低氧对斑马鱼细胞存活能力的影响
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摘要
【目的】明确低氧对斑马鱼细胞存活和抗氧化酶系统的影响,为揭示鱼类细胞低氧适应机理打下基础。【方法】采用不同浓度低氧处理斑马鱼胚胎上皮细胞(ZF-4细胞),检测低氧对其存活率的影响,并分析低氧应激下ZF-4细胞中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH)、一氧化氮合酶(NOS)活性及丙二醛(MDA)和一氧化氮(NO)含量的变化。【结果】1.0%O2处理可促进ZF-4细胞存活;SOD活性呈先降低后升高的变化趋势,在处理24 h时达最高值;CAT活性呈先降低后升高再降低的波动变化趋势,于处理8 h时达最低值;GSH活性在处理16 h时达最高值,随后开始下降;MDA含量与对照组无显著差异(P>0.05);NO含量和NOS活性在处理16 h时达最高值。经0.1%O2处理8 h后ZF-4细胞的存活率达最高值;SOD活性在处理24 h时显著高于对照组(P<0.05,下同);经0.1%O2处理后CAT活性极显著降低(P<0.01,下同),GSH活性则从处理8 h后极显著高于对照组;MDA含量显著或极显著降低;NO含量和NOS活性极显著降低。【结论】在极端缺氧环境下,斑马鱼细胞主要通过提高SOD和GSH等抗氧化酶活性以提高细胞的低氧适应能力,从而促进细胞存活。
【Objective】The study was to investigate the effects of hypoxic on cell survival and the antioxidative enzyme,and lay a foundation for the cell adaptive responses to hypoxia in fish.【Method】The epithelial cells of zebrafish(ZF-4 cells)were treated with different hypoxia concentrations,the cell survival were detected in the treatment. Then,the changes of activities of superoxide dismutase(SOD),catalase(CAT),glutathione reductase(GSH),nitric oxide synthase(NOS),and the contents of malondialdehyde(MDA),nitric oxide(NO)were analyzed.【Result】The study demonstrated that cell survival rate were elevated under 1% O2. The activity of SOD were decreased firstly and increased,and reached the highest value at the 24 thhour. The CAT activity went down firstly,then went up,and down again reaching minimum at the 8 thhour. The GSH levels gradually decreased,peak of activity was observed at the 16 thhour;but the content of MDA was not significantly changed compared to control(P>0.05). The content of NO and the activity of NOS increased and reached the peak at the 16 thhour. The ZF-4 cells had the highest survival rate under 0.1% oxygen after 8 hour. The activity of SOD wassignificantly higher than that in control at 24 thhour(P<0.05,the same below),but the activity of CAT was extremely reduced(P<0.01,the same below). The activity of GSH was extremely higher than that in control since the8 thhour;the contents of MDA significantly or extremely reduced under 0.1% oxygen;the content of NOS and NO were decreased significantly.【Conclusion】Under acute hypoxia condition,zebra fish mainly promote cell survival by improving the activity of SOD,GSH and other antioxidases to improve the hypoxia adaptation of cells,so that cells can survive.
【Objective】The study was to investigate the effects of hypoxic on cell survival and the antioxidative enzyme,and lay a foundation for the cell adaptive responses to hypoxia in fish.【Method】The epithelial cells of zebrafish(ZF-4 cells)were treated with different hypoxia concentrations,the cell survival were detected in the treatment. Then,the changes of activities of superoxide dismutase(SOD),catalase(CAT),glutathione reductase(GSH),nitric oxide synthase(NOS),and the contents of malondialdehyde(MDA),nitric oxide(NO)were analyzed.【Result】The study demonstrated that cell survival rate were elevated under 1% O2. The activity of SOD were decreased firstly and increased,and reached the highest value at the 24 thhour. The CAT activity went down firstly,then went up,and down again reaching minimum at the 8 thhour. The GSH levels gradually decreased,peak of activity was observed at the 16 thhour;but the content of MDA was not significantly changed compared to control(P>0.05). The content of NO and the activity of NOS increased and reached the peak at the 16 thhour. The ZF-4 cells had the highest survival rate under 0.1% oxygen after 8 hour. The activity of SOD wassignificantly higher than that in control at 24 thhour(P<0.05,the same below),but the activity of CAT was extremely reduced(P<0.01,the same below). The activity of GSH was extremely higher than that in control since the8 thhour;the contents of MDA significantly or extremely reduced under 0.1% oxygen;the content of NOS and NO were decreased significantly.【Conclusion】Under acute hypoxia condition,zebra fish mainly promote cell survival by improving the activity of SOD,GSH and other antioxidases to improve the hypoxia adaptation of cells,so that cells can survive.
