本文關(guān)鍵詞： 腺病毒 HIF-1α p21WAF1/CIP1 細胞增殖 細胞周期 細胞凋亡　出處：《南方醫(yī)科大學》2008年博士論文　論文類型：學位論文

【摘要】： 研究背景 哺乳動物細胞通過改變代謝狀態(tài)和生長速率以應(yīng)對不同的氧濃度的變化。越來越多證據(jù)提示,低氧以兩種不同的方式,即通過程序性細胞死亡或通過生長停滯,來改變細胞的增殖。在轉(zhuǎn)化細胞或永生細胞中,細胞凋亡是對低氧的主要反應(yīng),是通過激活p53腫瘤抑制基因產(chǎn)物依賴性通路發(fā)生的。這些改變主要是凋亡通路中p53直接作用產(chǎn)生的。然而,p53蛋白的蓄積和轉(zhuǎn)錄激活僅見于近似于缺氧的環(huán)境,而不是低氧的環(huán)境。 最近的研究提示,低氧/HIF-1α可抑制細胞生長,誘導(dǎo)細胞凋亡。HIF-1α可能通過兩個機制誘導(dǎo)細胞凋亡。第一,通過增強p53產(chǎn)物穩(wěn)定性。在環(huán)境應(yīng)激或DNA損傷時,p53通過調(diào)節(jié)Bax蛋白誘導(dǎo)凋亡,或通過p21WAF1/CIP1介導(dǎo),引起生長停滯。第二,低氧時,過度表達BNIP3結(jié)合到抗凋亡蛋白Bcl-2和Bcl-xL上,并抑制它們的活性,從而誘導(dǎo)細胞凋亡。由BNIP3介導(dǎo)的低氧誘導(dǎo)的細胞凋亡可能是HIF-1依賴性,因為缺乏HIF-1的細胞不能產(chǎn)生大量的BNIP3,細胞死亡率降低。先前的研究提示,低氧誘導(dǎo)的細胞周期停滯,常伴隨某些細胞周期蛋白依賴激酶(CDK)復(fù)合物的活性下降和視網(wǎng)膜母細胞瘤(Rb)蛋白的低磷酸化,抑制細胞周期的進展。然而,目前對HIF-1在細胞周期裝置中的作用的知之甚少。最近的研究闡述了HIF-1在低氧時對調(diào)節(jié)細胞周期的進展具有重要作用,并顯示HIF-1的誘導(dǎo)激活至少是通過兩個獨特的機制抑制G1/S轉(zhuǎn)折,第一,兩個細胞周期蛋白依賴激酶抑制因子(CKIs)p21WAF1/CIP1和p27KIP1呈HIF-1依賴性轉(zhuǎn)錄表達增高。這些CKIs持續(xù)表達見于野生型細胞,而未見于缺乏HIF-1α的細胞。p21WAF1/CIP1和p27KIP1抑制細胞周期蛋白/CDK2活性,引起細胞周期停滯于G1/S界面。第二,HIF-1可能調(diào)節(jié)細胞周期蛋白E,而不是調(diào)節(jié)細胞周期蛋白A的水平;這兩個蛋白與CDK2結(jié)合,并依據(jù)細胞周期的時期,調(diào)節(jié)細胞周期蛋白E/CDK2激酶的活性。然而,細胞分裂需要細胞周期蛋白和細胞周期蛋白依賴激酶的協(xié)調(diào)作用,促進細胞周期進入S期和有絲分裂。p21WAF1/CIP1是一個細胞周期蛋白依賴激酶抑制因子,通過抑制細胞周期蛋白依賴激酶的活性,抑制異常和過早的細胞增殖。在細胞損傷時,p21WAF1/CIP1表達增高。除了對細胞周期進展的調(diào)控外,p21WAF1/CIP1亦參與DNA修復(fù)和細胞凋亡的過程。 HIF-1是轉(zhuǎn)錄因子和低氧反應(yīng)時的重要調(diào)節(jié)因子。其大多數(shù)靶基因與細胞增殖、細胞存活、細胞凋亡、血管生成和葡萄糖代謝等密切相關(guān)。HIF-1由α和β亞基組成。β亞基為組成性表達,而α亞基的活性對氧水平的降低非常敏感。低氧主要在蛋白水平對HIF-1進行調(diào)節(jié)。在正常氧條件下,HIF-1α氧依賴降解結(jié)構(gòu)域(ODDD)的兩個脯氨酸殘基(人HIF-1α中的402位脯氨酸和564位脯氨酸,以564位脯氨酸為主)的羥化介導(dǎo)與VHL/E3復(fù)合物相互作用,使HIF-1α通過蛋白酶體破壞;HIF-1α的轉(zhuǎn)錄激活結(jié)構(gòu)域(TAD)的天冬酰胺殘基(人HIF-1α中的803位天冬酰胺)的羥化阻斷HIF-1α與轉(zhuǎn)錄輔助激活因子CBP/p300結(jié)合。 在常氧下,HIF-1α不穩(wěn)定并迅速被降解。先前對HIF-1α功能的研究通常在低氧下進行,而低氧本身可誘導(dǎo)細胞凋亡。一些策略已成功應(yīng)用于常氧下對HIF-1α的實驗性激活。例如,去除重要的ODDD可獲得有活性的重組HIF-1α分子。另一種方法是將HIF-1α的N末端的DNA和二聚體化結(jié)構(gòu)域與單純皰疹病毒VP16的轉(zhuǎn)錄激活結(jié)構(gòu)域相融合。此外,使用針對HIF-1α羥化酶的小分子抑制因子可穩(wěn)定HIF-1和激活轉(zhuǎn)錄反應(yīng)。但是,這些方法存在許多弊端,并不能完全模擬HIF-1α的特性。 在我們的實驗中,為了進一步研究常氧下HIF-1α調(diào)節(jié)細胞增殖和細胞凋亡的作用,并探討HIF-1α與p21WAF1/CIP1的相互關(guān)系,我們使用一個重組腺病毒載體,表達在564位脯氨酸和803位天冬酰胺均進行點突變的人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蛋白在常氧下幾乎維持全部的活性。 目的 研究重組腺病毒突變型人低氧誘導(dǎo)因子1α(Ad-HIF-1α-Ala564-Ala803)調(diào)節(jié)細胞增殖和細胞凋亡的分子機制。 方法 將重組腺病毒Ad-HIF-1α-Ala564-Ala803和對照病毒Ad-lacZ在HEK293A細胞中擴增,終點稀釋實驗法測定病毒滴度。X-gal染色檢測重組腺病毒對LoVo細胞的感染效率。重組腺病毒在常氧下以MOI=60感染LoVo細胞,用熒光定量PCR和Western blot分別檢測不同時間點HIF-1α與p21WAF1/CIP1的mRNA和蛋白表達水平,MTT檢測細胞增殖,流式細胞儀檢測細胞周期的變化,Hoechst染色檢測LoVo細胞的凋亡率。 結(jié)果 (1)重組腺病毒擴增后能獲得較高的滴度,Ad-HIF-1α-Ala564-Ala803和Ad-lacZ的滴度分別為4.0×10~9pfu/ml,6.3×10~9pfu/ml。重組腺病毒在LoVo細胞中具有很高的感染效率,感染效率與MOI成量效關(guān)系。