抗人CD20和CD34单抗的研制及其功能研究
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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
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。
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。
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
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
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
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。
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。
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
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
引文
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52 Bubien JK, Zhou LJ, Bell PD, et al Transfection of the CD20 cell-surface molecule into ectopic-cell types generates a Ca2+ conductance found constitutively in B lymphocytes J Cell Biol, 1993,121:1121-1132
53 Deans JP, Kalt L, Ledbetter JA, et al Association of 75/80 Kda phosphoproteins and the tyrosine kinases Lyn, Fyn and Lck with the B cell molecule CD20, evidence against involvement of the cytoplasmic regions of CD20 J Bio Chem, 1995,270:22632-22638
54 Kanzaki M, Nie L, Shibata H, et al Activation of a calcium-permeable cation channel CD20 expressed in Balb/c 3T3 cells by insulin-like growth factor-1 J Biol Chem, 1997,272(8):4964-4969
55 Golay JT, Clark EA, Beverley PC The CD20 (Bp35) antigen is involved in activation of B cells from the G0 to the G1 phase of the cell cycle J Immunol, 1985 , 135 (6):3795-3801
56 Deans JP, Schieven GL, Shu GL, et al Association of tyrosine and serine kinases with the B cell surface antigen CD20 Induction via CD20 of tyrosine phosphorylation and activation of phospholipase C-gamma 1 and PLC phospholipase C-gamma 2 J Immunol, 1993, 151(9):4494-4504
57 McConkey DJ, Orrenius S The role of calcium in the regulation of apoptosis J Leukoc Biol 1996, 59 (6):775-783 Review
58 Juin P, Pelletier M, Oliver L, et al Induction of a caspase-3-like activity by calcium in normal cytosolic extracts triggers nuclear apoptosis in a cell-free system J Biol Chem 1998 , 273 (28):17559-17564
59 Thormberry NA, Lazebnik Y Caspases: enemies within Science, 1998,281(5381):1312-1316
60 Harjunpaa A, Junnikkala S, Meri S, et al Rituximab (Anti-CD20) therapy of B-cell lymphomas: direct complement killing is superior to cellular effector mechanisms Scand J Immunol, 2000, 51:634-641
61 Arko Gorter, Seppo Meri Immune evasion of tumor cells using membrane-bound complement regulatory proteins Immunol Today, 1999, 20(12):576-582
62 Hooijberg E, Sein JJ, van den Berk PC, et al Eradication of large human B cell tumors in nude mice with unconjugated CD20 monoclonal antibodies and interleukin 2 Cancer Res 1995, 55 (12):2627-2634
63 Buchsbaum DJ, Wahl RL, Normolle DP, et al Therapy with unlabeled and 131I-labeled pan-B-cell monoclonal antibodies in nude mice bearing Raji Burkitt's lymphoma xenografts Cancer Res 1992, 52 (23):6476-6481
64 Byrd JC, Waselenko JK, Maneatis TJ, et al Rituximab therapy in hematologic malignancy patients with circulating blood tumour cells: association with increased infusion-related side effects and rapid blood tumour clearance J Clin Oncol, 1999,17:791-795
65 Tedder TF, Engel P CD: A regulator of cell-cycle progression of B lymphocytes Immunol Today, 1994,15(9):450-454
66 Clark EA, Shu G, Ledbetter JA Role of the Bp35 cell surface polypeptide in human B-cell activation Proc Natl Acad Sci U S A 1985，82(6):1766-1770
