[1]穆亚萌,江　湧,徐文仓.缺氧损伤对人脐静脉内皮细胞抗氧化能力、Ca依赖的三磷腺苷酶活性、Ca浓度及线粒体膜电位的影响[J].新乡医学院学报,2016,33(9):752-756.[doi:10.7683/xxyxyxb.2016.09.003]
　MU Ya-meng,JIANG Yong,XU Wen-cang.Effect of hypoxia on antioxidative capacity，Ca adenosine triphosphate enzyme activity，Ca concentration and mitochondiral membrane potential in ECV304 cells[J].Journal of Xinxiang Medical University,2016,33(9):752-756.[doi:10.7683/xxyxyxb.2016.09.003]
点击复制

缺氧损伤对人脐静脉内皮细胞抗氧化能力、Ca2+依赖的三磷腺苷酶活性、Ca2+浓度及线粒体膜电位的影响
分享到：

《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
33
期数:
2016年9
页码:
752-756
栏目:
基础研究
出版日期:
2016-08-23

文章信息/Info

Title:
Effect of hypoxia on antioxidative capacity，Ca2+ adenosine triphosphate enzyme activity，Ca2+ concentration and mitochondiral membrane potential in ECV304 cells
作者:
穆亚萌1; 江　湧2; 徐文仓1
（1.广州中医药大学，广东　广州　510405；2.广州中医药大学第一附属医院实验中心，广东　广州　510405）
Author(s):
MU Ya-meng1; JIANG Yong2; XU Wen-cang1
（1.Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China;2.Department of Experimental Center,the First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China)
关键词:
缺氧; ECV304细胞; 氧自由基; 钙离子浓度; 线粒体膜电位
Keywords:
hypoxia; ECV304 cells; oxygen radicals; Ca2+; mitochondiral membrane potential
分类号:
R543
DOI:
10.7683/xxyxyxb.2016.09.003
文献标志码:
A
摘要:
目的　探讨缺氧损伤对离体培养的人脐静脉内皮细胞（ECV304）抗氧化能力、Ca2+依赖的三磷腺苷酶（Ca2+-ATPase）活性、线粒体膜电位(MMP)和细胞内Ca2+浓度等的影响。方法　将ECV304细胞分为正常组和缺氧组。正常组用无血清培养基培养，缺氧组用连二亚硫酸钠（Na2S2O4）孵育ECV304细胞5 h，建立缺氧损伤模型。倒置显微镜下观察2组细胞形态的变化，细胞计数试剂盒（CCK-8）检测细胞活性，流式细胞仪测定细胞周期、细胞凋亡、细胞内Ca2+浓度及MMP的变化，化学比色法检测超氧化物歧化酶(SOD)活性、谷胱甘肽(GSH)和丙二醛(MDA)含量、一氧化氮(NO)分泌量及Ca2+-ATPase活性。结果　正常组细胞贴壁良好，细胞饱满、排列紧密，透明度好；缺氧组细胞出现皱缩,细胞间排列疏松、紊乱。缺氧组细胞吸光度值为0.91±0.12，低于正常组的1.30±0.25(P<0.05)。缺氧组G0/G1期细胞所占比例为（88.53±1.36）%，高于正常组的（81.27±0.25）%（P<0.05）；S期细胞所占比例为（5.17±0.21）%，低于正常组的（6.07±0.25）%（P<0.05）；缺氧组细胞凋亡率为（19.75±0.81）%，高于正常组的（9.83±1.77）%（P<0.05）。正常组MDA、GSH含量,一氧化氮（NO）分泌量,SOD、Ca2+-ATPase活性分别为（0.40±0.23）、（3.85±0.18）、（14.21±0.39）μmol·L-1、（64.61±0.05）×103 U·L-1、（18.90±1.09）U·mgprot-1；缺氧组以上各指标分别为（1.65±0.24）、（3.34±0.13）、（78.70±5.29）μmol·L-1、（35.33±0.04）×103 U·L-1、（5.80±0.31）U·mgprot-1；与正常组相比，缺氧组细胞SOD活性、GSH含量及Ca2+-ATPase活性均降低，MDA含量增加，NO分泌量升高（P<0.05）。缺氧组细胞内Ca2+荧光强度为（572.82±49.40）%，高于正常组的（505.97±19.40）%(P<0.05)。缺氧组细胞MMP为（182.07±6.00）%，低于正常组的（217.60±16.40）%(P<0.05)。结论　Na2S2O4作用于ECV304细胞引起细胞缺氧损伤，其机制可能与MMP降低、细胞内Ca2+浓度升高和细胞抗氧化能力及Ca2+-ATPase活性减弱相关。
Abstract:
