重组XIAP对H_2O_2诱导小肠上皮细胞凋亡的保护作用

Resistance of recombinant X-linked inhibitor of apoptosis protein to H_2O_2-induced apoptosis of intestinal epithelial cells

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,Westernblot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,Westernblot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,Westernblot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。

BACKGROUND： X-linked inhibitor of apoptosis protein （XIAP） plays an important role in cell apoptosis, and it is a potentia therapeutic target in apoptosis-related diseases. OBJECTIVE： To construct eukaryotic expression vector pXIAP-GFP expressing recombinant rat XIAP, and to investigate its resistance and potential mechanism to H202-induced apoptosis. METHODS： 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 pXlAP-GFP or pCMV6-AC-GFP, IEC-6 cells were stimulated by H202. Cell survival was analyzed by 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay. Expressions of XlAP, GFP, and Caspase-3 were detected by western blot. RESULTS AND CONCLUSION： DNA sequencing data verified pXlAP-GFP contained complete and accurate rat XIAP gene sequence, and western blot data verified pXIAP-GFP could express XIAP correctly. These results indicated pXlAP-GFP was constructed successfully. After transfection and H202 stimulation, compared with pCMV6-AC-GFP control group, absorbance value in pXlAP-GFP group increased, while expression of Caspase-3 protein decreased. These findings indicate recombinant XIAP increases resistance to H202-induced apoptosis by inhibiting Caspase-3 activation.