67 Funakoshi S, Longo DL, Beckwith M, et al Inhibition of human B-cell lymphoma growth by CD40 stimulation Blood 1994, 83(10):2787-2794
68 Press OW, Howell-Clark J, Anderson S, et al Retention of B-cell-specific monoclonal antibodies by human lymphoma cells Blood 1994,83 (5):1390-1397
69 Buchsbaum DJ, Wahl RL, Normolle DP, et al Therapy with unlabeled and 131I-labeled pan-B-cell monoclonal antibodies in nude mice bearing Raji Burkitt's
lymphoma xenografts Cancer Res 1992, 52(23):6476-6481
70 Anderson DR, Grillo-Lope ZA, Varns C, et al Targeted anti-cancer therapy using rituximab, a chimaeric anti-CD20 antibody (IDEC-C2B8) in the treatment of non-Hodgkin's B-cell lymphoma Biochem Soc Trans, 1997,25(2):705-708
71 Provvedini D M , Tsoukas D C , Deftos L J , et al 1,25-Dihydroxyvitamin D3 receptors in human leukocytes Science (Wash DC), 1983,221:1181-1183
72 Miyaura C, Abe E, Kuribayashi T, et al 1α-25-Dihydroxyvitamin D3 induces differentiation of human myeloid leukemia cells Biochem Biophys Res Commun, 1981,102:937-943
73 Mangelsdorf D J , Koeffler H P , Donaldson C A , et al 1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL60): receptor-mediated maturation to macrophage-like cells J Cell Biol, 1984, 98:391-398
74 Dodd R C , Newman S L , Citron S J , et al Vitamin D metabolites and retinoic acid induce the monoblastic U937 cells to become macrophages In: D V Cohn, T Fujita, J T Potts, and R V Talmage (Eds ): Endocrine Control of Bone and Calcium Metabolism, pp 342-345 New York: Elsevier Science Publishers BV, 1984
75 Manolagas S C , Deftos L J The vitamin D endocrine system and the hematolymphoporetic tissue Ann Int Med, 1984,100:144-146
76 Moore G E , Kitamura H Cell line derived from patients with myeloma N Y Stat J Med , 1968,68:2054-2060
77 Moore G E , Harbell J W , Woods L K , et al RPMI8226, a human myeloma cell line: an update (Abstract) Proc Am Assoc Cancer Res, 1982,23:33
78 Simmons DL, Satterthwaite AB, Tenen DG, et al Molecular cloning of a cDNA encoding CD34, a sialomucin of human hematopoietic stem cells J Immunol 1992, 148(1):267-271
79 He XY, Antao VP, Basila D, et al Isolation and molecular characterization of the human CD34 gene Blood, 1992, 79(9):2296-2302
80 Campos L, Guyotat D, Archimbaud E, et al Surface marker expression in adult acute myeloid leukaemia: correlations with initial characteristics, morphology and response to therapy Br J Haematol, 1989,72(2):161-166
81 Geller RB, Zahurak M, Hurwitz CA, et al Prognostic importance of immunophenotyping in adults with acute myelocytic leukaemia: the significance of the stem-cell glycoprotein CD34 (My10) Br J Haematol, 1990, 76(3):340-347
82 Civin CI, Strauss LC, Brovall C, et al Antigenic analysis of hematopoiesis III A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells J Immunol 1984,133(1):157-165
83 Tindle RW, Nichols RA, Chan L, et al A novel monoclonal antibody BI-3C5 recognises myeloblasts and non-B non-T lymphoblasts in acute leukaemias and CGL blast crises, and reacts with immature cells in normal bone marrow Leuk Res, 1985, 9(1):1-9
84 Hurwitz CA, Loken MR, Graham ML, et al Asynchronous antigen expression in B lineage acute lymphoblastic leukemia Blood, 1988, 72(1):299-307
85 Borowitz MJ, Gockerman JP, Moore JO, et al Clinicopathologic and cytogenic features of CD34 (My 10)-positive acute nonlymphocytic leukemia Am J Clin Pathol, 1989, 91(3):265-270
86 Azuma E, Umemoto M, Kubo M, et al CD34 antigen expression in children with Philadelphia chromosome-positive acute lymphoblastic leukemia Cancer, 1991, 67(6):1565-1569