Objective　To observe the effect of hypoxia on antioxidant capacity，Ca2+ adenosine triphosphate enzyme(Ca2+-ATPase) activity，Ca2+ concentration and mitochondiral membrane potential(MMP) of ECV304 cells cultured in vitro.Methods　ECV304 cells were divided into normal group which cultured in serum-free culture medium and hypoxia group which were cultured in Na2S2O4 for 5 hours to establish hypoxic injury model.Then the change of cell morphology in the two groups was observed by inverted microscope;the cytoactive was evaluated by cell counting kit-8(CCK-8);the cellcycle,apoptosis,intracellular Ca2+ concentration and MMP were assayed by flow cytometry;the activity of superoxide dismutase(SOD) and Ca2+-ATPase and the content of glutathione (GSH),malondialdehyde (MDA),nitric oxide (NO) secretion were measured by chemical colorimetry method.Results　The cells in normal group were well adherent,full of luster,closely spaced,and highly pellucidity;while the cells in hypoxia group appeared shrunken,and cell arranged loosely,disorder.The value of absorance in hypoxia group and normal group was 0.91±0.12,1.30±0.25 respectively;the value of OD in hypoxia group was significantly lower than that in normal group(P<0.05).The proportion of G0/G1 and S phase cells in normal group was (81.27±0.25)%,(6.07±0.25)%;which in hypoxia group was (88.53±1.36)%,(5.17±0.21)%;the proportion of G0/G1 phase cells was significantly higher and the S phase cells was significantly lower than that in normal group (P<0.05).The cell apoptosis rate in hypoxia group［(19.75±0.81)%］ was significantly higher than that in normal group［(9.83±1.77)%］（P<0.05）.The content of MDA and GSH,the content of NO secretion,the activity of SOD and Ca2+-ATPase in normal group was (0.40±0.23),(3.85±0.18),(14.21±0.39)μmol·L-1,(64.61±0.05) ×103 U·L-1,(18.90±1.09) U·mgprot-1 respectively;the above indexes in hypoxia group was (1.65±0.24),（3.34±0.13）,（78.70±5.29） μmol·L-1,（35.33±0.04）×103 U·L-1,（5.80±0.31） U·mgprot-1 respectively.Compared with normal group,the content of MDA,and content of NO secretion were increased(P<0.05);the activity of SOD,Ca2+-ATPase and the content of GSH were decreased(P<0.05) in hypoxia group.The fluorescence intensity of intracellular Ca2+ in hypoxia group ［(572.82±49.40)%］ was significantly higher than that in normal group ［(505.97±19.40)% ］(P<0.05).The MMP in hypoxia group ［(182.07±6.00)%］ was significantly lower than that in normal group ［(217.60±16.40)%］(P<0.05).Conclusion　The hypoxia injury of ECV304 cells being induced by Na2S2O4 is related with the decreasing of MMP and Ca2+-ATPase activity,and the increasing of the intracellular concentration of Ca2+and antioxidant capacity.

参考文献/References:

