[1]车选义,赵清侠,李迪.siRNA靶向沉默载脂蛋白2(Lcn 2)对缺氧诱导视网膜神经节细胞-5细胞凋亡的作用及机制[J].眼科新进展,2017,37(11):1027-1031.[doi:10.13389/j.cnki.rao.2017.0260]
　CHE Xuan-Yi,ZHAO Qing-Xia,LI Di.Effects of siRNA targeted Lcn2 gene silencing on cell apoptosis induced by hypoxia in RGC-5 cells and its mechanisms[J].Recent Advances in Ophthalmology,2017,37(11):1027-1031.[doi:10.13389/j.cnki.rao.2017.0260]
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siRNA靶向沉默载脂蛋白2(Lcn 2)对缺氧诱导视网膜神经节细胞-5细胞凋亡的作用及机制/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
37卷
期数:
2017年11期
页码:
1027-1031
栏目:
实验研究
出版日期:
2017-11-05

文章信息/Info

Title:
Effects of siRNA targeted Lcn2 gene silencing on cell apoptosis induced by hypoxia in RGC-5 cells and its mechanisms
作者:
车选义; 赵清侠; 李迪
710068　陕西省西安市，陕西省人民医院眼科，西安医学院（车选义）；710068　陕西省西安市，陕西省人民医院手术室二部（赵清侠）；710068　陕西省西安市，陕西省人民医院眼科（李迪）
Author(s):
CHE Xuan-Yi; ZHAO Qing-Xia; LI Di
Department of Ophtalmology，Shaanxi Provincial People’s Hospital，Xi’an Medical University（CHE Xuan-Yi），Xi’an 710068，Shaanxi Province，China；Department of Second Operating Room，Shaanxi Provincial People’s Hospital（ZHAO Qing-Xia），Xi’an 710068，Shaanxi Province，China；Department of Ophthalmology，Shaanxi Provincial People’s Hospital（LI Di），Xi’an 710068，Shaanxi Province，China
关键词:
载脂蛋白2; 细胞凋亡; 视网膜神经节细胞-5; 线粒体凋亡通路
Keywords:
lipocalin 2; cell apoptosis; retinal ganglion cells; mitochondrial apoptosis signaling pathway
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2017.0260
文献标志码:
A
摘要:
目的　研究载脂蛋白2（lipocalin 2，Lcn 2）对缺氧诱导视网膜神经节细胞-5（retinal ganglion cells，RGC-5）损伤的抑制作用及可能机制。方法　将RGC-5细胞置于缺氧环境下处理（0 h、4 h、8 h、12 h、24 h、48 h），采用实时定量PCR和Western blot法检测Lcn 2的表达水平。将细胞分为4组：对照组、缺氧组、siNC+缺氧组（转染siRNA阴性对照后进行缺氧处理24 h）和siLcn 2+缺氧组（细胞转染Lcn 2 siRNA后进行缺氧处理24 h）。ELISA检测细胞凋亡率和Caspase-3活性，DCFH-DA试剂盒检测活性氧（reactive oxygen speciesin，ROS）产生情况，线粒体膜电位检测试剂盒评价线粒体膜电势，Western blot检测半胱氨酸天冬氨酸蛋白酶3（cleaved-Caspase-3，c-Caspase-3）、聚腺苷二磷酸核糖聚合酶（cleaved-PARP，c-PARP）、Bax、Bcl-2和细胞色素C（cytochrome C，Cyto C）蛋白表达。结果　与0 h组相比，缺氧组（4 h、8 h、12 h、24 h和48 h）Lcn 2 mRNA表达水平均升高（均为P<0.05），同时缺氧组（12 h、24 h和48 h）的Lcn 2蛋白表达水平均较0 h组升高（均为P<0.05）。与对照组细胞凋亡率（99.66%±2.86%）比较，缺氧组（138.33%±13.76%）显著升高（P<0.05）；siLcn 2+缺氧组细胞凋亡率（105.02%±8.60%）较siNC+缺氧组（142.33%±6.54%）显著下降（P<0.05）。此外，缺氧组较对照组Caspase-3相对活性增强、c-Caspase-3和c-PARP表达均上调（均为P<0.05），与siNC+缺氧组比较，siLcn 2+缺氧组Caspase-3相对活性水平、c-Caspase-3和c-PARP相对表达水平均降低（均为P<0.05）。与对照组ROS相对荧光强度（1.03±0.11）比较，缺氧组（4.26±0.63）增强（P<0.05），siLcn 2+缺氧组ROS相对荧光强度（3.10±0.24）较siNC+缺氧组（4.73±0.26）显著减弱（P<0.05）。且siLcn 2+缺氧组较siNC+缺氧组线粒体膜电势增加、Cyto C蛋白表达水平及Bax和Bcl-2相对比率减少（均为P<0.05）。结论　沉默Lcn 2抑制缺氧诱导的细胞凋亡及ROS产生，可能是通过抑制线粒体凋亡信号通路实现的。
Abstract:
