【作者】 成中芹；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2009， 博士

【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。更多还原

【Abstract】 More attention was paid to CXCR4/SDF-1α,a pair of chemokine receptor and ligand currently.They are not only involved in cell growth,development,differentiation and proliferation,but also in a variety of pathological processes,such as cancer metastasis and inflammation.To be widely known,CXCR4/SDF-1αsignal regulates trafficking of leukocyte subsets to inflammatory sites through chemoattraction and by activating leukocyte integrins to bind their adhesion receptors on endothelial cells in the inflammatory response.Many studies had shown that tumor metastasis was similar with inflammatory cell infiltration,and cell rolling,adhesion and trans-endothelial migration are involved. More studies had reported that some tumor cells could express certain chemokines or chemokine receptors,and some chemokine signaling pathways were abnormal in tumor cells,suggesting that chemokine or its receptor may be involved in tumor metastasis by a similar mechanism of inflammatory cell infiltration.Currently CXCR4/SDF-1αsignal involved in cancer metastasis and proliferation,such as breast,lung,prostate cancer had been reported.Recently,it was found that in an effective immune response,CXCR4 /SDF-1αsignal not only chemotaxis T cells to the secondary lymphoid organs,but also had co-stimulatory effect on activation of T cells.This study aimed prepare mouse anti-human CXCR4 monoclonal antibody with CXCR4 gene transfected cell line(L929/CXCR4) as immunogen,and illustratedCXCR4/SDF-1αeffect on T cells as a co-stimulatory signal,and revealed biological significance of CXCR4/SDF-1αon glioma cells.PartⅠPreparation of mouse anti-human CXCR4 monoclonal antibodies and analysis of their biological characteristicsObjective:To prepare mouse anti-human CXCR4 monoclonal antibody for investigating CXCR4/SDF-1αbiological functions in vivo and the mechanisms.Methods:BALB/c mice were immunized with human CXCR4 transfectant L929/CXCR4 as an immungen.Mouse spleen B cells were fused with mouse plasmocytoma cells SP2/0.Hybridoma cells were screened with the transfectant L929/CXCR4.Fast-strip analysis was performed to identify Ig subclass of the generated monoclonal antibodies.The methods of immunophenotyping and western blot were designed to identify the specificity of generated mAbs.The epitopes recognized by mAbs were analyzed by competition assay.The expression of CXCR4 on subgroups of PBMCs was detected by FCM.Results:After multiple screening and subcloning,two monoclonal antibodies named 6H7 and 7D4 were obtained.Immunophenotyping showed that the two mAbs could bind to L929/CXCR4 transfected cells,but not to L929/mock,L929/CD133 and L929/HVEM cells,indicating that the two antibodies are specific for human CXCR4. The isotype of mAb 6H7 was proved to be IgG1 withκlight chain,while mAb 7D4 was IgG2a withκlight chain.Western blot showed mAb 7D4 could specifically bind to CXCR4 line-like molecule extracted from L929/CXCR4 cells,while 6H7 could not. The competition test conducted among the two mAbs evidenced that these mAbs were completely specific to different antigen binding sites of CXCR4.It was revealed that both mAbs could recognize CXCR4 in subgroups of PBMCs.Conclusion:Two anti-human CXCR4 monoclonal antibodies were generated, which recognize different epitopes.The successful preparation of mouse anti-human CXCR4 monoclonal antibody also provided materials to reveal CXCR4/SDF-1αbiological functions and the mechanisms. PartⅡProliferation and migration about glioma cell line mediated by CXCR4/SDF-1αsignal and blocking of monoclonal antibodyObjective:Preliminary analysis of CXCR4 expression on a few of tumor cell lines and glioma tissue and investigation about CXCR4/SDF-1αsignal biological function on glioma cell lines by monoclonal antibody against human CXCR4 preparedMethods:The expression of CXCR4 on tumor cell lines was analyzed by FCM with indirect