87 Borowitz MJ, Shuster JJ, Civin CI, et al Prognostic significance of CD34 expression in childhood B-precursor acute lymphocytic leukemia: a Pediatric Oncology Group study J Clin Oncol, 1990, 8(8):1389-1398
88 舒翠玲，汲言山，沈倍奋，等。 CD34基因克隆和表达。 中国免疫学杂志， 1995，11（2）：78-81
2 Houghton AN, Schneider-Gadicke E, Johnson JP Monoclonal antibodies in cancer therapy Curr Opon Immunol, 1993,5:732-739
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7 Maloney DG, Grillo-Lopez AJ, White CA, et al IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma Blood, 1997 , 90(6):2188-2195
8 Maloney DG, Grillo-Lopez AJ, Bodkin D, et al IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody: results of long-term follow-up of relapsed NHL Phase Ⅱ trial patients Blood, 1995, 86:54a (abstr )
9 Czuczman MS, Grillo-Lopez AJ, Jonas C, et al IDEC-C2B8 and CHOP chemoimmunotherapy of low-grade lymphoma Blood, 1995, 86:55a (abstr )
10 Reff ME, Carner K, Chambers KS, et al Depletion of B cells in vivo by a chemeric mouse human monoclonal antibody to CD20 Blood, 1994;83:435-445
11 Waldmann TA Monoclonal antibodies in diagnosis and therapy Science, 1991, 252:1657-1662
12 Grossbard ML, Press OW, Appelbaum FR, et al Monoclonal antibodies-based
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14 洪海燕，舒翠玲，沈倍奋。 CD20分子的研究进展。 细胞与分子免疫学杂志， 1999，15（增刊1）：7-9
15 Press OW, Howell-Clark J, Anderson S, et al Retention of B-cell-specific monoclonal antibodies by human lymphoma cells Blood 1994, 83(5):1390-1397
16 Press OW, Farr A G, Borroz K I, et al Endocytosis and degradation of monoclonal antibodies targeting human B-cell malignancies Cancer Res, 1989,49(17):4906-4912
17 Naomi PF IDEC Pharmaceutical Corp report positive final phase Ⅲ data on lymphoma antibody Genetic Engineering News, 1997,17:1-5
18 Lam KS, Zhao ZG Targeted therapy for lymphoma with peptides Hematol Oncol Clin North Am, 1997,11:1007-1019
19 Gopal AK, Press OW Clinical application of anti-CD20 antibodies J Lab Clin Med， 1999,134(5):445-450
20 Grillo-Lopez AJ, White CA, Chet Varns, et al Overview of the clinical development of rituximan: first monoclonal antibody approved for the treatment of lymphoma Seminars in Oncology, 1999,26(5) Suppl 14:66-73
21 Maloney D, Smith B, Appelbaum F The anti-tumour effect of monoclonal anti-CD20 antibody therapy includes direct anti-proliferative activity and induction of apoptosis in CD20 positive non-Hodgkin's lymphoma cell lines Blood (Abstract Supplement), 1996,1:88
22 Hirohumi Taji, Yoshitoyo Kagami, Yasutaka Okada, et al Growth Inhibition of CD20- positive B lymphomas cell lines by IDEC-C2B8 anti-CD20 monoclonal antibody Jpn J Cancer Res, 1998,89(7):748-756
23 Garcia-Conde J, Cabanillas F Mantle cell lymphoma: a lymphoproliferative disorder associated with aberrant function of the cycle Leukemia, 1996, (Suppl)2:78-83
24 Stashenko P, Nsdler L M, Hardy R, et al Characterization of a human B lymphocyte-specific antigen J Immun, 1980,125(4):1678-1685
25 Nsdler L M, Karsmeyer S J, Anderson K C, et al B cell origin of non-T cell acute lymphoblastic leukemia A model for discrete stages of neoplastic and normal pre-B cell differentiation J Clin Invest, 1984,74(2):332-340
26 Stashenko P, Nsdler L M, Hardy R, et al Expression of cell surface markers after human B lymphocyte activation Proc Natl Acad Sci, USA 1981,78(6):3848-3852