Objective　To investigate the roles and mechanisms of siRNA targeted lipocalin 2 (Lcn 2) gene silencing on hypoxia-induced cell apoptosis in retinal ganglion cells (RGC-5).Methods　RGC-5 cells were subjected to hypoxic condition for various time duration (0 h，4 h，8 h，12 h，24 h，48 h)，and quantitative real-time PCR and Western blot were used to evaluate the expression of Lcn 2 in mRNA and protein levels.Cells were divided into 4 groups：control group，hypoxia group，siNC+hypoxia group，in which cells were transfected with negative control for siRNA，and then subjected to hypoxic condition for 24 h，and siLcn 2+hypoxia group，in which cells were transfected with Lcn 2 siRNA，and then subjected to hypoxic condition for 24 h).Next，cell apoptotic rate and Caspase-3 activity were detected using ELISA.The fluorescence intensity of reactive oxygen species (ROS) was assayed using DCFH-DA kit，and mitochondrial membrane potential assay kit was used to evaluate the mitochondrial membrane potential.Finally，the expression levels of cleaved-Caspase-3 (c-Caspase-3)，cleaved-PARP (c-PARP)，Bax，Bcl-2 and cytochrome C (Cyto C) were detected using Western blot.Results　In the hypoxia group，Lcn 2 mRNA level at 4 h，8 h，12 h，24 h and 48 h was higher than that at 0 h (all P<0.05).Meanwhile，Lcn 2 protein level at 12 h，24 h and 48 h was also higher than that at 0 h (all P<0.05).Cell apoptotic rate in the hypoxia group (138.33%±13.76%) was significantly higher than that in the control group (99.66%±2.86%)(P<0.05)，and siLcn 2+hypoxia group (105.02%±8.60%) was lower than siNC+hypoxia group (142.33%±6.54%)(P<0.05).In addition，compared with the control group，the relative activity of Caspase-3 and the relative expression of c-Caspase-3 and c-PARP in the hypoxia group were all upregulated (all P<0.05)；whereas the relative activity of Caspase-3 and the relative expression of c-Caspase-3 and c-PARP in the siLcn2+hypoxia group were downregulated compared with the siNC+hypoxia group (all P<0.05).Moreover，the fluorescence intensity of ROS in the hypoxia group (4.26 0.63) was more enhanced than that in the control group (1.03±0.11) (P<0.05)，and the siLcn2+hypoxia group (3.10±0.24) was lower than siNC+hypoxia group (4.73±0.26)(P<0.05).Furthermore，mitochondrial membrane potential，Cyto C expression and the relative ratio of Bax to Bcl-2 in the siLcn2+hypoxia group was lower than those in the siNC+hypoxia group.Conclusion　Lcn 2 Lcn2 gene silencing can inhibit cell apoptosis and ROS production induced by hypoxia，which may be achieved by suppressing mitochondrial apoptosis signaling pathway.

