重组腺病毒突变型人低氧诱导因子1α调节细胞增殖和细胞凋亡的分子机制研究

发布时间：2018-01-29 20:35

本文关键词： 腺病毒 HIF-1α p21WAF1/CIP1 细胞增殖细胞周期细胞凋亡　出处：《南方医科大学》2008年博士论文　论文类型：学位论文

【摘要】： 研究背景哺乳动物细胞通过改变代谢状态和生长速率以应对不同的氧浓度的变化。越来越多证据提示,低氧以两种不同的方式,即通过程序性细胞死亡或通过生长停滞,来改变细胞的增殖。在转化细胞或永生细胞中,细胞凋亡是对低氧的主要反应,是通过激活p53肿瘤抑制基因产物依赖性通路发生的。这些改变主要是凋亡通路中p53直接作用产生的。然而,p53蛋白的蓄积和转录激活仅见于近似于缺氧的环境,而不是低氧的环境。最近的研究提示,低氧/HIF-1α可抑制细胞生长,诱导细胞凋亡。HIF-1α可能通过两个机制诱导细胞凋亡。第一,通过增强p53产物稳定性。在环境应激或DNA损伤时,p53通过调节Bax蛋白诱导凋亡,或通过p21WAF1/CIP1介导,引起生长停滞。第二,低氧时,过度表达BNIP3结合到抗凋亡蛋白Bcl-2和Bcl-xL上,并抑制它们的活性,从而诱导细胞凋亡。由BNIP3介导的低氧诱导的细胞凋亡可能是HIF-1依赖性,因为缺乏HIF-1的细胞不能产生大量的BNIP3,细胞死亡率降低。先前的研究提示,低氧诱导的细胞周期停滞,常伴随某些细胞周期蛋白依赖激酶(CDK)复合物的活性下降和视网膜母细胞瘤(Rb)蛋白的低磷酸化,抑制细胞周期的进展。然而,目前对HIF-1在细胞周期装置中的作用的知之甚少。最近的研究阐述了HIF-1在低氧时对调节细胞周期的进展具有重要作用,并显示HIF-1的诱导激活至少是通过两个独特的机制抑制G1/S转折,第一,两个细胞周期蛋白依赖激酶抑制因子(CKIs)p21WAF1/CIP1和p27KIP1呈HIF-1依赖性转录表达增高。这些CKIs持续表达见于野生型细胞,而未见于缺乏HIF-1α的细胞。p21WAF1/CIP1和p27KIP1抑制细胞周期蛋白/CDK2活性,引起细胞周期停滞于G1/S界面。第二,HIF-1可能调节细胞周期蛋白E,而不是调节细胞周期蛋白A的水平;这两个蛋白与CDK2结合,并依据细胞周期的时期,调节细胞周期蛋白E/CDK2激酶的活性。然而,细胞分裂需要细胞周期蛋白和细胞周期蛋白依赖激酶的协调作用,促进细胞周期进入S期和有丝分裂。p21WAF1/CIP1是一个细胞周期蛋白依赖激酶抑制因子,通过抑制细胞周期蛋白依赖激酶的活性,抑制异常和过早的细胞增殖。在细胞损伤时,p21WAF1/CIP1表达增高。除了对细胞周期进展的调控外,p21WAF1/CIP1亦参与DNA修复和细胞凋亡的过程。 HIF-1是转录因子和低氧反应时的重要调节因子。其大多数靶基因与细胞增殖、细胞存活、细胞凋亡、血管生成和葡萄糖代谢等密切相关。HIF-1由α和β亚基组成。β亚基为组成性表达,而α亚基的活性对氧水平的降低非常敏感。低氧主要在蛋白水平对HIF-1进行调节。在正常氧条件下,HIF-1α氧依赖降解结构域(ODDD)的两个脯氨酸残基(人HIF-1α中的402位脯氨酸和564位脯氨酸,以564位脯氨酸为主)的羟化介导与VHL/E3复合物相互作用,使HIF-1α通过蛋白酶体破坏;HIF-1α的转录激活结构域(TAD)的天冬酰胺残基(人HIF-1α中的803位天冬酰胺)的羟化阻断HIF-1α与转录辅助激活因子CBP/p300结合。在常氧下,HIF-1α不稳定并迅速被降解。先前对HIF-1α功能的研究通常在低氧下进行,而低氧本身可诱导细胞凋亡。一些策略已成功应用于常氧下对HIF-1α的实验性激活。例如,去除重要的ODDD可获得有活性的重组HIF-1α分子。另一种方法是将HIF-1α的N末端的DNA和二聚体化结构域与单纯疱疹病毒VP16的转录激活结构域相融合。此外,使用针对HIF-1α羟化酶的小分子抑制因子可稳定HIF-1和激活转录反应。但是,这些方法存在许多弊端,并不能完全模拟HIF-1α的特性。在我们的实验中,为了进一步研究常氧下HIF-1α调节细胞增殖和细胞凋亡的作用,并探讨HIF-1α与p21WAF1/CIP1的相互关系,我们使用一个重组腺病毒载体,表达在564位脯氨酸和803位天冬酰胺均进行点突变的人HIF-1α基因。其ODDD重要的脯氨酸和TAD重要的天冬酰胺均被丙氨酸替代,可使HIF-1α蛋白在常氧下几乎维持全部的活性。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)调节细胞增殖和细胞凋亡的分子机制。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,终点稀释实验法测定病毒滴度。X-gal染色检测重组腺病毒对LoVo细胞的感染效率。重组腺病毒在常氧下以MOI=60感染LoVo细胞,用荧光定量PCR和Western blot分别检测不同时间点HIF-1α与p21WAF1/CIP1的mRNA和蛋白表达水平,MTT检测细胞增殖,流式细胞仪检测细胞周期的变化,Hoechst染色检测LoVo细胞的凋亡率。结果 (1)重组腺病毒扩增后能获得较高的滴度,Ad-HIF-1α-Ala564-Ala803和Ad-lacZ的滴度分别为4.0×10~9pfu/ml,6.3×10~9pfu/ml。重组腺病毒在LoVo细胞中具有很高的感染效率,感染效率与MOI成量效关系。当MOI=60时,感染效率为90%以上。 (2)重组腺病毒常氧下感染LoVo细胞后,在不同时间点检测HIF-1α与p21WAF1/CIP1的mRNA表达水平,发现在Ad-HIF-1α-Ala564-Ala803组中,随HIF-1α表达的增高,p21WAF1/CIP1的表达也相应增高。HIF-1α蛋白表达维持在较高的水平,p21WAF1/CIP1的蛋白表达则逐渐增高。而Ad-lacZ组的HIF-1α和p21WAF1/CIP1蛋白均只有较低水平表达。 (3),Ad-HIF-1α-Ala564-Ala803组与Ad-lacZ组的MTT无统计学差异;但Ad-HIF-1α-Ala564-Ala803组细胞周期中G_1期的细胞增加,S期的细胞减少。 (4) Ad-HIF-1α-Ala564-Ala803组细胞的凋亡率高于Ad-lacZ组。结论 (1)重组腺病毒载体介导的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) HIF-1α在细胞周期和细胞凋亡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G_1期,并促进细胞凋亡。
[Abstract]:Background of the study In transformed cells or immortal cells , apoptosis is a major response to hypoxia by activating p53 tumor suppressor gene product - dependent pathways . These changes are primarily due to p53 direct effects in apoptotic pathways . However , accumulation and transcriptional activation of p53 proteins occurs only in an environment that approximates anoxic , rather than a hypoxic environment . It is suggested that hypoxia / HIF - 1伪 can inhibit the growth of cells and induce apoptosis . HIF - 1 is an important regulator of transcription factor and hypoxia response . Most of the target genes are closely related to cell proliferation , cell survival , apoptosis , angiogenesis and glucose metabolism . HIF - 1 is composed of 伪 and 尾 subunits . Under normoxia , HIF - 1伪 is unstable and rapidly degraded . Previous studies of HIF - 1伪 function are usually performed under hypoxia , while hypoxia itself can induce apoptosis . Some strategies have been successfully applied to the experimental activation of HIF - 1伪 . In addition , the use of small molecule inhibitors for HIF - 1伪 hydroxylase can stabilize HIF - 1 and activate transcription reactions . However , these methods have a number of drawbacks and do not fully mimic the characteristics of HIF - 1伪 . In our experiments , in order to further study the role of HIF - 1伪 in the regulation of cell proliferation and apoptosis , and to investigate the relationship between HIF - 1伪 and p21 ~ ( 1 / CIP1 ) , we used a recombinant adenovirus vector to express the human HIF - 1伪 gene with point mutation at 564 - position proline and 803 - position asparagine . The important proline of ODDD and asparagine - important asparagine are replaced by alanine , which can make HIF - 1伪 protein maintain almost all activity under normoxia . Purpose The molecular mechanism of regulating cell proliferation and apoptosis by recombinant adenovirus mutant hypoxia inducible factor 1伪 ( Ad - HIF - 1伪 - Ala564 - Ala803 ) was investigated . method The recombinant adenovirus Ad - HIF - 1伪 - Ala564 - Ala803 and the control virus Ad - lac were amplified by an end - dilution experiment to determine the infection efficiency of the recombinant adenovirus . The recombinant adenovirus was infected with LoVo cells under normal oxygen . The mRNA and protein expression levels of HIF - 1伪 and p21 ~ ( 1 / CIP1 ) were detected by fluorescence quantitative PCR and Western blot . The proliferation of cells was detected by MTT and flow cytometry . The apoptosis rate of LoVo cells was detected by flow cytometry . Results ( 1 ) After amplification of recombinant adenovirus , the titer of Ad - HIF - 1伪 - Ala564 - Ala803 and Ad - lac were 4.0 脳 10 ~ 9 and 6 . 3 脳 10 ~ 9 / ml , respectively . The recombinant adenovirus had high infection efficiency in LoVo cells . The efficiency of infection was related to the infection efficiency . When the infection rate was 60 , the infection efficiency was over 90 % . ( 2 ) In Ad - HIF - 1伪 - Ala564 - Ala803 group , it was found that the expression of HIF - 1伪 was higher in Ad - HIF - 1伪 - Ala564 - Ala803 than in Ad - HIF - 1伪 - Ala564 - Ala803 group . ( 3 ) In Ad - HIF - 1伪 - Ala564 - Ala803 group , there was no statistical difference between the groups of Ad - HIF - 1伪 and Ala564 - Ala803 , but the cells in Ad - HIF - 1伪 - Ala564 - Ala803 group increased in G1 phase and decreased in S phase . ( 4 ) The apoptosis rate of Ad - HIF - 1伪 - Ala564 - Ala803 group was higher than that in Ad - 1 group . Conclusion ( 1 ) The recombinant adenovirus vector - mediated HIF - 1伪 - Ala564 - Ala803 gene can effectively transfer LoVo cells into a certain dose - effect relationship . ( 2 ) HIF - 1伪 plays an important role in the regulation of cell cycle and cell apoptosis , which may induce cell cycle arrest in G _ 1 phase and promote apoptosis by up - regulating the expression of p21 ~ + / CIP1 .

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

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