27 Jean-Francois Rossi, Brian G M Durie, Christophe Duperray, et al Phenotypic and functional analysis of 1,25-dihydroxyvitamin D3 receptor mediated modulation of the human myeloma cell line RPMI8226 Cancer Research 1988,48:1213-1216
28 Ryan D, Kossover S, Mitchell S, et al Subpopulations of common acute lymphoblastic leukemia antigen-positive lymphoid cells in normal bone marrow identified by hematopoietic differentiation antigens Blood, 1986, 68(2):417-425
29 Civin CI, Loken MR Cell surface antigens on human marrow cells: dissection of hematopoietic development using monoclonal antibodies and multiparameter flow cytometry Review Int J Cell Cloning, 1987, 5(4):267-288
30 Baumheter S, Singer MS, Henzel W, et al Binding of L-selectin to the vascular sialomucin CD34 Science, 1993, 262(5132):436-438
31 Wagner JE Jr Isolation of primitive hematopoietic stem cells Semin Hematol, 1992, 29(2 Suppl 1):6-9
32 Okarma TB Stem cell selection for autologous bone marrow transplantation Semin Hematol, 1992, 29(2 Suppl 1):9-20
33 Chang KL, Arber DA, Weiss LM CD20: A Review Applied Immunohistochem, 1996,4:1-15
34 Rottenburger C, Kiel K, Bosing T, et al Clonotypic CD20+ and CD19+ B cells in peripheral blood of patient with multiple myeloma post high-dose therapy Br J Haematol, 1999, 106:540-542
35 Press OW, Appelbaum F, Ledbetter JA, et al Monoclonal antibody 1F5 (anti-CD20) serotherapy of human B cell lymphomas Blood 1987 ,69(2):584-591
36 Krause DS, Fackler MJ, Civin CI, et al CD34: structure, biology, and clinical utility Blood, 1996, 87(1):1-13
37 Tedder TF, McIntyre G, Schlossman SF Heterogengity in the B1(CD20) cell
surface molecule expressed by human B-lymphocyte Mol Immunol, 1988,25(12):1321-1330
38 Golay JT, Clark EA, Beverley PC, et al The CD20 (Bp35) antigen is involved in activation of B cells from the G0 to the G1 phase of the cell cycle J Immunol 1985, 135(6):3795-3801
39 Tedder TF, Klejman G, Disteche CM Cloning of complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homogous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19 J Immunol, 1988,141:4388-4394
40 洪海燕，孙英勋，郭燕翔，等。 人CD20基因在鼠NIH-3T3细胞膜上的表达。 生物化学与生物物理学报，2000，32（4）：430-433
41 Kanzaki M, Shibata H, Mogami H, et al Expression of calcium-permeable cation channel CD20 accelerates progression through the G1 phase in Balb/c 3T3 cells J Biol Chem, 1995,270（22）:13099-13104
42 Tedder TF, Streuli M, Schlossman SF, et al Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes Proc Natl Acad Sci U S A 1988 , 85(1):208-212
43 Stamenkovic I, Seed B Analysis of two cDNA clones encoding the B lymphocyte antigen CD20 (B1, Bp35), a type III integral membrane protein J Exp Med 1988,167(6):1975-1780
44 Reed JC Dysregulation of apoptisis in Non-Hodgkin's lymphomas: toward a molecular understanding of Bcl-2 Adv in Leukemia Lymphoma, 1998,7:1
45 Rosenberg J, Waldchuk C, Grillo-Lopez AJ, et al Pharmacokinetic analysis of serum concentrations of the chimeric anti-CD20 antibody IDEC-C2B8 in patients with relapsed B cell lymphoma Proc Am Soc Clin Oncol, 1996,15:418 (abstr )
46 Press OW, Eary JF, Appelbaum FR, et al Radiolabeled-antibody therapy of B-cell lymphoma with autologous bone marrow support N Engl J Med 1993, 329(17):1219-1224
47 Kaminski MS, Zasadny KR, Francis IR, et al Radioimmunotherapy of B-cell lymphoma with [131I]anti-B1 (anti-CD20) antibody N Engl J Med 1993, 329 (7):459-465
48 李爱玲，孙英勋，洪海燕，等。 利用基因重组抗原研制人CD20单克隆抗体及其功能研究。中华微生物学和免疫学杂志 (印刷中)
49 Daming Shan, Ledbetter JA, Press OW, et al Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies Blood, 1998, 91(5):1644-1652
