CAJ | 학술논문

目的:以本科室发现肿瘤细胞上表达的CD40 787AA突变为基础,研制识别肿瘤细胞上CD40突变体分子的单克隆抗体(mAb),并对其生物学特性作初步分析.方法:以转人CD40突变体转基因细胞L929-CD40mu为免疫原,免疫6～8周龄的雌性BALB/c小鼠;采用B淋巴细胞融合技术,将免疫小鼠脾脏细胞与Sp2/0融合,以L929-CD40mu转基因细胞为抗体筛选阳性细胞,免疫荧光标记法对杂交瘤进行反复筛选和多次的克隆化培养;采用快速定性试纸法及竞争抑制结合试验分析该mAb的亚类及抗原识别位点;免疫印迹法对该mAb进行鉴定;采用MTT法分析mAb在体外对肿瘤细胞的抑制增殖效应以及PI-annexin V方法进行细胞凋亡测定.结果:获得1株稳定分泌鼠抗人CD40mu mAb的杂交瘤细胞株(命名为10C5),该mAb能特异性地识别人肿瘤细胞株H08910表达的CD40突变体分子,而不识别正常扁桃体B淋巴细胞及血管内皮细胞表达的CD40分子,并且能够在体外促进肿瘤细胞凋亡.结论:成功地研制出1株特异性识别肿瘤细胞上CD40突变体分子的mAb,该mAb具有体外抑制肿瘤细胞生长并促进其凋亡的作用.
목적:이본과실발현종류세포상표체적CD40 787AA돌변위기출,연제식별종류세포상CD40돌변체분자적단극륭항체(mAb),병대기생물학특성작초보분석.방법:이전인CD40돌변체전기인세포L929-CD40mu위면역원,면역6～8주령적자성BALB/c소서;채용B림파세포융합기술,장면역소서비장세포여Sp2/0융합,이L929-CD40mu전기인세포위항체사선양성세포,면역형광표기법대잡교류진행반복사선화다차적극륭화배양;채용쾌속정성시지법급경쟁억제결합시험분석해mAb적아류급항원식별위점;면역인적법대해mAb진행감정;채용MTT법분석mAb재체외대종류세포적억제증식효응이급PI-annexin V방법진행세포조망측정.결과:획득1주은정분비서항인CD40mu mAb적잡교류세포주(명명위10C5),해mAb능특이성지식별인종류세포주H08910표체적CD40돌변체분자,이불식별정상편도체B림파세포급혈관내피세포표체적CD40분자,병차능구재체외촉진종류세포조망.결론:성공지연제출1주특이성식별종류세포상CD40돌변체분자적mAb,해mAb구유체외억제종류세포생장병촉진기조망적작용.
AIM: To prepare and characterize a mouse anti-human CD40 mutant monoclonal mAb. METHODS: Female BALB/c mice of 6-8 weeks old were immunized with CD40 mutant transfectant (L929-CD40mu) as immunogen. The spleen B cells of the mice were fused with Sp2/0 myeloma cells. The hybridoma cells were screened with CD40 mutant transfectant (L929-CD40mu) by FCM. Faststrip analysis was performed to identify Ig subclass of this mAb. The epitope recognized by this mAb was detected by Bio-5C11 competitive assay. Western blot technique was adopted to identify the mAb. The proliferation of tumor cells in vitro was analyzed by MTT assay and apoptosis of tumor cells in vitro was analyzed by PI-annexin V assay. RESULTS: One hybridoma cell line named 10C5 was obtained, which had the property of secreting anti-human CD40 mutant monoclonal antibody continuously and steadily. This mAb specifically recognized human CD40 mutant molecule and induced the apoptosis of tumor cells in vitro. CONCLUSION: One hybridoma cell line which can secret a mouse anti-human CD40 mutant mAb has been prepared successfully. This mAb can inhibit the growth of tumor cells expressing CD40 mutant and induce their apoptosis in vitro.