核仁小分子RNA宿主基因5对星形胶质细胞缺氧/复氧损伤的作用  

Effect of small nucleolar RNA host gene 5 on hypoxia/reoxygenation injury of astrocytes

作　　者：刘丹 于广周 宋景贵[2] Liu Dan;Yu Guangzhou;Song Jinggui(The third Department of Neurology,Zhumadian Central Hospital,Zhumadian 463000,China;Department of Neurology,the Second Affiliated Hospital of Xinxiang Medical College,Xinxiang 453000,China)

机构地区：[1]驻马店市中心医院神经内三科,463000 [2]新乡医学院第二附属医院神经内科,453000

出　　处：《中华行为医学与脑科学杂志》2021年第12期1069-1076,共8页Chinese Journal of Behavioral Medicine and Brain Science

基　　金：河南省医学科技攻关计划省部共建项目(SB201901063)。

摘　　要：目的探讨核仁小分子RNA宿主基因5(small nucleolar RNA host gene,SNHG5)对缺氧/复氧(hypoxia/reoxygenation,H/R)诱导的星形胶质细胞损伤的影响。方法(1)体外培养星形胶质细胞,采用先缺氧培养6 h,再复氧培养18 h的方法建立H/R细胞模型,Lipofectamine™2000脂质体法进行细胞lncRNA SNHG5转染,RT-qPCR检测H/R细胞及转染后lncRNA SNHG5的表达情况。(2)星形胶质细胞分为正常对照组、模型组、转染对照组(转染pcDNA-NC至细胞后,再建H/R细胞模型)和转染组(转染pcDNA-lncRNA SNHG5至细胞后,再建H/R细胞模型),观察过表达lncRNA SNHG5对星形胶质细胞的影响。(3)将转染lncRNA SNHG5并进行H/R干预的星形胶质细胞分为转染+溶媒组(0.1%的DMSO孵育)和转染+抑制剂组(20μmol/L的LY294002孵育),观察PI3K/Akt信号通路抑制剂LY294002对H/R星形胶质细胞的影响。(4)CCK-8检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测细胞中增殖蛋白(Cyclin D1、Cyclin E)、凋亡蛋白(Caspase-3、Bax)和p-PI3K、p-AKT的表达,ELISA检测细胞培养上清液中炎性因子IL-1β和TNF-α水平,比色法检测细胞培养上清液中乳酸脱氢酶(LDH)水平及细胞中丙二醛(MDA)和超氧化物歧化酶(SOD)水平。采用SPSS 22.0软件进行独立样本t检验和单因素方差分析,进一步两两比较用LSD-t检验。结果(1)RT-qPCR结果显示,经H/R干预后,星形胶质细胞中的lncRNA SNHG5水平较正常培养细胞低(t=33.28,P<0.05)。(2)lncRNA SNHG5过表达实验:模型组细胞增殖活性低于正常对照组[CCK-8 OD值:(0.64±0.02),(1.23±0.02),t=62.58,P<0.05],模型组细胞增殖蛋白Cyclin D1、Cyclin E表达均低于正常对照组(t=33.54,32.20,均P<0.05);转染组细胞增殖活性高于转染对照组[CCK-8 OD值:(1.49±0.02),(0.65±0.03),t=69.89,P<0.05],转染组细胞增殖蛋白Cyclin D1、Cyclin E表达均高于转染对照组(t=24.96,28.46,均P<0.05)。模型组细胞凋亡率高于对照组[流式细胞术结果:(25.33±1.13)%,(9.06±0.Objective To explore the effect of lncRNA SNHG5 on injury of astrocytes induced by hypoxia/reoxygenation(H/R).Methods(1)Astrocytes were cultured in vitro.The H/R cell model was established by hypoxia culture for 6 hours and then reoxygenaion culture for 18 hours.Lipofectamine™2000 liposome method was used to transfect lncRNA SNHG5 into astrocytes.RT-qPCR was used to detect the expression of lncRNA SNHG5 in H/R cells and after transfection.(2)Astrocytes were divided into normal control group,model group,transfection control group(pcDNA-NC was transfected first,then H/R cell model was established)and transfection group(pcDNA-lncRNA SNHG5 was transfected first,then H/R cell model was established).Then the effect of overexpression of lncRNA SNHG5 on astrocytes was observed.(3)The astrocytes transfected with lncRNA SNHG5 and H/R intervention were divided into transfection+vehicle group(0.1%DMSO incubation)and transfection+inhibitor group(20μmol/L LY294002 incubation),and then observe the effect of the inhibitor of PI3K/Akt signaling pathway LY294002 on H/R astrocytes was observed.(4)CCK-8 was used to detect cell proliferation.Flow cytometry was used to detect cell apoptosis.Western blot was used to detect the expression of cell proliferation proteins(Cyclin D1 and Cyclin E),apoptotic proteins(Caspase-3 and Bax),p-PI3K and p-AKT protein.ELISA was used to detect the levels of IL-1βand TNF-α.The colorimetric method was used to detect the level of lactate dehydrogenase(LDH)in cell culture supernatants and the level of malondialdehyde(MDA)and superoxide dismutase(SOD)in cells.SPSS 22.0 software was used for independent sample t-test and one-way ANOVA,and LSD-t test was used for further pairwise comparisons.Results(1)RT-qPCR results showed that the level of lncRNA SNHG5 in astrocytes induced by H/R was lower than that in the normal cultured cells(t=33.28,P<0.05).(2)lncRNA SNHG5 overexpression experiment:The cell proliferation activity of the model group was lower than that in the normal control group(CCK-8 OD value:(0.

关 键 词：核仁小分子RNA宿主基因5 长链非编码RNA 星形胶质细胞 缺氧/复氧 增殖 凋亡 

分 类 号：R743.3[医药卫生—神经病学与精神病学]