50 Daming Shan, Ledbetter JA, Press OW, et al Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells Cancer Immunol Immuother, 2000,48(12):673-683
51 Hofmeister JK, Cooney D, Coggeshall KM Clustered CD20 induced apoptosis: Src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis Blood Cells, Molecules, and Diseases, 2000,26(2):133-143
52 Bubien JK, Zhou LJ, Bell PD, et al Transfection of the CD20 cell-surface molecule into ectopic-cell types generates a Ca2+ conductance found constitutively in B lymphocytes J Cell Biol, 1993,121:1121-1132
53 Deans JP, Kalt L, Ledbetter JA, et al Association of 75/80 Kda phosphoproteins and the tyrosine kinases Lyn, Fyn and Lck with the B cell molecule CD20, evidence against involvement of the cytoplasmic regions of CD20 J Bio Chem, 1995,270:22632-22638
54 Kanzaki M, Nie L, Shibata H, et al Activation of a calcium-permeable cation channel CD20 expressed in Balb/c 3T3 cells by insulin-like growth factor-1 J Biol Chem, 1997,272(8):4964-4969
55 Golay JT, Clark EA, Beverley PC The CD20 (Bp35) antigen is involved in activation of B cells from the G0 to the G1 phase of the cell cycle J Immunol, 1985 , 135 (6):3795-3801
56 Deans JP, Schieven GL, Shu GL, et al Association of tyrosine and serine kinases with the B cell surface antigen CD20 Induction via CD20 of tyrosine phosphorylation and activation of phospholipase C-gamma 1 and PLC phospholipase C-gamma 2 J Immunol, 1993, 151(9):4494-4504
57 McConkey DJ, Orrenius S The role of calcium in the regulation of apoptosis J Leukoc Biol 1996, 59 (6):775-783 Review
58 Juin P, Pelletier M, Oliver L, et al Induction of a caspase-3-like activity by calcium in normal cytosolic extracts triggers nuclear apoptosis in a cell-free system J Biol Chem 1998 , 273 (28):17559-17564
59 Thormberry NA, Lazebnik Y Caspases: enemies within Science, 1998,281(5381):1312-1316
60 Harjunpaa A, Junnikkala S, Meri S, et al Rituximab (Anti-CD20) therapy of B-cell lymphomas: direct complement killing is superior to cellular effector mechanisms Scand J Immunol, 2000, 51:634-641
61 Arko Gorter, Seppo Meri Immune evasion of tumor cells using membrane-bound complement regulatory proteins Immunol Today, 1999, 20(12):576-582
62 Hooijberg E, Sein JJ, van den Berk PC, et al Eradication of large human B cell tumors in nude mice with unconjugated CD20 monoclonal antibodies and interleukin 2 Cancer Res 1995, 55 (12):2627-2634
63 Buchsbaum DJ, Wahl RL, Normolle DP, et al Therapy with unlabeled and 131I-labeled pan-B-cell monoclonal antibodies in nude mice bearing Raji Burkitt's lymphoma xenografts Cancer Res 1992, 52 (23):6476-6481
64 Byrd JC, Waselenko JK, Maneatis TJ, et al Rituximab therapy in hematologic malignancy patients with circulating blood tumour cells: association with increased infusion-related side effects and rapid blood tumour clearance J Clin Oncol, 1999,17:791-795
65 Tedder TF, Engel P CD: A regulator of cell-cycle progression of B lymphocytes Immunol Today, 1994,15(9):450-454
66 Clark EA, Shu G, Ledbetter JA Role of the Bp35 cell surface polypeptide in human B-cell activation Proc Natl Acad Sci U S A 1985，82(6):1766-1770
67 Funakoshi S, Longo DL, Beckwith M, et al Inhibition of human B-cell lymphoma growth by CD40 stimulation Blood 1994, 83(10):2787-2794
68 Press OW, Howell-Clark J, Anderson S, et al Retention of B-cell-specific monoclonal antibodies by human lymphoma cells Blood 1994,83 (5):1390-1397