［1］　TAKAHASHI K,SAWASAKI Y,HATA J,et al.Spontaneous transformation and immortalization of human endothelial cells［J］.In Vitro Cell Dev Biol,1990,26(1):265-274.
［2］　DREXLER H G,QUENTMEIER H,DIRKS W G,et al.Bladder carcinoma cell line ECV304 is not a model system for endothelial cells［J］.In Vitro Cell Dev Biol Anim,2002,38（4）:185-186.
［3］　KRUSE C,KURZ A R,PáLFI K,et al.Polarity protein scrib facilitates endothelial inflammatory signaling［J］.Arterioscler Thromb Vasc Biol,2015,35（9）：1954-1962.
［4］　CHATTERJEE S S.Venous ulcers of the lower limb:where do we stand［J］.Indian J Plast Surg,2012,45（2）:266-274.
［5］　TANG Q.Changes of human umbilical venous endothelium due to different degree ischemia-reperfusion injury and the interventional effect of qinglailing［J］.中国临床康复，2005，9（29）：199-201.
［6］　邓水秀,潘伟男.天然药物与血管内皮细胞的研究概况［J］.中国医院用药评价与分析,2015,15（5）：694-696.
［7］　ZHANG J,DENG H,LIU L,et al.α-Lipoic acid protects against hypoxia/reoxygenation-induced injury in human umbilical vein endothelial cells through suppression of apoptosis and autophagy［J］.Mol Med Rep,2015,12（1）:180-186.
［8］　GENIUS J,GEIGER J,BENDER A,et al.Creatine protects against excitoxicity in an in vitro model of neurodegeneration［J］.PLoS One,2012,7(2):e30554.
［9］　VANNUCCI R C,BRUCKLACHER R M,VANNUCCI S J.Intracellular calcium accumulation during the evolution of hypoxic-ischemia brain damage in the immaturerat［J］.Brain Res Dev Brain Res,2001,126(1):117-120.
［10］　STANIKA R I,PIVOVAROVA N B,BRANTNER C A,et al.Coupling diverse routes ofcalcium entry to mitochondrial dysfunction and glutamate excitotoxicity［J］.Proc Natl Acad Sci USA,2009,106(24):9854-9859.
［11］　田云云,刘宗智,张婷.胞浆型肌酸激酶cDNA 的克隆及其对兴奋毒性神经细胞钙超载的保护作用［J］.生物物理学报，2015,31(2):105-115.
［12］　江小萍,李海全,潘少霞,等.三七总皂苷对缺氧再给氧血管内皮细胞钙离子浓度及肌浆网Ca2+-ATP酶活性的影响［J］.中华中医药学刊，2011,29（4）:758-760.
［13］　陈慧莉.线粒体跨膜电位和细胞凋亡相关性的研究［J］.医学综述，2007,13(14):41-43.
［14］　HE X,BI X Y,LU X Z,et al.Mitochondria-endoplasmic reticulum interactions by acetylcholine protects human umbilical vein endothelial cells from hypoxia/reoxygenation injury［J］.Arterioscler Thromb Vasc Biol,2015,35(7):1623-1634.
［15］　HAEBERLEIN S L.Mitochondrial function in apoptotic neuronal cell death［J］.Neurochem Res,2004,29(3):521-530.
［16］　GALLUZZI L,MORSELLI E,KEPP O,et al.Targeting postmitochondrial effectors of apoptosis for neuroprotection［J］.Biochim Biophys Acta,2009,1787（5）:402-413.
［17］　LI M M,WU L Y,LEI T Z,et al.The protective role of 5-HMF against hypoxic injury［J］.Cell Stress Chaperones,2011,16（3）:267-273.
［18］　顾俊菲,封亮,袁嘉瑞,等.赤芍总苷、川芎总酚酸组分组成结构对缺氧损伤人脐静脉内皮细胞的影响［J］.中国中药杂志，2015,40(5):920-926.
［19］　熊建文,肖化,张镇西.MTT法和CCK-8法检测细胞活性之测试条件比较［J］.激光生物学报,2007,16(5):559-562.
［20］　ENDEMANN D H，SCHIFFRIN E L.Endothelial dysfunction［J］.J Am Soc Nephrol，2004，15(8):1983-1992.
［21］　VALUJSKIKH A,HEEGER P S.Emerging roles of endothelial cells in transplant rejection［J］.Curr Opin Immunol,2003,15(5):493-498.
