本文選題：CXCR4/SDF-la信號 + 單克隆抗體��； 參考：《蘇州大學(xué)》2009年博士論文

【摘要】： CXCR4/SDF-1α是目前最受關(guān)注的一對趨化因子配體和受體,相關(guān)研究揭示它們參與了細(xì)胞的生長、發(fā)育、分化和增殖等多種生理功能,并在多種病理過程中發(fā)揮重要作用。CXCR4/SDF-1α為人們廣為熟知的生物學(xué)功能是在炎癥反應(yīng)中趨化各種炎癥細(xì)胞到損傷部位,參與了炎癥細(xì)胞的滾動、黏附和跨內(nèi)皮遷移等一系列過程。許多研究表明,腫瘤轉(zhuǎn)移和炎癥細(xì)胞浸潤有著相似的形成過程,如均涉及到細(xì)胞滾動、黏附和跨內(nèi)皮遷移等。也有研究報道,腫瘤細(xì)胞可以限定性表達(dá)某些趨化因子或趨化因子受體,并且存在趨化因子信號途徑異常的狀態(tài),提示趨化因子或其受體可能通過與炎癥細(xì)胞浸潤相似的機(jī)制參與了腫瘤的轉(zhuǎn)移。目前CXCR4/SDF-1α參與多種腫瘤如乳腺癌、肺癌、前列腺癌的轉(zhuǎn)移和擴(kuò)散已見報道。 近來的還研究發(fā)現(xiàn),在有效的免疫應(yīng)答中,SDF-1α不僅能趨化T細(xì)胞到次級淋巴器官,而且對T細(xì)胞的活化還具有協(xié)同刺激作用。Nanki研究發(fā)現(xiàn),在風(fēng)濕性關(guān)節(jié)炎的關(guān)節(jié)腔內(nèi)有大量的記憶性CD4~+T的集聚,其細(xì)胞表面CXCR4的表達(dá)水平大大提高,關(guān)節(jié)腔內(nèi)SDF-1α表達(dá)水平也大大提高,推測CXCR4/SDF-1α的相互作用可能既可以聚集T細(xì)胞,而且為可T細(xì)胞活化提供一個潛在的協(xié)同刺激信號。 盡管目前已經(jīng)發(fā)現(xiàn)CXCR4/SDF-1α有多種不同的生物學(xué)功能,并且推測它在行使不同的功能時依靠不同的的信號途徑。但是這些功能的的作用機(jī)制,信號傳導(dǎo)差異以及影響因素還需要進(jìn)一步深入研究。 本研究旨在通過CXCR4基因轉(zhuǎn)染細(xì)胞株作為免疫原免疫小鼠研制獲得鼠抗人CXCR4單克隆抗體,并以抗人CXCR4單抗為材料研究揭示人CXCR4/SDF-1α在膠質(zhì)瘤細(xì)胞上表達(dá)的生物學(xué)意義和對T細(xì)胞的協(xié)同刺激作用。 一、鼠抗人CXCR4單克隆抗體的研制及生物學(xué)特性的鑒定 【目的】研制鼠抗人CXCR4單克隆抗體,為探討人CXCR4/SDF-1α的生物學(xué)功能及其機(jī)制提供必備的物質(zhì)手段。 【方法】利用高表達(dá)人CXCR4分子的基因轉(zhuǎn)染細(xì)胞株L929/CXCR4為免疫原,常規(guī)免疫BALB/C小鼠。采用B淋巴細(xì)胞雜交瘤融合技術(shù)研制鼠抗人CXCR4單克隆抗體,并以該基因轉(zhuǎn)染細(xì)胞L929/CXCR4作為陽性篩選細(xì)胞,以轉(zhuǎn)空質(zhì)粒的對照細(xì)胞L929/Mock作為陰性篩選細(xì)胞。經(jīng)間接免疫熒光標(biāo)記和流式細(xì)胞術(shù)分析、反復(fù)篩選和多次克隆化培養(yǎng),獲得特異分泌鼠抗人CXCR4分子單克隆抗體的雜交瘤細(xì)胞株;采用細(xì)胞核染色體計數(shù)、Ig亞型快速定性試紙法、競爭結(jié)合抑制以及Western blot等試驗,對獲得的雜交瘤細(xì)胞株及單克隆抗體進(jìn)行生物學(xué)特性的鑒定;用間接免疫熒光法初步分析單抗對免疫細(xì)胞表達(dá)CXCR4的識別作用。 【結(jié)果】成功獲得2株持續(xù)、穩(wěn)定分泌鼠抗人CXCR4單克隆抗體的雜交瘤細(xì)胞株,分別命名為6H7和7D4。核型分析顯示,雜交瘤細(xì)胞株的染色體數(shù)目在100條以上,超過小鼠B細(xì)胞和SP2/0細(xì)胞的染色體數(shù),表明為融合體;經(jīng)過快速定性試紙分析顯示,2株單抗輕鏈均為κ鏈,6H7重鏈為IgG1亞類,而7D7重鏈為IgG2a;Western blot分析結(jié)果顯示,單抗7D4能與CXCR4分子特異性結(jié)合,形成陽性條帶;單抗識別的抗原表位分析結(jié)果表明,單抗6H7和7D4與商品化的鼠抗人CXCR4單抗(克隆號12G5)識別不同的抗原表位。