本文選題：B7/CD28協(xié)同刺激信號　切入點：動物模型　出處：《蘇州大學》2011年碩士論文　論文類型：學位論文

【摘要】：CD28分子的基因定位于人2號染色體q33-34區(qū),由1120核苷酸組成,表達于大多數(shù)靜止及活化的T細胞,約95% CD4+T和50% CD8+T細胞表達該分子。此外,部分NK細胞和人惡性漿細胞也表達CD28分子。B7-(1CD80)和B7-(2CD86)是CD28的天然配體,CD28分子與表達在抗原呈遞細胞表面的相應(yīng)配體B7-1和B7-2結(jié)合,從而產(chǎn)生T細胞應(yīng)答所需的協(xié)同刺激信號。B7-CD28信號并非僅局限性地產(chǎn)生于抗原呈遞細胞與T細胞之間,已在某些腫瘤細胞如多發(fā)性骨髓瘤細胞,白血病細胞等也發(fā)現(xiàn)異常的B7-CD28信號通路,且該通路可能參與腫瘤的惡性增殖與轉(zhuǎn)移。通過特異性抗體封閉CD28分子,有可能對腫瘤細胞的生長發(fā)揮抑制作用。本研究旨在運用自行制備的CD28抗體,以轉(zhuǎn)染人CD28基因的小鼠T淋巴瘤細胞株及天然表達CD28分子的人白血病細胞株Jurkat為研究對象,在體外分析抗體對上述細胞生長與增殖影響的基礎(chǔ)上,通過建立腫瘤模型,探討抗體對腫瘤細胞的成瘤及腫瘤消長的影響,以期尋找具有抗瘤作用的生物制劑。 【目的】制備鼠抗人CD28分子單克隆抗體并鑒定其生物學功能。 【方法】采用本室建立的腹水誘生法制備單抗并經(jīng)Protein G親和層析分離純化。間接免疫熒光法分析單克隆抗體效價;FCM分析單克隆抗體識別的抗原位點;FCM分析單克隆抗體對不同細胞株人多發(fā)性骨髓瘤細胞(U266)、小鼠淋巴瘤細胞轉(zhuǎn)入CD28基因細胞株(CD28-T)和人白血病細胞株(Jurkat)膜型CD28分子的識別。 【結(jié)果】腹水形成率為90%以上,收獲量為5ml/只以上。腹水中抗體蛋白的得率達3mg/ml。純化抗體用于間接免疫熒光檢測的用量為0.5μg/5×105。競爭抑制試驗表明該單抗能完全阻斷標準鼠抗人CD28抗體與相應(yīng)抗原的結(jié)合,提示其識別的抗原表位與對照抗體相同或相似。FCM分析表明,CD28 mAb能良好地識別多種細胞表面膜型CD28分子。 【結(jié)論】制備了鼠抗人CD28分子mAb(命名為SQA-11),為探討CD28信號轉(zhuǎn)導在腫瘤生長與轉(zhuǎn)移過程中的作用及機制奠定了物質(zhì)基礎(chǔ)。 【目的】探討SQA-11體內(nèi)、外對CD28+腫瘤細胞的生長與增殖的影響作用,以期尋找具有抗瘤作用的生物制劑。 【方法】MTT法體外分析SQA-11對CD28-T和Jurkat生長和增殖的影響;建立CD28-T與Jurkat細胞的Balb/c裸鼠的腫瘤模型,FCM分析成瘤小鼠瘤體細胞CD28分子的表達;將CD28-T與Jurkat分別經(jīng)SQA-11預(yù)處理后再行建立腫瘤模型,觀察成瘤時間和成瘤率,同時與未經(jīng)抗體處理組進行比較。將腫瘤模型隨機分組,采用瘤內(nèi)注射法將SQA-11分2-3點、隔日一次共4次注入瘤內(nèi),抗體用量為50μg /mm3觀察腫瘤的消長及荷瘤鼠的生存狀態(tài)。 【結(jié)果】MTT檢測發(fā)現(xiàn)SQA-11能夠明顯抑制CD28-T和Jurkat細胞的體外增殖。CD28-T細胞于接種第3天即形成肉眼可見的腫瘤結(jié)節(jié),且成瘤率為100%(40/40)。Jurkat細胞于接種第14天后陸續(xù)形成肉眼可見的腫瘤結(jié)節(jié),成瘤率為80%(32/40)。對接種第40天的腫瘤結(jié)節(jié)中的細胞經(jīng)FCM進行分析,CD28的陽性率分別為CD28-T 90.4%、Jurkat74.3%。與未接種前體外培養(yǎng)的細胞無明顯差異。CD28-T與Jurkat細胞分別經(jīng)SQA-11共孵育后接種小鼠,CD28-T的成瘤時間推遲2天即第5天形成肉眼可見的腫瘤結(jié)節(jié),成瘤率仍為100%(20/20);Jurkat細胞的成瘤時間推遲5天即第19天開始形成腫瘤結(jié)節(jié),成瘤率為50%(10/20)。將CD28-T(第7天)及Jurkat(第30天)腫瘤模型用SQA-11瘤內(nèi)注射,觀察40天。Jurkat模型組部分(3/10)裸鼠的腫瘤結(jié)節(jié)變小,平均體積由注射時的56mm3減為32mm3;對CD28-T模型組腫瘤結(jié)節(jié)的生長無抑制作用。 【結(jié)論】SQA-11對天然表達CD28分子的人腫瘤具有一定的抑制作用;而對表達外源CD28分子的小鼠腫瘤無明顯影響。
[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.

【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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