69 Buchsbaum DJ, Wahl RL, Normolle DP, et al Therapy with unlabeled and 131I-labeled pan-B-cell monoclonal antibodies in nude mice bearing Raji Burkitt's
lymphoma xenografts Cancer Res 1992, 52(23):6476-6481
70 Anderson DR, Grillo-Lope ZA, Varns C, et al Targeted anti-cancer therapy using rituximab, a chimaeric anti-CD20 antibody (IDEC-C2B8) in the treatment of non-Hodgkin's B-cell lymphoma Biochem Soc Trans, 1997,25(2):705-708
71 Provvedini D M , Tsoukas D C , Deftos L J , et al 1,25-Dihydroxyvitamin D3 receptors in human leukocytes Science (Wash DC), 1983,221:1181-1183
72 Miyaura C, Abe E, Kuribayashi T, et al 1α-25-Dihydroxyvitamin D3 induces differentiation of human myeloid leukemia cells Biochem Biophys Res Commun, 1981,102:937-943
73 Mangelsdorf D J , Koeffler H P , Donaldson C A , et al 1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL60): receptor-mediated maturation to macrophage-like cells J Cell Biol, 1984, 98:391-398
74 Dodd R C , Newman S L , Citron S J , et al Vitamin D metabolites and retinoic acid induce the monoblastic U937 cells to become macrophages In: D V Cohn, T Fujita, J T Potts, and R V Talmage (Eds ): Endocrine Control of Bone and Calcium Metabolism, pp 342-345 New York: Elsevier Science Publishers BV, 1984
75 Manolagas S C , Deftos L J The vitamin D endocrine system and the hematolymphoporetic tissue Ann Int Med, 1984,100:144-146
76 Moore G E , Kitamura H Cell line derived from patients with myeloma N Y Stat J Med , 1968,68:2054-2060
77 Moore G E , Harbell J W , Woods L K , et al RPMI8226, a human myeloma cell line: an update (Abstract) Proc Am Assoc Cancer Res, 1982,23:33
78 Simmons DL, Satterthwaite AB, Tenen DG, et al Molecular cloning of a cDNA encoding CD34, a sialomucin of human hematopoietic stem cells J Immunol 1992, 148(1):267-271
79 He XY, Antao VP, Basila D, et al Isolation and molecular characterization of the human CD34 gene Blood, 1992, 79(9):2296-2302
80 Campos L, Guyotat D, Archimbaud E, et al Surface marker expression in adult acute myeloid leukaemia: correlations with initial characteristics, morphology and response to therapy Br J Haematol, 1989,72(2):161-166
81 Geller RB, Zahurak M, Hurwitz CA, et al Prognostic importance of immunophenotyping in adults with acute myelocytic leukaemia: the significance of the stem-cell glycoprotein CD34 (My10) Br J Haematol, 1990, 76(3):340-347
82 Civin CI, Strauss LC, Brovall C, et al Antigenic analysis of hematopoiesis III A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells J Immunol 1984,133(1):157-165
83 Tindle RW, Nichols RA, Chan L, et al A novel monoclonal antibody BI-3C5 recognises myeloblasts and non-B non-T lymphoblasts in acute leukaemias and CGL blast crises, and reacts with immature cells in normal bone marrow Leuk Res, 1985, 9(1):1-9
84 Hurwitz CA, Loken MR, Graham ML, et al Asynchronous antigen expression in B lineage acute lymphoblastic leukemia Blood, 1988, 72(1):299-307
85 Borowitz MJ, Gockerman JP, Moore JO, et al Clinicopathologic and cytogenic features of CD34 (My 10)-positive acute nonlymphocytic leukemia Am J Clin Pathol, 1989, 91(3):265-270
86 Azuma E, Umemoto M, Kubo M, et al CD34 antigen expression in children with Philadelphia chromosome-positive acute lymphoblastic leukemia Cancer, 1991, 67(6):1565-1569
87 Borowitz MJ, Shuster JJ, Civin CI, et al Prognostic significance of CD34 expression in childhood B-precursor acute lymphocytic leukemia: a Pediatric Oncology Group study J Clin Oncol, 1990, 8(8):1389-1398
88 舒翠玲，汲言山，沈倍奋，等。 CD34基因克隆和表达。 中国免疫学杂志， 1995，11（2）：78-81