引文
杜莉莉,吕润潇,杨晓漪,辛娜,马廷贤.2016.胎盘间充质干细胞低氧培养液对肠黏膜上皮细胞氧化应激损伤的保护作用[J].中国医科大学学报,45(2):131-135.[Du LL,LüR X,Yang X Y,Xin N,Ma T X.2016.Protection effect of placenta mesenchymal stem cells hypoxia culture medium on oxidative stress-induced injury of intestinal mucosal epithelial cells[J].Journal of China Medical University,45(2):131-135.]
郭会彩,熊晨,李军霞,张荣,赵丽娟,王永利.2012.间歇性低压低氧对过氧化氢心肌细胞损伤的保护作用[J].中国应用生理学杂志,28(3):199-202.[Guo H C,Xiong C,Li J X,Zhang R,Zhao L J,Wang Y L.2012.Protective effect of intermittent hypobaric hypoxia on cardiomyocytes injury induced by hydrogen peroxide[J].Chinese Journal of Applied Physiology,28(3):199-202.]
何云凌,吴丽颖,黄欣,赵彤,丁雪峰,吴奎武,范明,朱玲玲.2013.自噬对不同低氧条件下PC12细胞存活的影响[J].中国应用生理学杂志,29(3):193-196.[He Y L,Wu LY,Huang X,Zhao T,Ding X F,Wu K W,Fan M,Zhu LL.2013.The effects of autophagy on cell survival under different hypoxia[J].Chinese Journal of Applied Physiology,29(3):193-196.]
李根瑞,任利华,孙国华,杨建敏,韦秀梅,刘兆存,李尚俊,姜汉.2016.低溶氧胁迫对刺参(Apostichopus japonicus)氧化应激指标的影响[J].渔业科学进展,37(5):133-139.[Li G R,Ren L H,Sun G H,Yang J M,Wei X M,Liu ZC,Li S J,Jiang H.2016.Effects of hypoxic stress on oxidative stress indices in Apostichopus japonicas[J].Progress in Fishery Sciences,37(5):133-139.]
李艳丽,徐功玉,肖金文,李志,赵浩斌,周青春,桂建芳,钟雪萍.2017.血红素加氧酶1在斑马鱼低氧应激中的保护作用研究[J].水生生物学报,41(1):43-49.[Li Y L,Xu GY,Xiao J W,Li Z,Zhao H B,Zhou Q C,Gui J F,Zhong X P.2017.Studies on the protective role of zebrafish Ho1 in response to hypoxia[J].Acta Hydrobiologica Sinica,41(1):43-49.]
刘梅,李小刚,谭华.2012.大鼠脑缺血时脑组织NO含量的变化[J].现代临床医学,38(1):28-30.[Liu M,Li X G,Tan H.2012.The change of NO content on cerebral ischemia in rats[J].Journal of Modern Clinical Medicine,38(1):28-30.]
刘祥梅,吴宇强,严秋,唐瑶函,谭军.2008.力竭运动后24 h大鼠肾组织NO含量的变化[J].北京体育大学学报,31(10):1375-1377.[Liu X M,Wu Y Q,Yan Q,Tang Y H,Tan J.2008.Changes of nitric oxide content in rat’s kidney at 24 hours after exhaustive exercise[J].Journal of Beijing Sport University,31(10):1375-1377.]
区又君,陈世喜,王鹏飞,李加儿,温久福,王雯,谢木娇.2017.低氧环境下卵形鲳鲹的氧化应激响应与生理代谢相关指标的研究[J].南方水产科学,13(3):120-124.[Ou Y J,Chen S X,Wang P F,Li J E,Wen J F,Wang W,Xie M J.2017.Study on oxidative stress response and physiological metabolism related indices of Trachinotus ovatus under hypoxia stress[J].South China Fisheries Science,13(3):120-124.]
唐燕红,王剑,李金钢,谢惠春,李涛,何建平.2013.低氧应激下甘肃鼢鼠与SD大鼠心脏抗氧化酶和ATP酶活性的比较[J].兽类学报,33(2):178-185.[Tang Y H,Wang J,Li J G,Xie H C,Li T,He J P.2013.Comparision of antioxidase and ATPase activity in heart between Myospalax cansus and Rattus norvegicus under acute hypoxia[J].Acta Theriologica Sinica,33(2):178-185.]