流式細(xì)胞儀檢測結(jié)果表明,單抗6H7和7D4能檢測到PBMCs亞群上CXCR4分子的表達(dá)。 【結(jié)論】成功獲得兩株穩(wěn)定分泌特異性鼠抗人CXCR4單抗的雜交瘤細(xì)胞株,兩株單抗識別的抗原位點(diǎn)不同,與商品化單抗12G5識別的抗原位點(diǎn)也不同,是兩株新型的抗人CXCR4單克隆抗體。這兩株抗人CXCR4單抗的研制成功為探討CXCR4表達(dá)特性、CXCR4/SDF-1α多種生物學(xué)功能及其機(jī)制提供必備的物質(zhì)手段。 二、CXCR4/SDF-1α信號促進(jìn)膠質(zhì)瘤細(xì)胞遷移和增殖及其單抗阻斷作用的研究 【目的】利用研制的CXCR4單抗,初步分析了CXCR4在多種腫瘤細(xì)胞株和膠質(zhì)瘤組織上的表達(dá),以及探討了CXCR4/SDF-1α介導(dǎo)信號在單抗阻斷和非阻斷情況下對膠質(zhì)瘤細(xì)胞株U251體外生物物學(xué)行為的影響。 【方法】采用間接免疫熒光標(biāo)記法分析腫瘤細(xì)胞株表面CXCR4分子的表達(dá),用免疫組織化學(xué)法檢測膠質(zhì)瘤組織中CXCR4的表達(dá)。MTT法研究了SDF-1α刺激的膠質(zhì)瘤細(xì)胞株U251體外增殖及單抗7D4的阻斷作用。用趨化小室分析了CXCR4/SDF-1α信號對膠質(zhì)瘤細(xì)胞U251的體外遷移作用及單抗的阻斷作用。 【結(jié)果】流式細(xì)胞儀分析結(jié)果,顯示大多數(shù)腫瘤細(xì)胞株上都表達(dá)CXCR4分子,尤其是造血系統(tǒng)來源的腫瘤細(xì)胞株CXCR4表達(dá)較高,而在上皮源性的腫瘤細(xì)胞株M231、MCF-7和95D上表達(dá)相對偏低。免疫組化的結(jié)果表明,膠質(zhì)瘤組織上檢測到CXCR4的表達(dá)。增殖與抗體阻斷實驗結(jié)果表明,SDF-1α能夠刺激U251體外增殖,并且有濃度依賴性;CXCR4特異性的單抗7D4能阻斷SDF-1α的這一激發(fā)作用。遷移實驗結(jié)果顯示單抗7D4也能阻斷SDF-1α誘導(dǎo)的U251細(xì)胞的體外遷移作用,表明研制的抗人CXCR4單抗7D4具有阻斷作用。 【結(jié)論】本研究分析并證實了CXCR4在腫瘤細(xì)胞株表面的廣泛表達(dá)和在膠質(zhì)瘤組織上表達(dá);CXCR4/SDF-1α信號能夠作用于膠質(zhì)瘤細(xì)胞株的體外生長和轉(zhuǎn)移,推測該信號與膠質(zhì)瘤侵襲性生長有關(guān)。 三、CXCR4/SDF-1α信號對T細(xì)胞協(xié)同刺激及其單抗阻斷作用的研究 【目的】利用所獲得的抗人CXCR4單克隆抗體7D4和重組人SDF-1α,探討CXCR4/SDF-1α相互作用對T細(xì)胞協(xié)同刺激作用。 【方法】采用熒光單標(biāo)或雙標(biāo)記法通過流式細(xì)胞術(shù)分析DC誘導(dǎo)成熟過程中細(xì)胞表面及PHA活化的CD4~+、CD8~+T淋巴細(xì)胞上的CXCR4的表達(dá);用激發(fā)型抗人CD3單抗包板,重組人SDF-1α聯(lián)合或不聯(lián)合抗人CD28單抗處理,通過流式細(xì)胞術(shù)、MTT法和ELISA法分別測定了SDF-1α對T淋巴細(xì)胞活化、增殖及細(xì)胞因子分泌的作用;在此基礎(chǔ)上,用激發(fā)型抗人CD3單抗包板,選用特定的SDF-1α濃度和CD28單抗聯(lián)合處理,通過流式細(xì)胞術(shù)、MTT法和ELISA法分別測定單抗7D4對T淋巴細(xì)胞活化、增殖及細(xì)胞因子分泌的影響; 【結(jié)果】PHA活化條件下,CD4~+T細(xì)胞上CXCR4呈上調(diào)性表達(dá),隨后表達(dá)有所下降。