中的作用[A];中華醫(yī)學(xué)會(huì)第14次全國(guó)皮膚性病學(xué)術(shù)年會(huì)論文匯編[C];2008年

5 周琳;吳洪坤;張玲珍;仲人前;;強(qiáng)直性脊柱炎患者外周血中B淋巴細(xì)胞亞群、B細(xì)胞活化因子及其受體的表達(dá)檢測(cè)[A];中華醫(yī)學(xué)會(huì)第九次全國(guó)檢驗(yàn)醫(yī)學(xué)學(xué)術(shù)會(huì)議暨中國(guó)醫(yī)院協(xié)會(huì)臨床檢驗(yàn)管理專業(yè)委員會(huì)第六屆全國(guó)臨床檢驗(yàn)實(shí)驗(yàn)室管理學(xué)術(shù)會(huì)議論文匯編[C];2011年

6 章曉聯(lián);陳海丹;余剛;周霞;;C1GalT小分子干擾RNA能抑制CD8+T細(xì)胞活化并延長(zhǎng)皮膚移植存活[A];2008年全國(guó)糖生物學(xué)學(xué)術(shù)會(huì)議論文摘要[C];2008年

7 張鳳;儲(chǔ)以微;熊思東;;肝素抗炎機(jī)制研究——肝素抑制T細(xì)胞活化的機(jī)制研究[A];第六屆全國(guó)免疫學(xué)學(xué)術(shù)大會(huì)論文集[C];2008年

8 肖波;黃R

本文編號(hào)：1715204