當MOI=60時,感染效率為90%以上。 (2)重組腺病毒常氧下感染LoVo細胞后,在不同時間點檢測HIF-1α與p21WAF1/CIP1的mRNA表達水平,發(fā)現(xiàn)在Ad-HIF-1α-Ala564-Ala803組中,隨HIF-1α表達的增高,p21WAF1/CIP1的表達也相應(yīng)增高。HIF-1α蛋白表達維持在較高的水平,p21WAF1/CIP1的蛋白表達則逐漸增高。而Ad-lacZ組的HIF-1α和p21WAF1/CIP1蛋白均只有較低水平表達。 (3),Ad-HIF-1α-Ala564-Ala803組與Ad-lacZ組的MTT無統(tǒng)計學差異;但Ad-HIF-1α-Ala564-Ala803組細胞周期中G_1期的細胞增加,S期的細胞減少。 (4) Ad-HIF-1α-Ala564-Ala803組細胞的凋亡率高于Ad-lacZ組。 結(jié)論 (1)重組腺病毒載體介導(dǎo)的HIF-1α-Ala564-Ala803基因可有效地轉(zhuǎn)染LoVo細胞并成一定的量效關(guān)系。 (2) HIF-1α在細胞周期和細胞凋亡的調(diào)節(jié)中發(fā)揮重要的作用,可能通過上調(diào)p21WAF1/CIP1的表達,誘導(dǎo)細胞周期停滯于G_1期,并促進細胞凋亡。
[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偽 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 偽 and 尾 subunits . Under normoxia , HIF - 1偽 is unstable and rapidly degraded . Previous studies of HIF - 1偽 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偽 . In addition , the use of small molecule inhibitors for HIF - 1偽 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偽 . In our experiments , in order to further study the role of HIF - 1偽 in the regulation of cell proliferation and apoptosis , and to investigate the relationship between HIF - 1偽 and p21 ~ ( 1 / CIP1 ) , we used a recombinant adenovirus vector to express the human HIF - 1偽 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偽 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偽 ( Ad - HIF - 1偽 - Ala564 - Ala803 ) was investigated . method The recombinant adenovirus Ad - HIF - 1偽 - 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偽 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偽 - 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偽 - Ala564 - Ala803 group , it was found that the expression of HIF - 1偽 was higher in Ad - HIF - 1偽 - Ala564 - Ala803 than in Ad - HIF - 1偽 - Ala564 - Ala803 group . ( 3 ) In Ad - HIF - 1偽 - Ala564 - Ala803 group , there was no statistical difference between the groups of Ad - HIF - 1偽 and Ala564 - Ala803 , but the cells in Ad - HIF - 1偽 - Ala564 - Ala803 group increased in G1 phase and decreased in S phase . ( 4 ) The apoptosis rate of Ad - HIF - 1偽 - Ala564 - Ala803 group was higher than that in Ad - 1 group . Conclusion ( 1 ) The recombinant adenovirus vector - mediated HIF - 1偽 - Ala564 - Ala803 gene can effectively transfer LoVo cells into a certain dose - effect relationship . ( 2 ) HIF - 1偽 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 .

【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R363

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相關(guān)期刊論文 前1條

1 胡英芳;王月剛;謝宜軍;賴文巖;賴艷嫻;吳平生;;構(gòu)建雙突變型低氧誘導(dǎo)因子1α腺病毒載體的實驗[J];中國組織工程研究與臨床康復(fù);2007年33期

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本文編號：1474302