Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (28): 5212-5215.doi: 10.3969/j.issn.2095-4344.2012.28.017

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Resistance of recombinant X-linked inhibitor of apoptosis protein to H2O2-induced apoptosis of intestinal epithelial cells

Zhang Xian, Huang Yu, Zeng Xing   

  1. Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong Province, China
  • Received:2011-12-03 Revised:2012-01-19 Online:2012-07-08 Published:2012-07-08
  • About author:Zhang Xian☆, Doctor, Assistant researcher, Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong Province, China zhangx07@yahoo.com.cn

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Abstract:

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.
OBJECTIVE: To construct eukaryotic expression vector pXIAP-GFP expressing recombinant rat XIAP, and to investigate its resistance and potential mechanism to H2O2-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 pXIAP-GFP or pCMV6-AC-GFP, IEC-6 cells were stimulated by H2O2. Cell survival was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Expressions of XIAP, GFP, and Caspase-3 were detected by western blot.
RESULTS 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.

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