孙屹屹;杨丽华;李瑞俭;王景涛;

• 1:鹤壁市人民医院ICU
• 2:郑州大学第一附属医院神经内科

摘要(Abstract)：

目的研究siRNA下调星形胶质细胞中第10号染色体同源缺失性磷酸酶-张力蛋白(PTEN)表达对缺氧复氧诱导的细胞凋亡影响。方法分离培养乳鼠星形胶质细胞,用携带PTEN siRNA的慢病毒载体感染星形胶质细胞,经缺氧复氧处理后,用Real time PCR和Western blot检测细胞中PTEN表达变化。MTT测定细胞增殖活力,二硝基苯肼比色法检测乳酸脱氢酶(LDH)漏出率,流式细胞术测定周期和凋亡变化,Western blot检测细胞中细胞周期依赖性蛋白激酶4(Cdk4)、p21蛋白水平和胞质中细胞色素C(Cytochrome C)蛋白水平,JC-1法测定线粒体膜电位。结果携带PTEN siRNA的慢病毒载体感染后的星形胶质细胞中PTEN表达水平降低。缺氧复氧处理后的星形胶质细胞活力降低,细胞LDH漏出率升高,细胞凋亡率升高,细胞G1期比例升高,Cdk4蛋白水平降低,p21蛋白水平升高,胞质中Cytochrome C蛋白增多,线粒体膜电位降低。敲低PTEN可以提高缺氧复氧条件下星形胶质细胞活力,降低细胞LDH漏出率,减少G1期细胞比例,降低细胞凋亡率,促进细胞表达Cdk4蛋白,减少细胞表达p21,减少胞质中Cytochrome C蛋白,提高线粒体膜电位。结论敲低PTEN可以减少缺氧复氧诱导的星形胶质细胞凋亡,作用机制与线粒体凋亡途径有关。

关键词(KeyWords)： 星形胶质细胞;凋亡;PTEN;线粒体;缺氧复氧

Abstract:

Keywords:

基金项目(Foundation): 河南省科技攻关项目(编号:162104310248)

作者(Authors): 孙屹屹;杨丽华;李瑞俭;王景涛;

