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1、白细胞新单克隆抗体ZCH-2B8a的表达谱分析及其意义 作者：汤永民 郭莉, 杨世隆, 沈红强, 钱柏芹, 张怡, 张海忠【摘要】 ZCH-2B8a (IgG2a )是作者研究室最近采用髓细胞性白血病细胞系KG1a作为免疫原自行研制的鼠抗人造血细胞分化抗原的单克隆抗体，已提交第8届国际人类白细胞分化抗原协作组会议（HLDA8）鉴定。根据HLDA8总部反馈信息表明，该抗体识别的抗原性质不明，后者属国际上尚未认识的血细胞膜新的分化抗原。本研究旨在分析ZCH-2B8a单克隆抗体与正常人外周血细胞成分、骨髓及G-CSF动员的外周血CD34+造血干/祖细胞和血液肿瘤细胞系的反应规律及其意义。 采用多参数
2、流式细胞术分析2B8a抗体与正常及恶性肿瘤细胞表面的反应性，每种细胞成分重复实验3次，以阳性细胞数20%为阳性。结果表明： 2B8a抗原在外周血B细胞上表达（3/3例，平均阳性细胞数为26.29 ），而在T淋巴细胞和NK细胞上不表达（0/3例）；在粒细胞和单核细胞上阳性表达均为2/3例，平均阳性细胞数分别是23.72 和59.84 ；在DC细胞、红细胞和血小板上均不表达（0/3例）。2B8a抗原在骨髓CD34细胞上的阳性表达是3/3例，平均阳性细胞数39.33 ，而在G-CSF动员的外周血CD34+细胞上的阳性表达仅1/3例，平均阳性细胞数为1.25 。2B8a抗原在B系细胞系Raji、SMS
3、-SB、Nalm-6和Nall-1上的平均阳性细胞数分别为98.78 、98.61 、94.93 和5.68 ；在T系细胞系Molt-3上的平均阳性细胞数为31.40 ，而在Molt-4、JM和CCRF-CEM 细胞上不表达；在髓系细胞系U937、Meg-01、HL-60、K562、KG1a和HEL92.1.7上的平均阳性细胞数分别为67.78 、33.40 、29.70 、28.19 、16.23 和8.02 ；在神经母细胞瘤细胞系SK-N-SH、KCNR、BE、LAN-1和SK-N-AS细胞以及结肠癌细胞系HR8348细胞上均不表达，而在羊膜细胞系FL细胞上呈一定的阳性表达，平均阳性细胞数
4、为45.03。结论： 在正常外周血，2B8a抗体主要与B淋巴细胞和单核巨噬细胞反应，在骨髓和外周血CD34+造血干/祖细胞中也有不同程度的反应性；在细胞系上，2B8a抗体主要与B系细胞系、单核巨噬细胞系及上皮性肿瘤细胞系反应，提示该抗体有可能用于上述肿瘤的免疫学诊断与治疗。 【关键词】 ZCH-2B8a抗体； 白细胞单克隆抗体； 造血干/祖细胞； 白血病，淋巴瘤Reactivity of a Novel Monoclonal Antibody ZCH-2B8a on Normal Hematopoietic Cells and Malignant Cell Lines and Its Sign
5、ificanceAbstractsZCH-2B8a (IgG2a ) is a novel monoclonal antibody (McAb) generated in laboratory of Children Hospital of Medical College, Zhejiang University recently using human myeloblastic leukemia cell line KG1a as immunogen. This antibody has been submitted to the 8th International Workshop and
6、 Conference on Human Leukocyte Differentiation Antigens (HLDA8) and the results showed that the antibody recognized an unknown molecule on the surface of some blood cells. The aim of this study was to investigate the reactivity of this antibody on normal blood cells and malignant cell lines and to e
7、xplore its possible application in clinical practice. The multi-parameter flow cytometry was used to analyze the expression pattern of 2B8a antigen in triplicate on normal blood components including T cells, B cells , natural killers (NK), neutrophils, monocytes, dendritic cells (DC), red blood cell
8、s (RBC), platelets (Plt), hematopoietic stem/progenitor cells derived from either bone marrow or G-CSF mobilized peripheral blood CD34+ cells and malignant cell lines including 14 hematopoietic, 5 neuroblastoma, 1 colon cancer and 1 amniotic epithelium cell lines. The amount of positive cells 20% wa
