Cd28抗体与抗原结合的特性及介导的效应研究

发布时间：2018-03-07 22:02

本文选题：B7/CD28协同刺激信号　切入点：动物模型　出处：《苏州大学》2011年硕士论文　论文类型：学位论文

【摘要】：CD28分子的基因定位于人2号染色体q33-34区,由1120核苷酸组成,表达于大多数静止及活化的T细胞,约95% CD4+T和50% CD8+T细胞表达该分子。此外,部分NK细胞和人恶性浆细胞也表达CD28分子。B7-(1CD80)和B7-(2CD86)是CD28的天然配体,CD28分子与表达在抗原呈递细胞表面的相应配体B7-1和B7-2结合,从而产生T细胞应答所需的协同刺激信号。B7-CD28信号并非仅局限性地产生于抗原呈递细胞与T细胞之间,已在某些肿瘤细胞如多发性骨髓瘤细胞,白血病细胞等也发现异常的B7-CD28信号通路,且该通路可能参与肿瘤的恶性增殖与转移。通过特异性抗体封闭CD28分子,有可能对肿瘤细胞的生长发挥抑制作用。本研究旨在运用自行制备的CD28抗体,以转染人CD28基因的小鼠T淋巴瘤细胞株及天然表达CD28分子的人白血病细胞株Jurkat为研究对象,在体外分析抗体对上述细胞生长与增殖影响的基础上,通过建立肿瘤模型,探讨抗体对肿瘤细胞的成瘤及肿瘤消长的影响,以期寻找具有抗瘤作用的生物制剂。【目的】制备鼠抗人CD28分子单克隆抗体并鉴定其生物学功能。【方法】采用本室建立的腹水诱生法制备单抗并经Protein G亲和层析分离纯化。间接免疫荧光法分析单克隆抗体效价;FCM分析单克隆抗体识别的抗原位点;FCM分析单克隆抗体对不同细胞株人多发性骨髓瘤细胞(U266)、小鼠淋巴瘤细胞转入CD28基因细胞株(CD28-T)和人白血病细胞株(Jurkat)膜型CD28分子的识别。【结果】腹水形成率为90%以上,收获量为5ml/只以上。腹水中抗体蛋白的得率达3mg/ml。纯化抗体用于间接免疫荧光检测的用量为0.5μg/5×105。竞争抑制试验表明该单抗能完全阻断标准鼠抗人CD28抗体与相应抗原的结合,提示其识别的抗原表位与对照抗体相同或相似。FCM分析表明,CD28 mAb能良好地识别多种细胞表面膜型CD28分子。【结论】制备了鼠抗人CD28分子mAb(命名为SQA-11),为探讨CD28信号转导在肿瘤生长与转移过程中的作用及机制奠定了物质基础。【目的】探讨SQA-11体内、外对CD28+肿瘤细胞的生长与增殖的影响作用,以期寻找具有抗瘤作用的生物制剂。【方法】MTT法体外分析SQA-11对CD28-T和Jurkat生长和增殖的影响;建立CD28-T与Jurkat细胞的Balb/c裸鼠的肿瘤模型,FCM分析成瘤小鼠瘤体细胞CD28分子的表达;将CD28-T与Jurkat分别经SQA-11预处理后再行建立肿瘤模型,观察成瘤时间和成瘤率,同时与未经抗体处理组进行比较。将肿瘤模型随机分组,采用瘤内注射法将SQA-11分2-3点、隔日一次共4次注入瘤内,抗体用量为50μg /mm3观察肿瘤的消长及荷瘤鼠的生存状态。【结果】MTT检测发现SQA-11能够明显抑制CD28-T和Jurkat细胞的体外增殖。CD28-T细胞于接种第3天即形成肉眼可见的肿瘤结节,且成瘤率为100%(40/40)。Jurkat细胞于接种第14天后陆续形成肉眼可见的肿瘤结节,成瘤率为80%(32/40)。对接种第40天的肿瘤结节中的细胞经FCM进行分析,CD28的阳性率分别为CD28-T 90.4%、Jurkat74.3%。与未接种前体外培养的细胞无明显差异。CD28-T与Jurkat细胞分别经SQA-11共孵育后接种小鼠,CD28-T的成瘤时间推迟2天即第5天形成肉眼可见的肿瘤结节,成瘤率仍为100%(20/20);Jurkat细胞的成瘤时间推迟5天即第19天开始形成肿瘤结节,成瘤率为50%(10/20)。将CD28-T(第7天)及Jurkat(第30天)肿瘤模型用SQA-11瘤内注射,观察40天。Jurkat模型组部分(3/10)裸鼠的肿瘤结节变小,平均体积由注射时的56mm3减为32mm3;对CD28-T模型组肿瘤结节的生长无抑制作用。【结论】SQA-11对天然表达CD28分子的人肿瘤具有一定的抑制作用;而对表达外源CD28分子的小鼠肿瘤无明显影响。
[Abstract]:Gene mapping of CD28 molecule on human chromosome 2 q33-34, composed of 1120 nucleotides, expressed in most static and activation of T cells, about 95% CD4+T and 50% CD8+T cells express this molecule. In addition, some NK cells and malignant plasma cells also express CD28 molecule.B7- (1CD80) and B7- (2CD86) is a natural the ligand CD28, and the expression of CD28 molecule on the surface of antigen-presenting cells and B7-1 ligand binding to B7-2, resulting in T cell response to costimulatory signal.B7-CD28 signal not only between the limitation of real estate was born in antigen-presenting cells and T cells, in some tumor cells such as multiple myeloma cells, leukemia cells also found that B7-CD28 signal pathway, tumor proliferation and metastasis and the pathway may be involved in tumor. The closed CD28 molecules by specific antibodies, may have on the growth of tumor cells play inhibition. This study To use the self prepared CD28 polyclonal antibodies, the mouse lymphoma cell line T cells and natural CD28 expression of human CD28 gene transfection of human leukemia cell line Jurkat as the research object, analyzing the effect of antibody on the growth and proliferation of the cells in vitro, through the establishment of tumor model, to investigate the effect of tumor growth and tumor antibody formation the tumor cells, in order to find the biological agents have antitumor effect.
[Objective] to prepare mouse anti human CD28 monoclonal antibody and identify its biological function.