参考文献(References)：

• [1] CAHOY J D,EMERY B,KAUSHAL A,et al.A transcriptome database for astrocytes,neurons,and oligodendrocytes:a new resource for understanding brain development and function[J].Journal of Neurosci,2008,28(1):264-278.
• [2] STOGSDILL J A,RAMIREZ J,LIU D,et al.Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis[J].Nature,2017,551(7 679):192-197.
• [3] ALLEN N J,EROGLU C.Cell Biology of Astrocyte-Synapse Interactions[J].Neuron,2017,96(3):697-708.
• [4] PARK S Y,YOON S N,KANG M J,et al.Hippocalcin Promotes Neuronal Differentiation and Inhibits Astrocytic Differentiation in Neural Stem Cells[J].Stem Cell Reports,2017,8(1):95-111.
• [5] HORI O,MATSUMOTO M,MAEDA Y,et al.Metabolic and Biosynthetic Alterations in Cultured Astrocytes Exposed to Hypoxia/Reoxygenation[J].J Neurochem,2010,62(4):1 489-1 495.
• [6] YAN J W,TAN T Y,HUANG Q L.Protective effect of astrocyte-conditioned medium on neurons following hypoxia and mechanical injury[J].Chin J Traumatol,2013,16(1):3-9.
• [7] WEI X,WANG W,WANG L,et al.MicroRNA-21 induces 5-fluorouracil resistance in human pancreatic cancer cells by regulatingPTENandPDCD4[J].Cancer Med,2016,5(4):693-702.
• [8] AMORIM M A R D,GARCIA-SEGURA L M,GOYA R G,et al.Decrease in PTEN and increase in Akt expression and neuron size in aged rat spinal cord[J].Exp Gerontol,2010,45(6):457-463.
• [9] ZHOU H,LI X,WU Q,et al.shRNA against PTEN promotes neurite outgrowth of cortical neurons and functional recovery in spinal cord contusion rats[J].Regen Med,2015,10(4):411-429.
• [10] LING Y Z,LI X H,YU L,et al.Protective effects of parecoxib on rat primary astrocytes from oxidative stress induced by hydrogen peroxide[J].J Zhejiang Univ Sci B,2016,17(9):692-702.
• [11] OHTAKE Y,HAYAT U,LI S.PTEN inhibition and axon regeneration and neural repair[J].Neural Regen Res,2015,10(9):1 363-1 368.
• [12] WADEY A L,MUYDERMAN H,KWEK P T,et al.Mitochondrial glutathione uptake:characterization in isolated brain mitochondria and astrocytes in culture.[J].J Neurochem,2010,109(s1):101-108.
• [13] GABRYEL B,ADAMEK M,PUDE?KO A,et al.Piracetam and vinpocetine exert cytoprotective activity and prevent apoptosis of astrocytes in vitro in hypoxia and reoxygenation[J].Neurotoxicology,2002,23(1):19-31.
• [14] WEI N,TAN L U,CHUANG Y I.The influence of adenosine preconditioning on astrocytes after local cerebral ischemia-reperfusion injury in rats[J].J Apoplexy Nervous Dis,2013,30(4):298-301.
• [15] ZHANG Y,CHEN K,SLOAN SA,et al.An RNA-sequencing transcriptome and splicing database of glia,neurons,and vascular cells of the cerebral cortex.J Neurosci,2014,34(36):11 929-11 947.
• [16] ZHANG J H,LI J K,MA L L,et al.RNA interference-mediated silencing of S100B improves nerve function recovery and inhibits hippocampal cell apoptosis in rat models of ischemic stroke[J].J Cell Biochem,2018,119(10):8 095-8 111.
• [17] RADAK D,KATSIKI N,RESANOVIC I,et al.Apoptosis and Acute Brain Ischemia in Ischemic Stroke[J].Curr Vasc Pharmacol,2017,15(2):115-122.
• [18] CHEN B,WANG G,LI W,et al.Memantine attenuates cell apoptosis by suppressing the calpain-caspase-3 pathway in an experimental model of ischemic stroke[J].Exp Cell Res,2017,351(2):163-172.
• [19] BOLA?OS J P,HEALES S J R,LAND J M,et al.Effect of Peroxynitrite on the Mitochondrial Respiratory Chain:Differential Susceptibility of Neurones and Astrocytes in Primary Culture[J].J Neurochem,2010,64(5):1 965-1 972.
• [20] 田龙,黄月,丁娜.葛根素对大鼠星形胶质细胞增殖、凋亡和 β-catenin、cyclinD1、Bax蛋白表达的影响[J].郑州大学学报(医学版),2019,54(3):402-406.
• [21] CHEN X,LU M,HE X,et al.TRPC3/6/7 Knockdown Protects the Brain from Cerebral Ischemia Injury via Astrocyte Apoptosis Inhibition and Effects on NF-кB Translocation[J].Mol Neurobiol,2017,54(10):7 555-7 566.
• [22] CHANG D,WANG T Y,LI H C,et al.Prognostic significance of PTEN expression in esophageal squamous cell carcinoma from Linzhou City,a high incidence area of northern China[J].Dis Esophagus,2010,20(6):491-496.
• [23] MAGEE J,IKENOUE T,NAKADA D,et al.Temporal Changes in PTEN and mTORC2 Regulation of Hematopoietic Stem Cell Self-Renewal and Leukemia Suppression[J].Cell Stem Cell,2012,11(3):415-428.
• [24] TIAN T,NAN K J,GUO H,et al.PTEN inhibits the migration and invasion of HepG2 cells by coordinately decreasing MMP expression via the PI3K/Akt pathway.[J].Oncol Rep,2010,23(6):1 593-1 600.
• [25] MALANEY P,UVERSKY VN,DAVé V.PTEN proteoforms in biology and disease[J].Cell Mol Life Sci,2017,74(15):2 783-2 794.
• [26] SHI J,BEI Y,KONG X,et al.miR-17-3p Contributes to Exercise-Induced Cardiac Growth and Protects against Myocardial Ischemia-Reperfusion Injury[J].Theranostics,2017,7(3):664-676.
• [27] HOPKINS B D,HODAKOSKI C,BARROWS D,et al.PTEN function:the long and the short of it[J].Trends Biochem Sci,2014,39(4):183-90.
• [28] Toshio Nikaido,Li SF,Tanri Shiozawa,et al.Coabnormal expression of cyclin D1 and p53 protein in human uterine endometrial carcinomas[J].Cancer,78(6):1 248-1 253.
• [29] CHEN Y,HUANG WC,SéJOURNé J,et al.Pten Mutations Alter Brain Growth Trajectory and Allocation of Cell Types through Elevated β-Catenin Signaling[J].J Neurosci,2015,35(28):10 252-10 267.
• [30] SONG Z,HAN X,ZOU H,et al.PTEN-GSK3β-MOB1 axis controls neurite outgrowth in vitro and in vivo[J].Cell Mol Life Sci,2018,75(23):4 445-4 464.
• [31] DU F,YU Q,YAN S,et al.PINK1 signalling rescues amyloid pathology and mitochondrial dysfunction in Alzheimer's disease[J].Brain,2017,140(12):3 233-3 251.
• [32] WANG Z,YAN J,ZHENG Q,et al.CyclinD1,CDK4,and P21 expression by IEC-6 cells in response to NiTi alloy and polymeric biomaterials[J].Mater Sci Eng C,2012,32(8):2 183-2 189.
• [33] GULAPPA T,REDDY R S,SUMAN S,et al.Molecular interplay between cdk4 and p21 dictates G0/G1 cell cycle arrest in prostate cancer cells[J].Cancer Lett,2013,337(2):177-183.
• [34] DIROCCO D P,BISI J,ROBERTS P,et al.CDK4/6 inhibition induces epithelial cell cycle arrest and ameliorates acute kidney injury[J].Am J Physiol Renal Physiol,2014,306(4):F379-88.
• [35] LEE H L,LIN C S,KAO S H,et al.Gallic acid induces G1 phase arrest and apoptosis of triple-negative breast cancer cell MDA-MB-231 via p38 mitogen-activated protein kinase/p21/p27 axis[J].Anticancer Drugs,2017,28(10):1 150-1 156.
• [36] GONGPAN P,LU Y,WANG F,et al.AS160 controls eukaryotic cell cycle and proliferation by regulating the CDK inhibitor p21.Cell Cycle,2016,15(13):1 733-1 741.
• [37] BRANDMAIER A,HOU S Q,SHEN W H.Cell Cycle Control by PTEN[J].J Mol Biol,2017,429(15):2 265-2 277.
• [38] WANG L,YANG L,LU Y,et al.Osthole Induces Cell Cycle Arrest and Inhibits Migration and Invasion via PTEN/Akt Pathways in Osteosarcoma[J].Cell Physiol Biochem,2016,38(6):2 173-2 182.
• [39] DONG M L,ZHU Y C,HOPKINS J V.Oil A induces apoptosis of pancreatic cancer cells via caspase activation,redistribution of cell cycle and GADD expression[J].World J Gastroenterol,2003,9(12):2 745-2 750.
• [40] ZHAO Y,WANG X,SUN Y,et al.LYG-202 exerts antitumor effect on PI3K/Akt signaling pathway in human breast cancer cells[J].Apoptosis,2015,20(9):1 253-1 269.