【作者】 苟巧；

【导师】 米粲；

【作者基本信息】 重庆医科大学 ， 组织工程与细胞工程， 2009， 博士

【摘要】 目的:本文旨在研究抑癌基因PTEN缺失的小鼠胚胎成纤维细胞（MEFs）中抗氧化防御蛋白——过氧化物还原酶（Prx1,2,5,6）及铜锌-超氧化物歧化酶（Cu/Zn-SOD）表达下调对细胞抗氧化防御能力、细胞内活性氧（ROS）、脂质过氧化和DNA氧化损伤水平的影响,并探讨PTEN调控Prx1,2,5,6及Cu/Zn-SOD mRNA和蛋白表达的机制,以进一步完善PTEN的抑癌作用机理,对肿瘤的预防和治疗有一定的指导意义。方法:（1）用超氧化物歧化酶（SOD）检测试剂盒比较对照(PTEN^+╱+ MEFs)及PTEN缺失MEFs细胞(PTEN^-/- MEFs)内SOD活力水平;不同浓度过氧化氢（H₂O₂）作用后,中性彗星电泳检测PTEN^+╱+ MEFs及PTEN^-/- MEFs细胞中DNA双链断裂（DSBs）水平;Western blot检测不同浓度H₂O₂预处理后PTEN^+/+ MEFs及PTEN^-/- MEFs细胞内DNADSBs产物磷酸化组蛋白H2AX（γH2AX）表达水平。（2） 2’7’-二氯荧光素双乙酸盐（DCFH-DA）和二氢乙啶（DHE）荧光探针分别标记细胞内H₂O₂和超氧阴离子(O₂^·-)结合荧光显微镜及流式细胞学检测PTEN^+╱+ MEFs及PTEN^-/- MEFs细胞中基础ROS(H₂O₂和O₂^·-)水平。（3）用丙二醛（MDA）检测试剂盒比较PTEN^+/+MEFs及PTEN^-/-MEFs细胞中脂质过氧化产物MDA水平;免疫细胞化学检测PTEN^+/+MEFs及PTEN^-/-MEFs细胞内DNA氧化损伤产物8-羟基脱氧鸟苷（8-OH-dG）水平;Western blot及免疫荧光染色检测PTEN^+/+MEFs及PTEN^-/-MEFs细胞中DNA DSBs产物γH2AX蛋白表达水平;中性彗星电泳进一步检测PTEN^+/+MEFs及PTEN^-/-MEFs细胞内DNADSBs水平。（4）磷脂酰肌醇激酶（PI3K）抑制剂LY294002作用PTEN^-/-MEFs细胞后,Western blot检测丝氨酸/苏氨酸蛋白激酶AKT（或称PKB）磷酸化水平;Western blot和Northern blot分别检测LY294002作用后PTEN^-/-MEFs细胞中Prx1,2,5,6及Cu/Zn-SOD蛋白和mRNA表达水平;Western blot进一步检测PTEN^+/+MEFs及PTEN^-/-MEFs细胞中转录因子FOXO3a磷酸化水平及LY294002作用PTEN^-/-MEFs细胞后FOXO3a磷酸化水平。结果:（1）PTEN缺失MEFs细胞抗氧化防御能力降低:PTEN^+/+MEFs细胞的SOD活力是PTEN^-/-MEFs细胞的2.09倍（p＜0.01）,说明PTEN^-/-MEFs细胞中SOD清除O₂^·-能力降低。中性彗星电泳结合Western blot检测显示PTEN^-/-MEFs细胞在0.01mmol/LH₂O₂作用后就出现具有统计学意义的DNA DSBs水平升高（p＜0.01）,而PTEN^+/+MEFs细胞则要在H₂O₂作用浓度达到0.1mmol/L时,才出现具统计学意义的DNA DSBs损伤增强（p＜0.01）,说明PTEN^-/-MEFs细胞清除H₂O₂、防御其诱发的DNA氧化损伤的能力降低。（2）PTEN^-/-MEFs细胞内基础ROS水平升高:流式细胞学显示荧光探针孵育的不同时间点,PTEN^-/-MEFs细胞内2’7’-二氯荧光素（DCF）和氧化乙啶（Eth）荧光水平均明显高于PTEN^+/+MEFs细胞（p＜0.05和p＜0.01）,荧光显微图像显示PTEN^-/-MEFs细胞内DCF和Eth荧光强度均明显高于PTEN^+/+MEFs,均表明PTEN^-/-MEFs细胞内H₂O₂和O₂^-水平升高。（3）PTEN缺失MEFs细胞脂质过氧化和DNA氧化损伤增强:PTEN^+/+MEFs细胞内MDA水平明显低于PTEN^-/-MEFs细胞（p＜0.01）,分别为1.3±0.1 nmol/mgprot和1.75±0.05 nmol/mgprot,说明PTEN^-/-MEFs细胞脂质过氧化水平升高,氧化损伤增强。免疫细胞化学显示PTEN^-/-MEFs细胞内8-OH-dG水平是PTEN^+/+MEFs细胞的3.1倍（p＜0.01）,说明PTEN^-/-MEFs细胞DNA氧化损伤水平升高。Western blot和免疫荧光染色显示PTEN^-/-MEFs细胞中DNA DSBs产物γH2AX蛋白表达水平明显高于PTEN^+/+MEFs（p＜0.01）,中性彗星电泳显示PTEN^+/+MEFs细胞平均尾力矩（15.48）明显低于PTEN^-/-MEFs细胞（22.03）（p＜0.01）,均表明PTEN^-/-MEFs细胞DNA DSBs损伤增强。（4）PTEN可通过拮抗PI3K/AKT通路促进Prx1,2,5,6及Cu/Zn-SODmRNA和蛋白表达并抑制FOXO3a磷酸化:Western blot和Northern blot显示PI3K抑制剂LY294002作用后,PTEN^-/-MEFs细胞中AKT激酶活化明显抑制,而Prx1,2,5,6及Cu/Zn-SOD蛋白和mRNA均表达增强,说明PTEN可通过拮抗PI3K/AKT通路促进Prx1,2,5,6及Cu/Zn-SOD基因转录和蛋白表达。此外,Western blot显示PTEN^-/- MEFs细胞中转录因子FOXO3a磷酸化水平升高,LY294002作用PTEN^-/-MEFs细胞后FOXO3a磷酸化水平降低,说明PTEN可通过拮抗PI3K/AKT通路抑制下游转录因子FOXO3a磷酸化。结论:（1）PTEN作为一个重要的抑癌基因,其缺失可引起抗氧化防御蛋白Prx1,2,5,6及Cu/Zn-SOD表达下调,由此导致细胞抗氧化防御能力降低,细胞内基础ROS水平升高,氧化压力诱发的脂质过氧化和DNA氧化损伤增强。表明PTEN在维持细胞内氧化还原环境、保护细胞免于氧化损伤和维持基因组稳定性方面起着重要作用。（2）PTEN可通过拮抗PI3K/AKT通路促进抗氧化防御蛋白Prx1,2,5,6及Cu/Zn-SOD基因转录和蛋白表达,并且可能通过该通路下游的FOXO3a等FOXO家族（FOXOs）转录因子对其转录进行调控。更多还原

