BACKGROUND: X-linked inhibitor of apoptosis protein (XIAP) plays an important role in cell apoptosis, and it is a potential therapeutic target in apoptosis-related diseases.rnOBJECTIVE: To construct eukaryotic expression vector pXIAP-GFP expressing recombinant rat XIAP, and to investigate its resistance and potential mechanism to H2O2-induced apoptosis.rnMETHODS: XIAP ORF was amplified from IEC-6 mRNA by reverse transcription-PCR, pXIAP-GFP was constructed by inserting XIAP ORF into pCMV6-AC-GFP plasmid and verified by DNA sequencing. After transfection with pXIAP-GFP or pCMV6-AC-GFP, IEC-6 cells were stimulated by H2O2. Cell survival was analyzed byrn3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Expressions of XIAP, GFP, and Caspase-3 were detected by western blot.rnRESULTS AND CONCLUSION: DNA sequencing data verified pXIAP-GFP contained complete and accurate rat XIAP gene sequence, and western blot data verified pXIAP-GFP could express XIAP correctly. These results indicated pXIAP-GFP was constructed successfully. After transfection and H2O2 stimulation, compared with pCMV6-AC-GFP control group, absorbance value in pXIAP-GFP group increased, while expression of Caspase-3 protein decreased. These findings indicate recombinant XIAP increases resistance to H2O2-induced apoptosis by inhibiting Caspase-3 activation.%背景:XIAP在细胞抗凋亡中发挥关键作用,能作为与凋亡相关的多种疾病预防治疗的靶点.目的:构建大鼠重组XIAP表达质粒,观察其对H2O2诱导细胞凋亡的保护作用和机制.方法:采用RT-PCR从大鼠小肠上皮细胞IEC-6扩增出大鼠XIAP基因ORF序列,插入pCMV6-AC-GFP载体,重组成pXIAP-GFP表达质粒,测序鉴定.以重组pXIAP-GFP与对照质粒pCMV6-AC-GFP转染IEC-6细胞,Western blot检测细胞重组XIAP的表达,同时在H2O2诱导细胞凋亡后,MTT法检测细胞存活,Western blot检测细胞Casepase-3表达.结果与结论:经测序证实pXIAP-GFP重组序列正确,Western blot证实将其转染IEC-6细胞后细胞正确表达XIAP,表明pXIAP-GFP重组质粒构建成功;在转染质粒和H2O2诱导凋亡后,与对照质粒pCMV6-AC-GFP组比较,pXIAP-GFP组细胞 A值增高,Caspase- 3表达降低.表明重组XIAP能通过抑制Caspase-3的表达抵抗H2O2诱导的细胞凋亡.
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