缺氧诱导Canstatin基因表达实验研究

目的构建Canstatin基因缺氧表达增强体系，观察其在常氧与缺氧条件的基因表达量和生物学效应。方法定向克隆法构建含有缺氧反应元件（HRE）的Canstatin基因表达载体，荧光定量PCR法检测转染细胞在常氧和缺氧条件下Canstatin mRNA表达量，流式细胞术检测细胞周期变化及细胞凋亡，电镜下观察内皮细胞超微结构的改变。结果成功构建Canstatin缺氧增强表达体系。缺氧条件下该体系转染的细胞Canstatin mRNA的表达显著增强。该体系转染后人脐静脉内皮细胞出现G0-G1期阻滞并伴有大量细胞凋亡，电镜下观察到典型的凋亡改变。结论Canstatin缺氧表达体系在缺氧条件下表达量及生物学效应显著增强。

Hypoxia induced Canstatin high level expression system

Objective To establish hypoxia induced Canstatin high level expression system and study its biological effect under oxic and anoxic condition. Methods Hypoxia response elements （HRE） were inserted into the upper stream of Canstatin of recombinant vector pCMV Scrlpt-Cans we constructed in former research, and the new recombinant vector named pCMV-Script-3HRE-Cans. Real time RT-PCR method, flow cytometry and electron microscope were used in this research. Results The expression of Canstatin mRNA in pCMV-Script-3HRE-Cans transformed tumor cells and endothelial cells were increased greatly in anoxic condition than in oxic condition. G0-G1 phase blocking, significant apoptosis and typical ultrastructure were observed in the HUVEC transformed with recombinant vector pCMV-Script-3HRE-Cans. Conclusion The hypoxia induced Canstatin expression system can increase Canstatin mRNA expression and enhance the biological effects of Canstatin under anoxic condition.