[method] the relationship induced ascites and preparation of monoclonal antibodies by Protein G affinity chromatography. Analysis of monoclonal antibody titer by indirect immunofluorescence; FCM analysis of antigenic sites recognized by monoclonal antibody against FCM; monoclonal antibody analysis of different cell strains of human multiple myeloma cells (U266), CD28 gene cell line mouse lymphoma cells into (CD28-T) and human leukemia cell line (Jurkat) identification of membrane CD28 molecule.
[results] the ascites formation rate is above 90%, the harvest is 5ml/ or more. The antibody in ascites was purified 3mg/ml. antibody for indirect immunofluorescence detection was 0.5 g/5 * 105. competitive inhibition test showed that the monoclonal antibody could block with standard mouse anti human CD28 antibody and the corresponding antigen, suggesting that the identification of antigenic epitope and control antibody to the same or similar.FCM analysis showed that CD28 mAb can be a good way to identify a variety of cell surface CD28 molecules.
[Conclusion] the mouse anti human CD28 molecule mAb (named SQA-11) has been prepared, which lays a solid foundation for exploring the role and mechanism of CD28 signal transduction in the process of tumor growth and metastasis.
[Objective] to investigate the effect of SQA-11 in vivo and in vitro on the growth and proliferation of CD28+ tumor cells in order to find a biological agent with antitumor effect.
[method] MTT method in the analysis of effect of SQA-11 on growth and proliferation of CD28-T and Jurkat; CD28-T and Jurkat tumor model of Balb/c cells in nude mice, FCM expression analysis of mice tumor cell CD28 molecule; CD28-T and Jurkat were treated with SQA-11 to establish the tumor model, observe tumor formation time and the rate of tumor formation, and at the same time without antibody treatment group. The tumor models were randomly divided into two groups, the intratumoral injection of SQA-11 2-3, every other day for a total of 4 times the amount of antibody injection in tumor, 50 g /mm3 to observe the tumor growth and tumor bearing mice survival state.
[results] MTT assay showed that SQA-11 could significantly inhibit CD28-T and Jurkat cell proliferation in.CD28-T cells after third days, that the formation of visible tumor nodules, and the tumor was 100% (40/40).Jurkat cells in Fourteenth days after inoculation were visible tumor nodules, tumor formation rate was 80% (32/40). Analysis of tumor nodules after fortieth days in the cells treated with FCM, the positive rate of CD28 was 90.4% CD28-T, Jurkat74.3%. and non inoculated cells before in vitro showed no significant difference between.CD28-T and Jurkat cells were treated with SQA-11 after incubation with mice, tumor CD28-T time delayed 2 days or fifth days form the naked eye visible tumor nodules, tumor formation rate is 100% (20/20); tumor cell Jurkat time delayed 5 days or nineteenth days began to form tumor nodules, tumor formation rate was 50% (10/20). CD28-T (seventh days) and Jurkat (thirtieth days) tumor model The tumor nodules in partial.Jurkat 3/10 rats were reduced by SQA-11 intratumoral injection for 40 days. The average volume was reduced from 56mm3 to 32mm3 at the time of injection, which had no inhibitory effect on the growth of tumor nodules in the CD28-T model group.
[Conclusion] SQA-11 has a certain inhibitory effect on human tumors that naturally express CD28 molecules, but has no significant effect on the tumor expressing exogenous CD28 molecules.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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