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目的:本研究旨在研制抗人食管癌侵袭转移功能性单克隆抗体,确定其抑制食管癌转移的特性。方法:用5例新鲜肺转移性食管癌原发瘤标本中分离的有核细胞免疫6只BALB/c小鼠。选择高血清效价小鼠脾细胞与S/P20细胞融合,采用HAT在甲基纤维素中选择培养。使用活细胞免疫荧光,迁移、侵袭、黏附、细胞增殖等方法筛选和鉴定有治疗价值的功能性单克隆抗体。结果:细胞融合挑选单个集落1260个。经活细胞免疫荧光筛选获得545株识别食管癌细胞膜蛋白的单抗。再通过免疫组化筛选获得了485株不与正常人食管组织反应的相对特异的单抗。通过对迁移、侵袭、黏附、增殖等功能筛选获得70株抑制迁移、32株抑制肺内皮黏附的相关单抗。结论:采用高通量制备、筛选、鉴定功能性单克隆抗体技术,获得了多株能够显著抑制食管癌细胞转移相关生物学行为和功能的单抗。
OBJECTIVE: The purpose of this study was to develop functional monoclonal antibodies against esophageal cancer invasion and metastasis, and to determine its characteristics of inhibiting esophageal cancer metastasis. Methods: Six BALB / c mice were immunized with nucleated cells isolated from 5 fresh primary lung metastatic esophageal carcinomas. High-titer mouse spleen cells were selected for fusion with S / P20 cells and selectively cultured in methylcellulose using HAT. The use of living cells immunofluorescence, migration, invasion, adhesion, cell proliferation and other methods to screen and identify functional monoclonal antibody has therapeutic value. RESULTS: 1260 single colonies were picked by cell fusion. 545 strains of esophageal cancer cell membrane protein monoclonal antibodies were obtained by living cell immunofluorescence screening. And then by immunohistochemistry to obtain 485 strains not normal esophageal tissue reaction specific monoclonal antibody. Totally 70 anti-migration and 32 anti-lung mAbs were screened by functional screening of migration, invasion, adhesion and proliferation. Conclusion: The high-throughput preparation, screening and identification of functional monoclonal antibodies have led to the discovery of multiple monoclonal antibodies that significantly inhibit the biological behaviors and functions of esophageal cancer metastasis.