【作者】 李爱玲；

【导师】 赵法伋； 郭俊生； 黎燕；

【作者基本信息】 第二军医大学 ， 军事预防医学， 2002， 博士

【摘要】 白血病是最具威胁人类生命的恶性肿瘤之一，对于这类疾病的治疗，传统的化疗、放疗、免疫调节等治疗方法，难以取得令人满意的疗效。近年来，针对细胞表面分子的抗原靶向性治疗已取得了巨大进展，并成为一种非常有发展前途的治疗方法。抗人CD20单抗（CD20- McAb）在临床上治疗B细胞淋巴瘤白血病中，已获得显著疗效；而抗人CD34单抗（CD34- McAb）用于造血干细胞的富集对于大大提高造血干细胞移植的成功率也是非常重要的。这两种单抗目前正广泛应用于临床，且有较好的发展前景。国内目前还没有商品化的抗CD20和CD34工程抗体，临床使用的抗体均购自国外，且价格昂贵。基于上述的研究背景和实际的应用情况，本课题旨在制备出有功能活性的CD20- McAb和CD34- McAb，并进一步阐明它们可能的效应机理，为临床上研制、应用工程抗体提供物质基础。由于活性维生素D（1,25-(OH)2VD3）能够诱导细胞表面CD20分子的表达，故本研究还观察了所获CD20-McAb与1,25-(OH)2VD3对骨髓瘤细胞增殖抑制的协同作用，可望为临床提高CD20- McAb的疗效和治疗骨髓瘤提供新的思路。方法：1、抗人CD20单克隆抗体的研制、鉴定、功能及其机理研究以克隆的抗原cDNA转染鼠源性NIH3T3细胞为重组抗原，采用细胞融合技术来制备CD20- McAb；以间接免疫荧光法筛选杂交瘤上清；免疫沉淀法用于鉴定McAb识别抗原的分子量；双扩实验鉴定了McAb的亚类；以流式细胞术（FCM）检测McAb的特异性、细胞凋亡及胞内游离钙的水平；MTT法测定McAb对细胞增殖的影响；光镜和电镜下观察细胞的形态学改变； Western Blot检测凋亡相关蛋白表达的变化。2、CD20单抗与1,25-(OH)2VD3协同杀伤骨髓瘤细胞的作用研究台盼蓝拒染法用于观察1,25-(OH)2VD3对细胞存活的影响；FCM检测<WP=8>1,25-(OH)2VD3对细胞表型的改变情况；MTT法测定CD20- McAb与1,25-(OH)2VD3协同杀伤骨髓瘤细胞的作用。3、CD34- McAb的制备、鉴定及功能研究以表达人CD34全长cDNA的重组痘苗病毒为抗原免疫动物，其它方法与制备CD20- McAb类同。结果：1、CD20-McAb的产生及鉴定经融合、筛选及反复克隆化，得到了一株能够稳定分泌抗人CD20单抗的杂交瘤细胞株1-28，测定其亚类为IgM。1-28具有CD20-McAb特异的细胞反应谱，其识别的抗原分子量为33kD，它与pcDNA3 1/CD20+转染的NIH-3T3细胞反应为阳性，并且能明显竞争标准CD20-FITC McAb与Daudi细胞表面CD20分子的结合。这些结果不仅证实了1-28是抗CD20的特异性单抗，而且是与标准单抗有共同活性部位的功能性抗体。2、1-28的功能及其抑瘤机理MTT、PI染色、光镜及电镜结果显示1-28具有抑制B淋巴瘤细胞增殖和直接诱导其凋亡的功能，而临床上使用的进口CD20- McAb（商品名为美罗华）没有观察到对B淋巴瘤细胞的直接促凋亡作用，这可能是1-28抑制B淋巴瘤细胞增殖的优势之一。1-28作用Daudi细胞后使其胞内游离钙离子浓度明显升高(P<0 05)，Bcl-2、Caspase-3酶原和 Caspase-9酶原表达量下降，而Bax、Caspase-3和 Caspase-9的表达显著升高，提示1-28的促凋亡机理与调节胞内这些信号分子有关。1-28还能通过介导补体发挥溶解肿瘤细胞的作用，表明补体依赖的溶解作用（CDC）是1-28杀伤B淋巴瘤细胞的又一重要效应机制。3、1-28与1,25-(OH)2VD3协同杀伤骨髓瘤细胞的作用单独的1,25-(OH)2VD3能够抑制骨髓瘤细胞RPMI8226的增殖和促进细胞表面CD20分子的表达，加入1-28可协同增强1,25-(OH)2VD3对RPMI8226细胞的杀伤作用。这一结果在国内外文献中未见类似报道，这是1-28作为CD20- McAb发挥抑瘤作用的另一优势和新的探索。<WP=9>4、CD34- McAb的产生及鉴定表达人CD34的重组痘苗病毒经过纯化、扩增及效价测定后，用于制备CD34-McAb，筛选得到两株能分泌CD34-McAb的杂交瘤细胞5D41C12（++）和18-15（+），但在以后的鉴定中发现18-15转为阴性，而5D41C12细胞株的特异性还有待于进一步鉴定。结论：根据上述研究，可得出以下结论：1、所制备的1-28是一个有功能活性的CD20-McAb，对B淋巴瘤细胞具有明显杀伤作用。2、1-28的杀瘤机制与直接诱导细胞凋亡和通过补体介导的细胞溶解作用有关，而细胞内游离钙离子浓度的增加和凋亡相关蛋白的改变参与了1-28的促凋亡过程。3、1-28可以增强1,25-(OH)2VD3对骨髓瘤细胞的增殖抑制作用。4、筛选得到一株CD34阳性的杂交瘤细胞，其特异性有待于进一步鉴定。更多还原

