重组腺病毒突变型人低氧诱导因子1α对细胞凋亡调节机制的研究

Mechanism of recombinant adenovirus-mediated mutations of hypoxia inducible factor 1α in modulation of cell apoptosis

目的研究重组腺病毒突变型人低氧诱导因子1α(Ad-HIF-1α-Ala564-Ala803)对细胞凋亡的调节机制。方法将重组腺病毒Ad-HIF-1α-Ala564-Ala803和对照病毒Ad-lacZ感染常氧下LoVo细胞,荧光定量PCR检测不同时间点HIF-1α,p21WAF1/CIP1的mRNA表达水平,Westernblot检测HIF-1α,p21WAF1/CIP1的蛋白表达水平,及Hoechst染色检测loVo细胞的凋亡率。结果Ad-HIF-1α-Ala564-Ala803感染LoVo细胞后随着HIF-1αmRNA和蛋白表达水平的增高,p21WAF1/CIP1的mRNA和蛋白表达水平也相应增高,Hoechst染色示Ad-HIF-1α-Ala564-Ala803组细胞的凋亡率(16.2%)明显高于对照组(5.5%)(P=0.00)。结论HIF-1α可通过上调p21WAF1/CIP1的表达,诱导细胞周期停滞,促进细胞凋亡。

Objective To investigate the mechanism by which recombinant adenovirus （Ad）-mediated mutations ofhypoxia inducible factor lot （Ad-HIF-1α-Ala564-Ala803） regulates cell apoptosis. Methods LoVo cells were infected with recombinant Ad-HIF-1α-Ala564-Ala803 and control virus Ad-lacZ under normoxia condition. Real-time PCR was used to detect HIF-1α and p21WAF1/CIP1 mRNA expressions at different time points. Western blotting was employed to verify HIF-1α and p21WAF1/CIP1 protein expression. Hoechst 33342 ftourescein staining was performed to observe the ratio of apoptotic LoVo cells. Results The expression levels ofHIF-1α mRNA and protein increased after infection with Ad-HIF-1α- Ala564-Alag03, accompanied by an increase in p21WAF1/CIP1 mRNA and protein expressions. The apoptotic ratio was significantly higher in LoVo cells infected with recombinant Ad-HIF-1α-Ala564-Ala803 （16.2%） than in the control ceils （5.5%, P=0.00）. Conclusion HIF-1α may induce cell cycle arrest by up-regulating p21WAF1/CIP1 expressions to promote LoVo cell apoptosis.