【摘要】 目的 研究纤溶酶原激活剂抑制物2型(PAI-2)的两种不同突变体抵抗肿瘤坏死因子-α(TNF-α)诱导Hela细胞凋亡的活性差异,探讨PAI-2CD螺旋间区的空间构象对其抗凋亡功能的影响。方法 分别构建突变体PAI-2D和PAI-2M真核表达质粒。转染Hela细胞,RT-PCR鉴定,筛选出能稳定表达PAI-2D和PAI-2M的阳性细胞株。用MTT法检测PAI-2的这两个突变体抗凋亡的活性,并利用计算机同源模建,比较PAI-2的这两个突变体空间构象的变化。结果 突变体PAI-2M能抑制TNF-α诱导的Hela细胞凋亡,而PAI-2D则不能。PAI-2M较好地保持了野生型PAI-2的空间构象,而PAI-2D CD螺旋间区的空间构象则发生了较大的变化。结论 PAI-2抑制TNF-α诱导的细胞凋亡依赖于其CD螺旋间区空间构象的完整性,而与PENF结构域(PENF73～76)无关。更多还原

【Abstract】 Purpose To investigate different protective activities of two PAI-2 mutants against TNF-α mediated apopotosis and to explore the effect of the spatial structure of CD interhelical region of PAI-2 on its anti-apoptotic activity. Methods Two PAI-2 mutants,PAI-2D and PAI-2M,were cloned into eukaryotic expression vector respectively and were transfected into Hela cells. After being analyzed by RT-PCR, the positive cell strains were obtained. The anti-apoptotic effects of these two mutants were compared by MTT assay and computer-aided homology modeling revealed some changes of their spatial structures. Results PAI-2M could protect cells from the cytotoxic effects of TNF-α, while PAI-2D lost its protection function. Mostly PAI-2M retained the spatial structure of wild type PAI-2,while PAI-2D showed obvious difference, especially in the CD interhelical region. Conclusions The protection of PAI-2 from TNF-α induced apop-tosis depends on the spatial integrality of CD interhelical region of PAI - 2, and the PENF domain （PENF73₇6）in the CD interhelical region is not so important that it could be substituted.更多还原