鼠抗人CXCR4单克隆抗体的研制及其对肿瘤细胞和T细胞生物学作用的研究

发布时间：2018-05-20 10:18

本文选题：CXCR4/SDF-la信号 + 单克隆抗体　；参考：《苏州大学》2009年博士论文

【摘要】： CXCR4/SDF-1α是目前最受关注的一对趋化因子配体和受体,相关研究揭示它们参与了细胞的生长、发育、分化和增殖等多种生理功能,并在多种病理过程中发挥重要作用。CXCR4/SDF-1α为人们广为熟知的生物学功能是在炎症反应中趋化各种炎症细胞到损伤部位,参与了炎症细胞的滚动、黏附和跨内皮迁移等一系列过程。许多研究表明,肿瘤转移和炎症细胞浸润有着相似的形成过程,如均涉及到细胞滚动、黏附和跨内皮迁移等。也有研究报道,肿瘤细胞可以限定性表达某些趋化因子或趋化因子受体,并且存在趋化因子信号途径异常的状态,提示趋化因子或其受体可能通过与炎症细胞浸润相似的机制参与了肿瘤的转移。目前CXCR4/SDF-1α参与多种肿瘤如乳腺癌、肺癌、前列腺癌的转移和扩散已见报道。近来的还研究发现,在有效的免疫应答中,SDF-1α不仅能趋化T细胞到次级淋巴器官,而且对T细胞的活化还具有协同刺激作用。Nanki研究发现,在风湿性关节炎的关节腔内有大量的记忆性CD4~+T的集聚,其细胞表面CXCR4的表达水平大大提高,关节腔内SDF-1α表达水平也大大提高,推测CXCR4/SDF-1α的相互作用可能既可以聚集T细胞,而且为可T细胞活化提供一个潜在的协同刺激信号。尽管目前已经发现CXCR4/SDF-1α有多种不同的生物学功能,并且推测它在行使不同的功能时依靠不同的的信号途径。但是这些功能的的作用机制,信号传导差异以及影响因素还需要进一步深入研究。本研究旨在通过CXCR4基因转染细胞株作为免疫原免疫小鼠研制获得鼠抗人CXCR4单克隆抗体,并以抗人CXCR4单抗为材料研究揭示人CXCR4/SDF-1α在胶质瘤细胞上表达的生物学意义和对T细胞的协同刺激作用。一、鼠抗人CXCR4单克隆抗体的研制及生物学特性的鉴定【目的】研制鼠抗人CXCR4单克隆抗体,为探讨人CXCR4/SDF-1α的生物学功能及其机制提供必备的物质手段。【方法】利用高表达人CXCR4分子的基因转染细胞株L929/CXCR4为免疫原,常规免疫BALB/C小鼠。采用B淋巴细胞杂交瘤融合技术研制鼠抗人CXCR4单克隆抗体,并以该基因转染细胞L929/CXCR4作为阳性筛选细胞,以转空质粒的对照细胞L929/Mock作为阴性筛选细胞。经间接免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养,获得特异分泌鼠抗人CXCR4分子单克隆抗体的杂交瘤细胞株;采用细胞核染色体计数、Ig亚型快速定性试纸法、竞争结合抑制以及Western blot等试验,对获得的杂交瘤细胞株及单克隆抗体进行生物学特性的鉴定;用间接免疫荧光法初步分析单抗对免疫细胞表达CXCR4的识别作用。【结果】成功获得2株持续、稳定分泌鼠抗人CXCR4单克隆抗体的杂交瘤细胞株,分别命名为6H7和7D4。核型分析显示,杂交瘤细胞株的染色体数目在100条以上,超过小鼠B细胞和SP2/0细胞的染色体数,表明为融合体;经过快速定性试纸分析显示,2株单抗轻链均为κ链,6H7重链为IgG1亚类,而7D7重链为IgG2a;Western blot分析结果显示,单抗7D4能与CXCR4分子特异性结合,形成阳性条带;单抗识别的抗原表位分析结果表明,单抗6H7和7D4与商品化的鼠抗人CXCR4单抗(克隆号12G5)识别不同的抗原表位。流式细胞仪检测结果表明,单抗6H7和7D4能检测到PBMCs亚群上CXCR4分子的表达。【结论】成功获得两株稳定分泌特异性鼠抗人CXCR4单抗的杂交瘤细胞株,两株单抗识别的抗原位点不同,与商品化单抗12G5识别的抗原位点也不同,是两株新型的抗人CXCR4单克隆抗体。这两株抗人CXCR4单抗的研制成功为探讨CXCR4表达特性、CXCR4/SDF-1α多种生物学功能及其机制提供必备的物质手段。