王剑.2010.急性低氧下甘肃鼢鼠心脏抗氧化酶及ATP酶活性研究[D].西安:陕西师范大学.[Wang J.2010.Study on antioxidant enzymes and ATPase activities in heart of Gansu zokor in acute hypoxia[J].Xi’an:Shaanxi Normal University.]
韦日明,高雅,张可锋,李迎迎.2016.杠板归总黄酮对小鼠酒精性肝损伤的保护作用[J].南方农业学报,47(4):664-669.[Wei R M,Gao Y,Zhang K F,Li Y Y.2016.Protective effect of total flavonoids extracted from Polygonum knotweed L.on mice with alcoholic liver injury[J].Journal of Southern Agriculture,47(4):664-669.]
毋亚男,宋丽华,郭春花,来丽娜,张晓一,张晓京.2017.NOS/NO在白藜芦醇抗大鼠心肌缺血再灌注损伤中的作用[J].长治医学院学报,31(5):321-323.[Wu Y N,Song L H,Guo C H,Lai L N,Zhang X Y,Zhang X J.2017.The role of NOS/NO on myocardial in the protective effect of resveratrol against myocardial ischemia-reperfusion injury in rats[J].Journal of Changzhi Medical College,31(5):321-323.]
吴志昊,尤锋,王英芳,文爱韵,马得友,徐永立,张培军.2011.低氧和高氧对大菱鲆幼鱼红细胞核异常及氧化抗氧化平衡的影响[J].上海海洋大学学报,20(6):808-813.[Wu Z H,You F,Wang Y F,Wen A Y,Ma D Y,Xu Y L,Zhang P J.2011.The effects of hypoxia and hyperoxia on nucleus anomaly,SOD,CAT activities and MDA content in juvenile turbot Scophthalmus maximus[J].Journal of Shanghai Ocean University,20(6):808-813.]
谢惠春.2016.三种小型哺乳动物低氧适应生理生化机制研究[D].西安:陕西师范大学.[Xie H C.2016.Three kinds of small mammals to adapt to the physiological and biochemical mechanisms of hypoxia[D].Xi’an:Shaanxi Normal University.]
谢印芝,杨曦明,马子敏,尹昭云.2000.低氧大鼠脑、肺组织NO、NOS变化及其机理研究[J].高原医学杂志,10(1):4-6.[Xie Y Z,Yang X M,Ma Z M,Yin Z Y.2000.Changes of nitric oxide and nitric oxide synthase in rats during acuta and intermittent hypoxia[J].Journal of High Altitude Medicine,10(1):4-6.]
杨明阳.2017.低氧和高氧对斑马鱼胚胎心血管发育的影响[D].重庆:西南大学.[Yang M Y.2017.The influence of hypoxia and hyperoxia on cardiovascular development in zebrafish embryo[D].Chongqing:Southwest University.]
杨文君,钟梅芳,张宁,陈昉里,吴秀凤,周兆年,陈红.2010.不同强度短期间歇性减压低氧对血液及心血管组织抗氧化物质的影响[J].上海交通大学学报(医学版),30(6):656-659.[Yang W J,Zhong M F,Zhang N,Chen FL,Wu X F,Zhou Z N,Chen H.2010.Effects of different levels of short-term intermittent hypobaric hypoxia on antioxidants of blood and cardiovascular tissues in rats[J].Journal of Shanghai Jiaotong University(Medical Edition),30(6):656-659.]
赵燕静,狄桂兰,孔祥会,张杰,李莉,聂国兴,李学军.2016.低氧环境下poly I:C刺激对淇河鲫肝胰脏抗氧化防护的影响[J].水产学报,40(12):1830-1841.[Zhao Y J,Di G L,Kong X H,Zhang J,Li L,Nie G X,Li X J.2016.Effect on antioxidant defense in hepatopancreas of Carassius auratus after poly I:C injection under hypoxia[J].Journal of Fisheries of China,40(12):1830-1841.]
Galvin D J,Watson R W G,O’Neill A,Coffey R N T,Taylor C,Gillespie J I,Fitzpatrick J M.2005.Hypoxia inhibits human bladder smooth muscle cell proliferation:A potential mechanism of bladder dysfunction[J].Neurourology and Urodynamics,23(4):342-348.