而在CD8~+T細(xì)胞上沒有明顯的變化;CXCR4分子在單核細(xì)胞誘導(dǎo)至mDC過程中,呈現(xiàn)逐步上調(diào)性表達(dá)。SDF-1α單獨(dú)作用時,并不能明顯促進(jìn)T細(xì)胞的體外增殖、活化和上調(diào)細(xì)胞因子的分泌,而與CD28信號協(xié)同作用時,SDF-1α則能促進(jìn)T細(xì)胞的增殖和細(xì)胞因子的分泌,并且有一定的劑量依賴性。但是,對T細(xì)胞上CD25和CD69的表達(dá)無顯著影響。單抗7D4可阻斷SDF-1α對T細(xì)胞的協(xié)同刺激作用,并呈現(xiàn)一定的劑量依賴性。而且單抗7D4能不同程度地下調(diào)CD4~+T細(xì)胞上CD25和CD69的表達(dá),但對CD8~+T上的CD25和CD69影響并不明顯。 【結(jié)論】以研制獲得的抗人CXCR4單抗7D4和重組人SDF-1α為手段,揭示了CXCR4在免疫細(xì)胞上的調(diào)節(jié)性表達(dá)。同時,CXCR4/SDF-1α信號在與CD28信號共同作用時,對T細(xì)胞的活化具有協(xié)同刺激作用,單抗7D4能抑制SDF-1α的協(xié)同刺激作用。 綜上所述,本研究成功獲得了兩株穩(wěn)定分泌特異性鼠抗人CXCR4單抗的雜交瘤細(xì)胞株,進(jìn)一步對單抗的生物學(xué)特性進(jìn)行了鑒定。在此基礎(chǔ)上分析了PBMCs亞群上CXCR4的表達(dá)譜。通過對腫瘤表面CXCR4表達(dá)的檢測和在膠質(zhì)瘤細(xì)胞株上的功能分析,證實了CXCR4是腫瘤組織中廣泛表達(dá)的分子,并且對膠質(zhì)瘤細(xì)胞的增殖、侵襲和轉(zhuǎn)移都有一定的作用。同時還發(fā)現(xiàn)CXCR4/SDF-1α信號在與CD28聯(lián)合作用時,對T細(xì)胞的活化具有一定的協(xié)同刺激作用,研制的抗體對SDF-1α的協(xié)同刺激作用呈現(xiàn)出阻斷效應(yīng)。
[Abstract]:CXCR4/SDF-1 alpha is one of the most concerned chemokine ligands and receptors. The related research reveals that they are involved in many physiological functions, such as cell growth, development, differentiation and proliferation, and play an important role in various pathological processes..CXCR4/SDF-1 A is widely known for its biological function in the inflammatory response. A number of studies have shown that tumor metastasis and inflammatory cell infiltration have a similar formation process, such as cell rolling, adhesion and transendothelial migration. Factor or chemokine receptor, and there is an abnormal state of chemokine signaling pathway, suggesting that chemokine or its receptor may participate in tumor metastasis through a mechanism similar to inflammatory cell infiltration. CXCR4/SDF-1 alpha is currently involved in the metastasis and diffusion of a variety of tumors such as breast cancer, lung cancer, prostaglandin adenocarcinoma.
