【摘要】 目的 为了进一步了解纤溶酶原激活剂抑制物2型(PAI-2)在细胞内的定位对其抗凋亡活性的影响。方法 构建绿色荧光蛋白质(EGFP)与PAI-2入核突变体融合蛋白质的表达质粒;转染HeLa细胞,荧光显微镜下比较野生型PAI-2与PAI-2的入核突变体在HeLa细胞内分布的差异;用MTT法检测PAI-2入核突变体抗肿瘤坏死因子-α(TNF-α)诱导细胞凋亡的活性。结果 在核定位序列的引导下,几乎所有的PAI-2都进入了细胞核,而野生型PAI-2则弥散分布于HeLa细胞内。MTT法检测发现,当PAI-2被定位于细胞核内时,丧失其抗凋亡的功能。结论 PAI-2在细胞内的定位能影响其抗凋亡的活性,并且当用TNF-α诱导HeLa细胞凋亡时,野生型PAI-2在细胞内的分布没有发生明显的改变。更多还原

【Abstract】 Purpose To study the effect of cellular location of PAI-2 on its antiapoptotic activity. Methods The cDNA of PAI-2-NLS mutant,3’-terminus of which a nuclear localization sequence was in-serted downstream,was cloned in-frame into eukaryotic expression vector,pEGFPC3,producing pEGFPC3-PAI2-NLS. After transfected into HeLa cells, different distributions of PAI-2 and PAI2-NLS mutant in HeLa cells were observed through fluorescence microscopy,and MTT assay was used to analyze the antiapoptotic activity of this PAI-2 mutant. Results Due to the nuclear localization sequence, almost all PAI-2 mutants entered nuclei, while wide type PAI-2 distributed evenly in the cytoplasm and nucleus. The results of MTT assay indicated that if PAI-2 was located in the nuclei, it would lose its antiapoptotic activity against TNF-α. Conclusions All the results demonstrate that antiapoptotic activity of PAI-2 is affected by its cellular localization. In addition, the treatment of TNF-α would not affect the distribution of PAI-2 in HeLa cells.更多还原