【Abstract】 Objective:This study aims to investigate the effect of the deregulated expression of several antioxidant defense enzymes,including peroxiredoxin（Prx） 1,2, 5,6 and copper/zinc superoxide dismutase（Cu/Zn-SOD）,on the antioxidant defense ability and basal levels of reactive oxygen species （ROS）,lipid peroxidation and oxidative DNA damage in PTEN-deleted mouse embryonic fibroblasts(PTEN^-/- MEFs).The regulation mechanism of PTEN on the protein and mRNA expression of Prx1,2,5,6 and Cu/Zn-SOD was also explored to supplement and perfect the anti-tumor action principles of PTEN which may have potential in prevention and therapy of cancer.Methods:（1） SOD Assay Kit-WST was used to test the superoxide dismutase （SOD） enzyme activities in control group(PTEN^+/+ MEFs) and PTEN^-/- MEFs.The comet assay was applied for the detection of various concentrations of H₂O₂-induced DNA double-strand breaks（DSBs）.The expression of phosphorylation of histone H2AX（γH2AX）,a widely used marker for DNA DSBs,was detected by western blot treated by H₂O₂ with different dose.（2） Using 2’,7’-dichlorofluorescein diacetate（DCHF-DA） and dihydroethidium（DHE）,the intracellular generation of H₂O₂ and superoxide anion(O₂^·-) was monitored by flow cytometry and fluorescence microscope in PTEN^+/+ MEFs and PTEN^-/- MEFs.（3） To detect the effect of PTEN on lipid peroxide,the levels of malonaldehyde（MDA） in PTEN^+/+ MEFs and PTEN^-/- MEFs was tested by MDA Assay Kit.To analyze the influence of PTEN deletion on oxidative DNA damage,immunostaining assay was used to detect the expression of 8-OH-dG.The expression ofγH2AX in PTEN^+/+ MEFs and PTEN^-/- MEFs was monitored by western blot and immunofluorescence.The comet assay was also applied to further detect the levels of DNA DSBs.（4） To explore whether PTEN regulate the expression of antioxidant enzymes through inhibiting PI3K/AKT signal pathway,western blot and northern blot were used to test the protein and mRNA expression of P-AKT,Prx1,2,5,6 and Cu/Zn-SOD in PTEN^-/- MEFs treated by LY294002,a inhibitor of phosphatidylinositol-3-kinase（PI3K）.Western blot was further applied to analyze the basal expression of FOXO3a in PTEN^+/+ MEFs and PTEN^-/- MEFs and the expression levels of FOXO3a treated by LY294002 with various time in PTEN^-/- MEFs to identify if PTEN regulate the phosphorylation of the transcription factor FOXO3a through inhibiting PI3K/AKT signaling pathway.Results:（1） Decreased antioxidant defense ability in PTEN-deleted MEFs:SOD enzyme activity data revealed a 2.09-fold decrease in PTEN^-/- MEFs（p＜0.01）.The comet assay and western blot showed that exposure to each concentration of H₂O₂ resulted in a highly statistically significant increase in the frequency of DNA DSBs in PTEN^-/- MEFs（p＜0.01） compared to H₂O₂-untreated control at each concentration,whereas this increase was seen only in the 0.1 mmol/L H₂O₂-treated PTEN^+/+ MEFs （p＜0.01 compared to H₂O₂-untreated control）,indicating the decreased antioxidant defense ability in PTEN^-/- MEFs.