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目的探讨人CD40-IgG1Fc段融合蛋白对EB病毒(EBV)转化的B淋巴细胞在细胞生长及凋亡方面的影响。方法应用流式细胞术检测EBV转化的B淋巴细胞膜表面异位表达CD40L;应用本研究所构建的CD40-IgG1Fc段融合蛋白CHO稳定表达株的培养上清与EBV转化的B淋巴细胞共孵育,通过MTT法检测B细胞的生长变化,吖啶橙(AO)、溴化乙锭(EB)双染色法和DNA ladder法检测融合蛋白对B细胞凋亡的影响。结果EBV转化的B细胞膜表面异位表达CD40L;CD40-IgG1Fc段融合蛋白可有效抑制EB病毒转化的B细胞生长,抑制程度与蛋白浓度呈正相关;AO与EB双染色法及DNA ladder法均检测到B细胞凋亡明显增加,与共孵育时间呈正相关。结论CD40-CD40L的相互作用是EBV转化的B细胞异常激活的重要机制,人CD40-IgG1Fc段融合蛋白能阻断CD40-CD40L的相互作用,抑制EB病毒转化的B细胞的生长,促进其凋亡,为下一步自身免疫性疾病的临床实验治疗奠定了基础。
Objective To investigate the effect of human CD40-IgG1Fc fusion protein on the growth and apoptosis of EBV-transformed B lymphocytes. Methods Flow cytometry was used to detect the ectopic expression of CD40L on the surface of EBV-transformed B lymphocytes. The supernatant of CD40-IgG1Fc fusion protein CHO stable expression strain constructed in this study was incubated with EBV-transformed B lymphocytes The growth of B cells was detected by MTT assay. Apoptosis of B cells was detected by acridine orange (AO), ethidium bromide (EB) double staining and DNA ladder method. Results CD40L was expressed on the surface of EBV-transformed B cell membrane. CD40-IgG1Fc fusion protein could effectively inhibit the growth of EBV-transformed B cells. The inhibition was positively correlated with the protein concentration. Both AO and EB double staining and DNA ladder B cell apoptosis increased significantly, and co-incubation time was positively correlated. Conclusions The interaction of CD40-CD40L is an important mechanism of abnormal activation of EBV-transformed B cells. The human CD40-IgG1 Fc fragment fusion protein can block the interaction of CD40-CD40L and inhibit the growth of EBV-transformed B cells and promote their apoptosis , Which laid the foundation for the clinical experimental treatment of autoimmune diseases in the next step.