人骨肉瘤融合瘤的快速制备及其体外的免疫效应  

Experimental research on rapid production of hybridoma of human osteosarcoma and a preliminary investigation of in vitro immunogenicity effect

作　　者：王臻[1] 吕昌伟[1] 李立文[1] 彭磊[1] 郭刚[1] 曹云新[2] 李雪松[2] 

机构地区：[1]第四军医大学西京医院全军骨科研究所,西安710033 [2]基础部免疫教研室

出　　处：《中国骨肿瘤骨病》2002年第6期329-332,328,共5页Chinse Journal Of Bone Tumor And Bone Disease

基　　金：国家自然科学基金资助(39770749)

摘　　要：目的 研究人骨肉瘤抗原进入提呈细胞的有效途径和方法,探索筛选制备人骨肉瘤与活化B淋巴细胞融合瘤的方法,并研究其体外免疫原性和作为人骨肉瘤免疫疫苗的可能性,为进一步临床应用做有意义的前期实验。方法抗人Ig M、金黄色葡萄球菌A蛋白及少量IL-2和PMA活化的人脾脏来源B淋巴细胞,以500mg/ml聚乙二醇为融合剂,使骨肉瘤细胞与活化B淋巴细胞进行化学融合,贴壁后去除未融合淋巴细胞,特异性抗B细胞抗体预包被平板捕捉与B细胞融合的骨肉瘤。条件培养基体外筛选扩增,检测融合效率。紫外线灭活,同时设单纯灭活的未融合骨肉瘤组作为对照,记数法测量各组刺激淋巴细胞增殖能力,各组培养所得淋巴细胞进行体外杀伤实验(效靶比50:1),改良MTT法测量淋巴细胞对亲源之骨肉瘤细胞的杀伤指数。结果 人B淋巴细胞体外激活后可以与人骨肉瘤细胞化学融合,可利用贴壁性质和特异性B淋巴细胞抗体等方法部分纯化融合细胞并适当扩增浓集。融合细胞于3周和4周表达B淋巴细胞的标志性抗原效率分别为60.6％和45.4％。紫外线灭活融合瘤明显提高其体外刺激淋巴细胞增殖能力(P<0.01),诱发淋巴细胞对亲源骨肉瘤细胞的杀伤作用(11.60％且P<0.01)。结论 利用人骨肉瘤细胞和活化B淋巴细胞可以制备融合瘤。Objectives The research intended to investigate some effective methods for the preparation of hybridoma of human osteosarcoma PL-11 with activated B lymphocytes and the in vitro immunogentcity of these fused tumor cells, for the potentials of osteosarcoma vaccine production.Methods Sarcoma cells PL-11 was fused with B - lymphocyte activated by anti -human Ig - M antibody, Staphylovoccus aurous protein A, and morsel dose of IL - 2 and phorbel - 12 - myristate - 13 - acetate by 500 mg/ml PEG. The fused cells was separated from nonfused B cells by adhesion method, and from unwanted sarcoma cells by B - lymphocyte - specific - antibody pre - coated plate, the content of fused cells were measured at 3 and 4 weeks , and ultraviolet - radiation inactivated hybridomas were cultured with lymphocytes, the nonfused PL - 11 cells as control. The proliferation and lymphocytotoxicity of both groups were measured by counting method and modified MTT method respectively. Results Osteosarcoma hybridma can be effectively produced by PEG fusion and selected by adhesion method and subsepuently antibody pre - coated plates, and then properly proliferated. The hybridomas percentage at 3 and 4 week were 60.6% and 45.5 % respectively, and inactivated hybridomas of human osteosarcoma significantly increased the proliferation of autologous lymphocytes ( P < 0.01= and induced their cytotoxicity (1.1.6% )on parental osteosarcoma. Conclusions rapidly - prepared hybridoma of human osteosarcoma with activated B - lymphocytes could be a new way to induce effective anti - sarcoma immuno - reaction, which have important potentials for the production of effective individual human osteosarcoma vaccine.

关 键 词：骨肉瘤 细胞融合 淋巴细胞 癌症疫苗 

分 类 号：R738.1[医药卫生—肿瘤]