immunofluorescence labeling.Immunohistochemistry was used to detect CXCR4 expression in glioma tissue.The proliferation of U251 cells stimulated with SDF-1αwas analyzed by MTT method.Blocking effect of 7D4 on proliferation of U251 cells was analyzed by MTT method.Migration of U251 glioma cells mediated by CXCR4/SDF-1αsignal was investigated with transwell chambers.Results:FCM analysis revealed that CXCR4 was expressed on the majority of tumor cell lines.In particular,CXCR4 expressed higher on tumor cell lines from hematopoietic system,while CXCR4 expressed relatively low on umor cell lines from epithelial-derived,such as M231,MCF-7 and 95D.Immunohistochemistry results showed CXCR4 expression on glioma tissue.Proliferation results of U251 cell line showed that,SDF-1αcould stimulate the proliferation of U251 with a concentration-dependent manner in vitro.CXCR4-specific monoclonal antibody 7D4 could block Proliferation of U251 cell line stimulated by SDF-1α.The transwell chambers results showed that monoclonal antibody 7D4 could block migration of U251 cells by SDF-1α-mediated.Conclusion:CXCR4 expression in a wide range of tumor cell lines and glioma tissue were analyzed and confirmed.CXCR4/SDF-1αsignals could affected glioma cell line U251 growth and metastasis in vitro,suggesting that the signal was related to the invasive growth pattern of glioma in vivoPartⅢCo-stimulatory function of human CXCR4 on T cells and the intervention effect of monoclonal antibodyObjective:To investigate CXCR4/SDF-1αco-stimulatory function on the by mAb and recombinant human SDF-1α,on the base of analysis of CXCR4 expression in immune cells.Methods:The expression of CXCR4 on subgroups of T cell stimulated with PHA was respectively analyzed by FCM.Human monocytes were cultured in the medium containing GM-CSF and IL-4 to be induced to imDCs and further to develop into mDCs driven by LPS.In this course CXCR4 expression was analyzed by FCM.With or without anti-CD28 mAb,human T cells were stimulated with anti-CD3 mAb in presence of SDF-1α,then the activation was detected by FCM,the proliferation of T cells was analyzed by MTT method,and the production of cytokines was measured by ELISA method.Furthermore,blocking function of monoclonal antibody 7D4 on co-stimulatory induced by SDF-1αwas detected.Results:When T cells were stimulated by PHA,CXCR4 expression on CD4~+T increased gently at the beginning and then decreased from 72 h.,while CXCR4 expression on CD8~+T showed no significant changes.In the process of monocytes being induced to DCs,CXCR4 expression was up-regulated.It was demonstrated that SDF-1αcould stimulate T cell proliferation and cytokines secretion,such as IL-2,IL-10 and IFN-γwith a dose dependent manner when anti-CD28 mAb was present,while it showed not without anti-CD28 mAb.With mAb 7D4, upregulation of proliferation and cytokine production mediated by SDF-1αwas suppressed.Even,CD25 and CD69 expression was decreased by mAb 7D4.Conclusion:By means of mAb 7D4,it was revealed that CXCR4 expression was regulated on immune cells.CXCR4/SDF-1αsignals together with CD28 signals on T cell activation had a co-stimulatory effect,mAb 7D4 could block CXCR4/SDF-1αsignals co-stimulatory effect.In summary,this paper has successfully accomplished the investigations as follows: Using the transfectant L929/CXCR4 as immungen,two specific anti-human CXCR4 monoclonal antibodies were generated.Further investigations indicated CXCR4 was expressed on the majority of tumor cell lines,and CXCR4/SDF-1αsignals could promoted glioma growth and migration in vitro,which suggested participation of the signal in the invasive growth pattern of glioma in vivo.Besides,Co-stimulatory function of human CXCR4 on T cells and the effect of monoclonal antibody were explored.It was proved that CXCR4/SDF-1αstimulated T cell proliferation and cytokines(IL-2, IL-10 and IFN-γ) secretion with a dose dependent manner when anti-CD28 mAb was present,while mAb 7D4 could block CXCR4/SDF-1αsignals co-stimulatory effect.更多还原