重组腺病毒突变型人低氧诱导因子1α调节细胞周期的分子机制  

Molecular mechanism of recombinant adenovirus-mediated mutations of human hypoxia inducible factor 1 alpha for regulating cell cycle

作　　者：韦莉莉[1] 王月刚[1] 陈娓[1] 唐其东[1] 赖艳娴[1] 胡英芳[1] 吴平生[1] 

机构地区：[1]南方医科大学南方医院心内科,广东省广州市510515

出　　处：《中国组织工程研究与临床康复》2008年第20期3814-3817,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30370987)~~

摘　　要：背景:目前研究显示低氧诱导因子1α可诱导细胞周期停滞,但其机制尚不清楚。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)调节细胞周期的分子机制。设计、时间及地点:对照实验,于2007-03/12在南方医科大学中医实验中心完成。材料:人结肠腺癌细胞株LoVo细胞由南方医院消化内科实验室提供;重组腺病毒Ad-HIF-1α-Ala564-Ala803和对照病毒Ad-lacZ载体为自行构建。方法:将重组腺病毒Ad-HIF-1α-Ala564-Ala803和对照病毒Ad-lacZ在HEK293A细胞中扩增,测定病毒滴度,然后在常氧下以感染复数10,20,30,40,50,60,70,80,90,100感染LoVo细胞。主要观察指标:X-gal染色检测重组腺病毒对LoVo细胞的感染效率;荧光定量PCR检测不同时间点低氧诱导因子1α与p21WAF1/CIP1的mRNA表达水平;MTT检测细胞增殖;流式细胞仪检测细胞周期的变化。结果:①重组腺病毒扩增后能获得较高的滴度,在LoVo细胞中具有很高的感染效率,感染效率与感染复数成量效关系,当感染复数为60时,感染效率为90%以上。②随低氧诱导因子1α表达的增高,p21WAF1/CIP1的表达也相应增高,两者存在正相关(r=0.945,P<0.05)。③与对照病毒相比,重组腺病毒感染后LoVo细胞增殖轻度受抑;细胞周期示G1期细胞明显增加,S期的细胞显著减少(P<0.05)。结论:①重组腺病毒载体介导的人HIF-1α-Ala564-Ala803基因可有效地转染LoVo细胞并成一定的量效关系。②低氧诱导因子1α在细胞周期的调节中发挥重要的作用,可能通过上调p21WAF1/CIP1的表达,诱导细胞周期停滞于G1期。BACKGROUND： Present study demonstrated that hypoxia inducible factor 1α （HIF-1α ） can induce cell cycle arrest, but its mechanism is unclear. OBJECTIVE： To investigate the molecular mechanism of recombinant adenovirus-mediated mutations of human hypoxia inducible factor 1α （Ad-HIF- 1α -Ala564-Ala803） for regulating cell cycle. DESIGN, TIME AND SETTING： A control experiment was conducted at the Experimental Center of Traditional Chinese Medicine of Southern Medical University from March to December 2007. MATERIALS： Human adenocarcinoma of colon cell strain LoVo cell was provided by Laboratory of Department of Gastroenterology of Southern Medical University. Recombinant adenovirus Ad-HIF-1α-Ala564-Ala803 and Ad-lacZ were self-constructed. METHODS： Recombinant adenovirus Ad-HIF-1α -Ala564-Ala803 and Ad- lacZ were amplified in HEK 293A cells and adenoviral titer was determined. LoVo cells were infected at multiplicity of infection （MOI） of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 in normoxia. MAIN OUTCOME MEASURES： The infection efficiency was observed with X-gal staining. The expression of mRNA level of HIF-1α and p21WAF1/CIP1 in LoVo cells was performed by fluorescent quantitation polymerase chain reaction （PCR）. The cell proliferation was assessed by MTT assay. Flow cytometer was used to analyze the changes in cell cycle. RESULTS： High-titer adenoviruses were produced after amplified. LoVo cells could be effectively infected by recombinant adenovirus in vitro; the infection efficiency had the dose-effect relationship with MOI. When MOI was 60, the infection efficiency was more than 90%. With the increase of mRNA level of HIF-1α, that of p21WAF1/CIP1 was increased. There was positive correlation between the mRNA level of HIF-1α and that of p21WAF1/CIP （r=-0.945, P 〈 0. 05）. Compared with Ad-lacZ group, cell proliferation was slightly inhibited, and the ratio of cells in G1 phase increased and meanwhile that in S phase decreased significantly （P 〈 0.05） in

关 键 词：腺病毒 HIF-1Α P21WAF1/CIP1 细胞周期停滞 

分 类 号：R392.114[医药卫生—免疫学]