87位氨基酸在TM-TNF-α诱导靶细胞凋亡中的作用

Function of 87^(th) position amino acid residue in target cell apoptosis indu ced by TM-TNF-α

目的 研究TM TNF α分子结构与功能的关系 ,阐明其诱导靶细胞凋亡的分子机制。方法 构建定点突变的 87/PheTM TNF pcDNA3.0 ,并瞬时转染Cos 7细胞 ,利用MTT法 ,annexinⅤ ,Westernblot检测TM TNF α突变体的胞毒效应 ,对靶细胞死亡方式及对核转录因子NF κB转位的影响。结果 87位氨基酸置换后 ,TM TNF α对靶细胞的杀伤率无明显改变 ,但TM TNF α突变体通过促进NF κB核转位 ,诱导靶细胞死亡的方式由凋亡转为坏死。结论 87位氨基酸是TM TNF α诱导靶细胞凋亡的关键氨基酸残基 。

Objective The structure-function of TM-TNF-α w as studied to illustrate the molecular mechanism of target cell apoptosis inductio n. Methods Site-directed mutant -87/Phe TM-TNF-α w as reconstructed into eukocyte expression vector pcDNA3.0 and transiently transf ected into Cos-7 cells. The mutein on transfected cells surface was tested for its cytotoxicity, the effect on the induction of target cell death manner and NF -κB translocation using MTT method, annexin Ⅴ and Western blot. Re sults The substitution at position of 87 had no effect on the cytotoxicity of TM-TNF, but the mutein could induce the change of target cell death manner, from apoptosis to necrosis, and promote NF-κB translocation from the cytoplasm into nucleus. Conclusion The result s suggested that the amino acid at position 87 may be one of the key residues to mediate apoptosis. The domain containing this residue may be the function regio n inducing apoptosis.[