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目的 研究纤溶酶原激活剂抑制物 2型 (PAI 2 )抑制细胞凋亡的分子机制。方法 用TNFα与放线菌酮协同诱发HeLa细胞建立HeLa细胞凋亡模型 ;将人PAI 2cDNA与真核表达载体 pcDNA3重组 ,构建PAI 2真核表达质粒并转染HeLa细胞 ,经G4 18筛选 ,NorthernBlot、WesternBlot鉴定筛选出稳定表达PAI 2的细胞株 ;运用结晶紫染色和MTT法检测表达PAI 2的HeLa细胞株对TNFα诱发细胞凋亡作用的抵抗作用 ,并检测胞外添加PAI 2对TNFα作用的影响和PAI 2对有H2 O2 引起的细胞损伤作用的影响。结果 HeLa细胞经TNFα诱导后发生凋亡 ;而PAI 2转染HeLa细胞后获得的三株表达克隆对TNFα诱导的细胞凋亡有明显的抵抗作用 ,对由H2 O2 引起的细胞凋亡无明显影响 ;胞外添加PAI 2对TNFα的作用无影响。结论 细胞内PAI 2的表达能抑制由TNFα引起的细胞凋亡 ,而对由H2 O2 引起的细胞凋亡无影响。
Objective To investigate the molecular mechanism of PAI 2 inhibition of apoptosis. Methods HeLa cells were induced by TNFα in combination with cycloheximide to establish a HeLa cell apoptosis model. Human PAI 2 cDNA was recombined with eukaryotic expression vector pcDNA3 to construct PAI 2 eukaryotic expression plasmid and transfected into HeLa cells. After G418 selection, Northern Blot , And the cell lines stably expressing PAI 2 were screened by Western Blot. The resistance of PAI 2-expressing HeLa cells to TNFα-induced apoptosis was detected by crystal violet staining and MTT assay, and the effect of PAI 2 on TNFα Effect of PAI 2 on H2O2-induced cellular injury. Results The apoptosis of HeLa cells was induced by TNFα. However, the three clones obtained after HeLa cells were transfected with PAI 2 showed obvious resistance to TNFα-induced apoptosis and had no effect on the apoptosis induced by H2O2 ; Extracellular addition of PAI 2 had no effect on TNFα. Conclusion The intracellular expression of PAI 2 can inhibit the apoptosis induced by TNFα, but has no effect on the apoptosis induced by H2O2.