参考文献/References:

［1］　葛坚.我国近五年青光眼临床与基础研究进展［J］.中华眼科杂志，2005，41（8）：710-716.
GE J.Recent advances of clinical and basic studies in glaucoma in China in the last five years［J］.Chin J Ophthalmol，2005，41（8）：710-716.
［2］　THAM YC，LI X，WONG TY，QUIGLEY HA，AUNG T，CHENG CY.Global prevalence of glaucoma and projections of glaucoma burden through 2040：a systematic review and meta-analysis［J］.Ophthalmology，2014，121（11）：2081.
［3］　李晓鹏，高建伟，王爽，李彦，刘静.白内障超声乳化联合人工晶状体植入术治疗原发性闭角型青光眼合并白内障临床疗效观察［J］.新乡医学院学报，2015，35（2）：169-170.
LI XP，GAO JW，WANG S，LI Y，LIU J.Clinical effect of phacoemulsification combined with intraocular lens implantation in management of primary angle-closure glaucoma with cataract［J］.J Xinxiang Med Univ，2015，35（2）：169-170.
［4］　任朋亮，范雪娇，杨晓龙，刘明佳，刘戟，黄晶晶.SIRT1增强青光眼小梁网细胞DSBs修复能力及抗细胞衰老的研究［J］.四川大学学报医学版，2014，45（4）：572-577.
REN PL，FAN XJ，YANG XL，LIU MJ，LIU J，HUANG JJ.SIRT1 promote GTM cell DSBs repair and resist cellular senescence［J］.J Sichuan Univ （Med Sci Edi），2014，45（4）：572-577.
［5］　MOSCHEN AR，ADOLPH TE，GERNER RR，WIESER V，TILG H.Lipocalin-2：a master mediator of intestinal and metabolic inflammation［J］.Trends Endocrinol Metab，2017，28（5）：388-397.
［6］　STEELE MR，INMAN DM，CALKINS DJ，HORNER PJ，VETTER ML.Microarray analysis of retinal gene expression in the DBA/2J model of glaucoma［J］.Invest Ophthalmol Vis Sci，2006，47（3）：977-985.
［7］　GUO Y，JOHNSON EC，CEPURNA WO，DYCK JA，DOSER T，MORRISON JC.Early gene expression changes in the retinal ganglion cell layer of a rat glaucoma model［J］.Invest Ophthalmol Vis Sci，2011，52（3）：1460-1473.
［8］　JIANG W，CONSTANTE M，SANTOS MM.Anemia upregulates lipocalin 2 in the liver and serum［J］.Blood Cells Mol Dis，2008，41（2）：169-174.
［9］　NELSON AM，ZHAO W，GILLILAND KL，ZAENGLEIN AL，LIU W，THIBOUTOT DM.Neutrophil gelatinase-associated lipocalin mediates 13-cis retinoic acid-induced apoptosis of human sebaceous gland cells［J］.J Clin Invest，2008，118（4）：1468-1478.
［10］　XU G，AHN J，CHANG S，EGUCHI M，OGIER A，HAN S，et al.Lipocalin-2 induces cardiomyocyte apoptosis by increasing intracellular iron accumulation［J］.J Biol Chem，2012，287（7）：4808-4817.
［11］　CHIEN MH，YING TH，YANG SF.Lipocalin-2 induces apoptosis in human hepatocellular carcinoma cells through activation of mitochondria pathways［J］.Cell Biochem Biophys，2012，64（3）：177-186.
［12］　LEE S，LEE WH，LEE MS，MORI K，SUK K.Regulation by lipocalin-2 of neuronal cell death，migration，and morphology［J］.J Neurosci Res，2012，90（3）：540-550.
［13］　ERAWAN BK，EDDY VDL，ZIMMERMANN HW，KARLIN RAJA K，LIDIA T，UTE H，et al.Protective effects of lipocalin-2 （Lcn 2） in acute liver injury suggest a novel function in liver homeostasis［J］.Biochim Biophys Acta，2013，1832（5）：660-673.
［14］　GUOLIANG WANG XL，LIUKUN MENG，SHENGHUA LIU，LI WANG，JUN LI，CHUANJUE CUI，et al.Up-Regulated Lipocalin-2 in Pulmonary Hypertension Involving in Pulmonary Artery SMC Resistance to Apoptosis［J］.Int J Biol Sci，2014，10（7）：798-806.
［15］　王玲，单保恩，桑梅香，连易水，王彬，丁春艳.靶向沉默p65基因对人乳腺癌细胞MDA-MB-231增殖及凋亡的影响［J］.南方医科大学学报，2011，31（10）：1742-1747.
WANG L，SHAN BE，SANG MX，LIAN YS，WANG B，DING CY.Effect of micro RNA-mediated p65 gene silencing on the proliferation and apoptosis of human breast cancer MDA-MB-231 cells［J］.J South Med Univ，2011，31（10）：1742-1747.
［16］　吕娟娟，陈志江，郑贵浪，王斌，陶少华，项丹，等.脓毒症相关性脑病远期脑线粒体损伤与氧化应激的关系［J］.中华实用儿科临床杂志，2015，30（11）：860-862.
LV JJ，CHEN ZJ，ZHEGN GL，WANG B，TAO SH，XIANG D，et al.Study of long-term mitochondrial dysfuction and oxidative stress in a sepsis-associated encephalopathy model［J］.Chin J Appl Clin Pediatr，2015，30（11）：860-862.
［17］　易健，舒徐.活性氧对细胞凋亡和增殖的调控作用［J］.基础医学与临床，2013，33（10）：1341-1344.
YI J，SHU X.The regulation of cell apoptosis and proliferation by reactive oxygen species［J］.Bas & Clin Med，2013，33（10）：1341-1344.
［18］　WU L，DU Y，LOK J，LO EH，XING C.Lipocalin-2 enhances angiogenesis in rat brain endothelial cells via reactive oxygen species and iron-dependent mechanisms［J］.J Neurochem，2015，132（6）：622-628.

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更新日期/Last Update: 2017-11-01