（2） Increased ROS in PTEN-deleted MEFs:A flow-cytometry-based DCHF-DA and DHE analysis revealed that the levels of both DCF and Eth fluorescence in PTEN^-/- MEFs were higher than in control wild-type cells during 15-to 60-min incubation.（p＜0.05 and p＜0.01）.Fluorescence photomicrographs also suggested an increased H₂O₂ and O₂^·- in PTEN^-/- MEFs.（3） Increased lipid peroxide and oxidative DNA damage in PTEN-deleted MEFs: The level of MDA in PTEN^-/- MEFs was higher than in PTEN^+/+ MEFs（1.75±0.05 nmol/mgprot vs 1.3±0.1 nmol/mgprot,p＜0.01）. Immunostaining assay data revealed that PTEN-deleted MEFs displayed a significant increase in the levels of 8-OH-dG（p＜0.01）.The frequency of DNA DSBs was estimated by immunofluorescence assay or western blotting forγH2AX.Compared to PTEN^+/+ MEFs,intenseγH2AX staining or increasedγH2AX level was observed in PTEN^-/- MEFs（p＜0.01）.The comet assay showed that mean tail moment in PTEN^-/- MEFs was significantly higher than in control（22.03 vs 15.48,p＜0.01）,indicating that there is extensive spontaneous DNA DSBs in PTEN^-/- MEFs.（4） PTEN may increase the expression of Prx1,2,5,6 and Cu/Zn-SOD and decrease the phosphorylation of FOXO3a through inhibiting PI3K/AKT signaling pathway:Western blot and northern blot displayed that LY294002 successfully inhibited the activation of PI3K/AKT signaling pathway and the protein and mRNA expression of Prx1,2,5,6 and Cu/Zn-SOD up-regulated in PTEN^-/- MEFs treated by LY294002,indicating that PTEN may increase the expression of Prx1,2,5,6 and Cu/Zn-SOD through inhibiting PI3K/AKT signal pathway.Meanwhile,western blot also showed that the protein expression of P-FOXO3a in PTEN^-/- MEFs was higher than in PTEN^+/+ MEFs and deregulated in PTEN^-/- MEFs treated by LY294002, indicating that PTEN may decrease the phosphorylation of FOXO3a through inhibiting PI3K/AKT signaling pathway.Conclusion:（1） PTEN,as an important tumor suppressor gene,may up-regulate the expression of several antioxidant enzymes,including Prx1,2,5,6 and Cu/Zn-SOD,which results in increased antioxidant defense ability and decreased ROS levels,lipid peroxide and oxidative DNA damage.These findings suggest an essential role for PTEN in maintaining normal redox state and genomic stability against oxidative damage.（2） PTEN may up-regulate the expression of Prx1,2,5,6 and Cu/Zn-SOD through inhibiting the PI3K/AKT signaling pathway.Its mechanism may be to deregulate the phosphorylation of the PI3K/AKT downstream Forkhead transcription factors（FOXOs）.更多还原