Recent studies have also found that in an effective immune response, SDF-1 alpha not only converges T cells to secondary lymphoid organs, but also has synergistic stimulation of the activation of T cells. It is found that a large number of memory CD4~+T concentrations in the articular cavity of rheumatoid arthritis are gathered, and the expression level of CXCR4 on the surface of the cells is greatly improved. The expression level of SDF-1 alpha in the lumen is also greatly improved. It is speculated that the interaction of CXCR4/SDF-1 alpha may not only accumulate T cells, but also provide a potential synergistic stimulation signal for the activation of T cells.
Although there are many different biological functions of CXCR4/SDF-1 alpha, it is speculated that it relies on different signaling pathways in the exercise of different functions. However, the mechanism of the function, the difference of signal transmission and the influencing factors need to be further studied.
The aim of this study was to obtain mouse anti human CXCR4 monoclonal antibodies by using CXCR4 gene transfected cell lines as immunogenic immune mice. The study revealed the biological significance of the expression of human CXCR4/SDF-1 alpha on glioma cells and the synergistic stimulation of T cells by anti human CXCR4 monoclonal antibody.
Preparation of a mouse anti human CXCR4 monoclonal antibody and identification of its biological characteristics
[Objective] to prepare mouse anti human CXCR4 monoclonal antibody and provide essential material for exploring the biological function and mechanism of human CXCR4/SDF-1 alpha.
[Methods] using the gene transfected cell line L929/CXCR4 of high surface CXCR4 molecule as immunogen and immunization with BALB/C mice, the mouse anti human CXCR4 monoclonal antibody was developed by B lymphocyte hybridoma fusion technique, and the transfected cell L929/CXCR4 was used as the positive screening cell, and the control cell L929/Mock was used as the Yin of the empty plasmid. Through indirect immunofluorescence labeling and flow cytometry analysis, repeated screening and multiple cloning, the hybridoma cell lines that specifically secreted the monoclonal antibodies against human CXCR4 molecules were obtained. The results were obtained by using the cell nuclear chromosome count, Ig subtype rapid qualitative test paper, competition binding inhibition and Western blot test. The biological characteristics of hybridoma cell lines and monoclonal antibodies were identified, and indirect immunofluorescence was used to identify the role of McAbs for the identification of CXCR4 in immune cells.
[results] 2 hybridoma cell lines, which secrete the anti human CXCR4 monoclonal antibody, were successfully obtained. The karyotype analysis of 6H7 and 7D4. showed that the number of chromosomes of the hybridoma cell lines was more than 100, exceeding the number of chromosomes of B and SP2/0 cells in mice, indicating that the number of chromosomes of the mouse and SP2/0 cells was a fusion body. The light chain of 2 monoclonal antibodies was kappa chain, 6H7 heavy chain was IgG1 subclass and 7D7 heavy chain was IgG2a, and Western blot analysis showed that McAb 7D4 could combine with CXCR4 molecules to form positive bands. The antigen epitope analysis of monoclonal antibody identified that the monoclonal antibody 6H7 and 7D4 are different from the commercialized mouse anti human CXCR4 monoclonal antibody (clone number 12G5). The results of flow cytometry showed that McAb 6H7 and 7D4 could detect the expression of CXCR4 molecules on PBMCs subsets.
[Conclusion] two hybridoma cell lines that secrete the specific anti human CXCR4 monoclonal antibody were successfully obtained. The antigen loci identified by two monoclonal antibodies were different, and the antigen loci identified by commercial monoclonal antibody 12G5 were different, and two new anti human CXCR4 monoclonal antibodies. The successful development of these two anti human CXCR4 monoclonal antibodies was to explore the expression characteristics of CXCR4. CXCR4/SDF-1 alpha provides essential material means for various biological functions and mechanisms.
Two, CXCR4/SDF-1 alpha signaling promotes the migration and proliferation of glioma cells and the blocking effect of McAb.
[Objective] to preliminarily analyze the expression of CXCR4 in a variety of tumor cell lines and glioma tissues by using the developed CXCR4 monoclonal antibody, and to explore the effect of CXCR4/SDF-1 alpha mediated signal on the in vitro biological physical behavior of glioma cell line U251 in the case of monoclonal antibody blocking and non blocking.
[Methods] the expression of CXCR4 molecules on the surface of tumor cell lines was analyzed by indirect immunofluorescence. The expression of CXCR4 in glioma tissues was detected by immunohistochemical staining and.MTT method was used to study the proliferation of SDF-1 alpha stimulated glioma cell line U251 in vitro and the blocking effect of monoclonal antibody 7D4. The CXCR4/SDF-1 alpha signal was analyzed by the chemotactic chamber. The migration of U251 in vitro and the blocking effect of McAb.