二、CXCR4/SDF-1α信号促进胶质瘤细胞迁移和增殖及其单抗阻断作用的研究【目的】利用研制的CXCR4单抗,初步分析了CXCR4在多种肿瘤细胞株和胶质瘤组织上的表达,以及探讨了CXCR4/SDF-1α介导信号在单抗阻断和非阻断情况下对胶质瘤细胞株U251体外生物物学行为的影响。【方法】采用间接免疫荧光标记法分析肿瘤细胞株表面CXCR4分子的表达,用免疫组织化学法检测胶质瘤组织中CXCR4的表达。MTT法研究了SDF-1α刺激的胶质瘤细胞株U251体外增殖及单抗7D4的阻断作用。用趋化小室分析了CXCR4/SDF-1α信号对胶质瘤细胞U251的体外迁移作用及单抗的阻断作用。【结果】流式细胞仪分析结果,显示大多数肿瘤细胞株上都表达CXCR4分子,尤其是造血系统来源的肿瘤细胞株CXCR4表达较高,而在上皮源性的肿瘤细胞株M231、MCF-7和95D上表达相对偏低。免疫组化的结果表明,胶质瘤组织上检测到CXCR4的表达。增殖与抗体阻断实验结果表明,SDF-1α能够刺激U251体外增殖,并且有浓度依赖性;CXCR4特异性的单抗7D4能阻断SDF-1α的这一激发作用。迁移实验结果显示单抗7D4也能阻断SDF-1α诱导的U251细胞的体外迁移作用,表明研制的抗人CXCR4单抗7D4具有阻断作用。【结论】本研究分析并证实了CXCR4在肿瘤细胞株表面的广泛表达和在胶质瘤组织上表达;CXCR4/SDF-1α信号能够作用于胶质瘤细胞株的体外生长和转移,推测该信号与胶质瘤侵袭性生长有关。三、CXCR4/SDF-1α信号对T细胞协同刺激及其单抗阻断作用的研究【目的】利用所获得的抗人CXCR4单克隆抗体7D4和重组人SDF-1α,探讨CXCR4/SDF-1α相互作用对T细胞协同刺激作用。【方法】采用荧光单标或双标记法通过流式细胞术分析DC诱导成熟过程中细胞表面及PHA活化的CD4~+、CD8~+T淋巴细胞上的CXCR4的表达;用激发型抗人CD3单抗包板,重组人SDF-1α联合或不联合抗人CD28单抗处理,通过流式细胞术、MTT法和ELISA法分别测定了SDF-1α对T淋巴细胞活化、增殖及细胞因子分泌的作用;在此基础上,用激发型抗人CD3单抗包板,选用特定的SDF-1α浓度和CD28单抗联合处理,通过流式细胞术、MTT法和ELISA法分别测定单抗7D4对T淋巴细胞活化、增殖及细胞因子分泌的影响; 【结果】PHA活化条件下,CD4~+T细胞上CXCR4呈上调性表达,随后表达有所下降。而在CD8~+T细胞上没有明显的变化;CXCR4分子在单核细胞诱导至mDC过程中,呈现逐步上调性表达。SDF-1α单独作用时,并不能明显促进T细胞的体外增殖、活化和上调细胞因子的分泌,而与CD28信号协同作用时,SDF-1α则能促进T细胞的增殖和细胞因子的分泌,并且有一定的剂量依赖性。但是,对T细胞上CD25和CD69的表达无显著影响。单抗7D4可阻断SDF-1α对T细胞的协同刺激作用,并呈现一定的剂量依赖性。而且单抗7D4能不同程度地下调CD4~+T细胞上CD25和CD69的表达,但对CD8~+T上的CD25和CD69影响并不明显。【结论】以研制获得的抗人CXCR4单抗7D4和重组人SDF-1α为手段,揭示了CXCR4在免疫细胞上的调节性表达。同时,CXCR4/SDF-1α信号在与CD28信号共同作用时,对T细胞的活化具有协同刺激作用,单抗7D4能抑制SDF-1α的协同刺激作用。综上所述,本研究成功获得了两株稳定分泌特异性鼠抗人CXCR4单抗的杂交瘤细胞株,进一步对单抗的生物学特性进行了鉴定。在此基础上分析了PBMCs亚群上CXCR4的表达谱。通过对肿瘤表面CXCR4表达的检测和在胶质瘤细胞株上的功能分析,证实了CXCR4是肿瘤组织中广泛表达的分子,并且对胶质瘤细胞的增殖、侵袭和转移都有一定的作用。同时还发现CXCR4/SDF-1α信号在与CD28联合作用时,对T细胞的活化具有一定的协同刺激作用,研制的抗体对SDF-1α的协同刺激作用呈现出阻断效应。
[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.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

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