9、s considered as positivity. The results showed that 2B8a antibody reacted to 3/3 specimens of blood B cells with a positive rate of 26.29% and 2/3 specimens of monocytes with an average positive rate of 59.84%. 2B8a was weakly reactive to neutrophils (23.72%) and negative for T cells, NK, DC, RBC an
10、d Plt. The antibody reacted to all 3 marrow CD34+ cells with an average positive rate of 39.33% while it was negative for G-CSF-mobilized CD34+ peripheral blood stem/progenitor cells (PBSC, 1.25%). Cell line analysis showed that the antibody notably reacted to three out of 4 cell lines (Raji, SMS-SB
11、, Nalm-6 and Nall-1) with the positive rates of 98.78%, 98.61%, 94.93% respectively and weakly to one of them with 5.68% in B lineage cell lines and monoblastic cell line (U937, 67.78%) while it was only weakly positive or negative for other myeloid leukemia cell lines including Meg01 (33.40%), HL-6
12、0 (29.70%)，K562 (28.19%), KG1a (16.23 %) and HEL92.1.7 (8.02%). Among 4 T lineage leukemia，5 neuroblastoma and 1 colon cancer cell lines tested, only Molt-3 was found weakly positive (31.40%) for 2B8a, while the remaining 3 T cell lines (Molt4, JM and CCRF-CEM), 5 neuroblastoma cell lines (LA-N1, KC
13、NR, BE, SK-N-SH, SK-N-AS) and the colon cancer cell line (HR8348) tested were negative. An amniotic epithelium cell line (FL) was showed positive for the antibody (45.03%). It is concluded that 2B8a antibody primarily reacts to B lineage and monocytic lineage cells which may bear the diagnostic and
14、therapeutic applications among different types of hematopoietic malignancies.Key wordsZCH-2B8a antibody; leukocyte monoclonal antibody; hematopoietic stem /progenitor cell; leukemia; lymphoma白细胞膜抗原抗体研究对血液系统肿瘤的诊断、分型、微小残留病（MRD）的监测以及靶向治疗均具有十分重要的意义和广阔的应用前景1-7。ZCH(Zhejiang Children Hospital)-2B8a是本科研究室自行
15、研制的鼠抗人造血细胞分化抗原的单克隆抗体，已经提交第8届国际人类白细胞分化抗原协作组会议（HLDA8）鉴定。根据HLDA8总部大量研究的反馈信息表明，该抗体识别的抗原性质不明，属国际上尚未认识的血细胞膜新的分化抗原8。为了进一步明确2B8a抗体的抗原识别特性，我们应用多色流式细胞术检测2B8a抗原在正常人外周血细胞、外周血和骨髓CD34+造血干/祖细胞以及各种细胞系细胞上的表达，现将初步结果总结报道如下。材料和方法ZCH-2B8a杂交瘤细胞株的建立以人原始髓细胞白血病细胞系KG1a作为免疫原，采用经典杂交瘤技术9将小鼠脾脏细胞与小鼠骨髓瘤细胞系SP2/0融合，经选择培养液培养后和KG1a细胞筛
16、选，获得1株与KG1a细胞呈弱阳性反应的细胞系，命名为ZCH-2B8a（简称2B8a）。经3次以上亚克隆培养，建立了能稳定分泌2B8a抗体的杂交瘤细胞系。该抗体经用抗小鼠免疫球蛋白亚类试剂盒和流式细胞仪（FCM）分析，证实为IgG2a 抗体。以培养上清（滴度116）作为抗体来源进行研究。正常细胞来源正常外周血细胞成分的分离选择本实验室身体健康的年轻志愿者3名，分别抽取其外周静脉血15 ml, 肝素钠抗凝。经180g离心5分钟分离血浆和血细胞，取血浆部分移入另一试管中，经1800 g离心20分钟，弃上清，沉淀物作为血小板的来源，悬浮于适量的含5小牛血清的RPMI 1640 培养液中备用。血细胞部分用10 ml培养液稀释后，分别加入终浓度为1.5%的羟乙基淀粉（Hespan, 美国Baxter公司产品），放入37水浴锅中孵浴1小时，使之自然沉降，取上层液体部分作为白细胞的来源，经磷酸盐