[results] the results of flow cytometry showed that most of the tumor cell lines expressed CXCR4 molecules, especially the tumor cell lines derived from the hematopoietic system, CXCR4, which were relatively low in the epithelial derived tumor cell lines, M231, MCF-7 and 95D. The immunohistochemical results showed that the CXCR4 was detected in the glioma tissue. The results of proliferation and antibody blocking showed that SDF-1 alpha could stimulate the proliferation of U251 in vitro, and had a concentration dependence; CXCR4 specific monoclonal antibody 7D4 could block the excitation of SDF-1 alpha. The migration experiment showed that McAb 7D4 could also block the in vitro migration of U251 cells induced by SDF-1 alpha, indicating the development of anti human CXCR4 McAb 7D4. It has a blocking effect.
[Conclusion] this study analyses and confirms the extensive expression of CXCR4 on the surface of the tumor cell lines and the expression on the glioma tissue, and the CXCR4/SDF-1 alpha signal can act on the growth and metastasis of glioma cell lines in vitro. It is presumed that the signal is related to the invasive growth of glioma.
Three, the effect of CXCR4/SDF-1 alpha signal on T cell co stimulation and its blocking effect on McAb
[Objective] to explore the synergistic effect of CXCR4/SDF-1 alpha interaction on T cells by using the anti human CXCR4 monoclonal antibody 7D4 and recombinant human SDF-1 alpha.
[Methods] the fluorescence single or double labeling method was used to analyze the expression of CXCR4 on the cell surface and the PHA activated CD4~+, the CD8~+T lymphocyte in the DC induced maturation process by flow cytometry, and the recombinant human CD3 mono antibody cladding, recombinant human SDF-1 alpha combined or not combined with the anti human CD28 single resistance treatment, through flow cytometry, MTT method and ELISA method. The effects of SDF-1 alpha on activation, proliferation and cytokine secretion of T lymphocytes were measured respectively. On this basis, the activation, proliferation and cytokine secretion of McAb were measured by flow cytometry, MTT and ELISA methods, using specific SDF-1 alpha concentration and CD28 monoclonal antibody combined with stimulated anti human CD3 monoclonal antibody cladding. Influence;
[results] under the activation of PHA, the expression of CXCR4 on CD4~+T cells was up-regulated and then decreased, but there was no obvious change on CD8~+T cells. The CXCR4 molecule could not obviously promote the proliferation, activation and up-regulation of T cells in the process of monocyte induction to mDC during the process of gradually up regulation of.SDF-1 a. In conjunction with CD28 signal, SDF-1 alpha can promote the proliferation of T cells and the secretion of cytokines, and has a dose dependence. However, there is no significant effect on the expression of CD25 and CD69 on T cells. McAb 7D4 can block the synergistic stimulation of SDF-1 a to T cells, and present a certain dose dependence. McAb 7D4 could regulate the expression of CD25 and CD69 on CD4~+T cells in different degrees, but had little effect on CD25 and CD69 on CD8~+T.
[Conclusion] the anti human CXCR4 monoclonal antibody 7D4 and recombinant human SDF-1 alpha have been developed to reveal the regulatory expression of CXCR4 on the immune cells. At the same time, the CXCR4/SDF-1 alpha signal has a synergistic stimulation effect on the activation of T cells in conjunction with the CD28 signal, and the McAb 7D4 can inhibit the synergistic stimulation of SDF-1 alpha.
To sum up, two hybridoma cell lines that secrete the specific anti human CXCR4 monoclonal antibody were successfully obtained, and the biological characteristics of McAbs were further identified. On this basis, the expression profiles of CXCR4 on the PBMCs subgroup were analyzed. The detection of the CXCR4 expression on the tumor surface and the function analysis on the glioma cell line were analyzed. It is confirmed that CXCR4 is a widely expressed molecule in tumor tissue and plays a role in the proliferation, invasion and metastasis of glioma cells. At the same time, it is found that CXCR4/SDF-1 alpha signal has a synergistic stimulation effect on the activation of T cells in combination with CD28, and the synergistic stimulation of SDF-1 alpha is blocked by the developed antibody. Effect.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392

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相關(guān)期刊論文 前1條

1 邱玉華，張學(xué)光，謝煒，，朱學(xué)東;一種顯著提高小鼠生產(chǎn)單抗腹水產(chǎn)量的新方法[J];中國免疫學(xué)雜志;1995年06期

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