【Abstract】 At present, the monoclonal antibodies (McAbs) against the molecules on the cell surface are being used in clinics for the diagnosis and treatment of various diseases Anti-CD20 McAb have been successfully employed in the clinical treatment of B-cell lymphomas in both unmodified and radiolabeled forms Clinical studies of anti-CD20 McAb have demonstrated considerable anti-tumor activity and safety The CD34 McAb is now commonly used for enrichment of hemopoietic progenitors for bone marrow transplantation In the present study, the recombinant antigens were expressed by NIH3T3 cells either transfected with the plasmids containing CD20 cDNA or infected with the recombinant vaccinia virus containing CD34 cDNA to obtain the anti-CD20 and anti-CD34 McAbs, which are being widely used, but not commercially made in China The anti-CD20 McAb named 1-28 was identified by indirect immunofluorescence with Daudi cells The specificity of 1-28 was determined by immunoprecipitation and flow cytometry assay (FCM) The results showed that 33kD molecules on the surface of Daudi cells were recognized by 1-28 The specific reaction with different cell lines was tested to be accorded with standard anti-CD20 McAb 1-28 could compete with standard anti-CD20 McAb to bind to CD20 molecules on the cytoplasmic membrane of Daudi cells The growth inhibition and apoptosis induced by 1-28 were confirmed by several different assays, including MTT or trypan blue staining, PI staining in FCM, electron and optical microscopy The signaling events involved in anti-CD20-induced apoptosis were investigated Increased intracellular Ca2+ concentration, caspase activation, and cleavage of caspase substrates were observed The results also indicated that complement-dependent cytotoxicity (CDC) was another important effector mechanism in Daudi cell killing 1,25-(OH)2VD3 was found to inhibit proliferation of human myeloma cell line RPMI8226 and stimulate the expression of CD20 molecules on the cytoplasmic membrane of RPMI8226 cells 1-28 was proved to have a cooperative effect on growth inhibition of the RPMI8226 cells by 1,25-(OH)2VD3 Further studies of the<WP=11>anti-tumor effect of 1-28 should provide new insights into more effective therapies for B-cell lymphomas Two hybridoma cell lines 5D41C12 and 18-15 secreting anti-CD34 McAb were selected by indirect immunofluorescence with KG1-a cells However, 18-15 turned negative to KG1-a cells later The specificity of 5D41C12 are in research The preparation of anti-CD34 McAb is important not noly for autologous marrow reinfusion, but also for the exploration of the regulation and function of CD34 molecules更多还原