［22］　XU T X,ZHAO S Z,DONG M,et al.Hypoxia responsive miR-210 promotes cell survival and autophagy of endometriotic cells in hypoxia［J］.Eur Rev Med Pharmacol Sci，2016,20(3):399-406.
［23］　GRAEBER T G,OSMANIAN C,JACKS T,et al.Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours［J］.Nature,1996,379(6560):88-91.
［24］　薛大喜，张毅，杜玲玲，等.超氧化物歧化酶与年龄相关性白内障关系的研究［J］.眼科新进展，2013，33(8):740-742.
［25］　ZHANG Y Y,ZHOU H F,YANG J H,et al.Effects of Danhong injection and its main components on anticoagulation and fibrinolysis in cultured vein endothelial cells［J］.Chin J Integr Med,2016,22(4):276-283.
［26］　KASTRUG A,THOMAS C,HARTMANN C，et al.Cerembral blood flow and CO2 reactivity in interictal migraineurs:a transcranial Doppler study［J］.Headache，1998,38（8）:608-613.
［27］　MRCIO A G，MARCELO A V，FBIO E M，et al.Effects of hypoxia and reoxygenation on the antioxidant defense system of the locomotor muscle of the crab Neohelice granulate(Decapoda,Varunidae)［J］.J Comp Physiol B,2016.［Epub ahead of print］.
［28］　秦波,成洪波,黄丽娜,等.牵拉性视神经损伤后视神经中MDA、SOD水平的改变［J］.眼科新进展,2007,27(4):283-284.
［29］　HUANG H,LAI S,WAN Q,et al.Astragaloside IV protects cardiomyocytes from anoxia/reoxygenation injury by upregulating the expression of Hes1 protein［J］.Can J Physiol Pharmacol，2016,94（5）:542-553.
［30］　张铭，黄跃生，张琼.不同浓度NO对缺氧心肌细胞损害的影响［J］.第三军医大学学报,2007,29（11）:1017-1019.
［31］　郭斌,范钦华,牛超,等.缺氧条件下移植培养视网膜分泌NO、ET-1和8-iso-PGF2α的变化［J］.眼科新进展,2010,30(1):24-27.
［32］　王晓丽,李玉梅,张大方.附子人参不同配伍比例对缺糖缺氧/复糖复氧损伤大鼠心肌细胞的保护作用［J］.中国新药与临床药理,2015,26（2）:148-152.
［33］　CHIARELLO D I,MARíN R,PROVERBIO F，et al.Effect of hypoxia on the calcium and magnesium content,lipid peroxidation level,and Ca2+-ATPase activity of syncytiotrophoblast plasma membranes from placental explants［J］.Biomed Res Int,2014，2014:597357.
［34］　朱英伟，张晶.运动性心肌肥厚与病理性肥厚心肌大鼠模型中线粒体膜电位的差异 ［J］.新乡医学院学报，2015，32(8):718-721.
［35］　辛晓蓉,巩天祥.缺氧对脑脊液-视神经屏障中脑膜上皮细胞内质网和线粒体膜电位的影响［J］.眼科新进展,2014,34(11):1009-1012.
［36］　POPOVA T G,MILLIS B,CHUNG M C,et al.Anthrolysin O and fermentation products mediate the toxicity of Bacillus anthracis to lung epithelial cells under microaerobic conditions［J］.FEMS Immunol Med Microbiol,2011,61(1):15-27.
［37］　PARK S I,PARK E J,KIM N H,et al.Hypoxia delays the intracellular Ca2+ clearance by Na+-Ca2+ exchanger in human adult cardiac myocytes［J］.Yonsei Med J,2001,42(3):333-337.
［38］　陈红勤,陈鹏慧.缺氧对大鼠心肌细胞钙通道的影响［J］.高原医学杂志，2001，11(2)：12-15.
［39］　WAN J,YAMAMURA A,ZIMNICKA A M,et al.Chronic hypoxia selectively enhances L- and T-type voltage-dependent Ca2+ channel activity in pulmonary artery by upregulating Cav1.2 and Cav3.2［J］.Am J Physiol Lung Cell Mol Physiol,2013,305(2):L154-L164.

相似文献/References:

[1]马永昌,苏全玉,翁蔓荣,等.十三例肺部疾病缺氧原因分析[J].新乡医学院学报,1987,4(01):042.
[2]刘玉兰． 赵志霞.婴儿闷热综合征26例临床分析[J].新乡医学院学报,2001,18(01):060.
[3]高连英,李小英.活血抗凝佐治慢性肺心病心衰40 例[J].新乡医学院学报,2003,20(04):292.
[4]高连英,李小英.活血抗凝佐治慢性肺心病心衰40例[J].新乡医学院学报,2003,20(04):292.

更新日期/Last Update: 2016-09-05