Genbacev O,Krtolica A,Kaelin W,Fisher S J.2001.Human cytotrophoblast expression of the von hippel-lindau protein is downregulated during uterine invasion in situ and upregulated by hypoxia in vitro[J].Developmental Biology,233(2):526-536.
Ivanovic Z.2009.Hypoxia or in situ normoxia:The stem cell paradigm[J].Journal of Cellular Physiology,219(2):271-275.
Min J,Jin Y M,Moon J S,Sung M S,Jo S A,Jo I.2006.Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells[J].Hypertension,47(6):1189-1196.
Morrison S J.2001.Neuronal potential and lineage determination by neural stem cells[J].Current Opinion in Cell Biology,13(6):666-672.
Tzeng Y W,Lee L Y,Chao P L,Lee H C,Wu R T,Lin A M.2010.Role of autophagy in protection afforded by hypoxic preconditioning against MPP+-induced neurotoxicity in SH-SY5Y cells[J].Free Radical Biology&Medicine,49(5):839-846.
郭会彩,熊晨,李军霞,张荣,赵丽娟,王永利.2012.间歇性低压低氧对过氧化氢心肌细胞损伤的保护作用[J].中国应用生理学杂志,28(3):199-202.[Guo H C,Xiong C,Li J X,Zhang R,Zhao L J,Wang Y L.2012.Protective effect of intermittent hypobaric hypoxia on cardiomyocytes injury induced by hydrogen peroxide[J].Chinese Journal of Applied Physiology,28(3):199-202.]
何云凌,吴丽颖,黄欣,赵彤,丁雪峰,吴奎武,范明,朱玲玲.2013.自噬对不同低氧条件下PC12细胞存活的影响[J].中国应用生理学杂志,29(3):193-196.[He Y L,Wu LY,Huang X,Zhao T,Ding X F,Wu K W,Fan M,Zhu LL.2013.The effects of autophagy on cell survival under different hypoxia[J].Chinese Journal of Applied Physiology,29(3):193-196.]
李根瑞,任利华,孙国华,杨建敏,韦秀梅,刘兆存,李尚俊,姜汉.2016.低溶氧胁迫对刺参(Apostichopus japonicus)氧化应激指标的影响[J].渔业科学进展,37(5):133-139.[Li G R,Ren L H,Sun G H,Yang J M,Wei X M,Liu ZC,Li S J,Jiang H.2016.Effects of hypoxic stress on oxidative stress indices in Apostichopus japonicas[J].Progress in Fishery Sciences,37(5):133-139.]
李艳丽,徐功玉,肖金文,李志,赵浩斌,周青春,桂建芳,钟雪萍.2017.血红素加氧酶1在斑马鱼低氧应激中的保护作用研究[J].水生生物学报,41(1):43-49.[Li Y L,Xu GY,Xiao J W,Li Z,Zhao H B,Zhou Q C,Gui J F,Zhong X P.2017.Studies on the protective role of zebrafish Ho1 in response to hypoxia[J].Acta Hydrobiologica Sinica,41(1):43-49.]
刘梅,李小刚,谭华.2012.大鼠脑缺血时脑组织NO含量的变化[J].现代临床医学,38(1):28-30.[Liu M,Li X G,Tan H.2012.The change of NO content on cerebral ischemia in rats[J].Journal of Modern Clinical Medicine,38(1):28-30.]
刘祥梅,吴宇强,严秋,唐瑶函,谭军.2008.力竭运动后24 h大鼠肾组织NO含量的变化[J].北京体育大学学报,31(10):1375-1377.[Liu X M,Wu Y Q,Yan Q,Tang Y H,Tan J.2008.Changes of nitric oxide content in rat’s kidney at 24 hours after exhaustive exercise[J].Journal of Beijing Sport University,31(10):1375-1377.]
区又君,陈世喜,王鹏飞,李加儿,温久福,王雯,谢木娇.2017.低氧环境下卵形鲳鲹的氧化应激响应与生理代谢相关指标的研究[J].南方水产科学,13(3):120-124.[Ou Y J,Chen S X,Wang P F,Li J E,Wen J F,Wang W,Xie M J.2017.Study on oxidative stress response and physiological metabolism related indices of Trachinotus ovatus under hypoxia stress[J].South China Fisheries Science,13(3):120-124.]
唐燕红,王剑,李金钢,谢惠春,李涛,何建平.2013.低氧应激下甘肃鼢鼠与SD大鼠心脏抗氧化酶和ATP酶活性的比较[J].兽类学报,33(2):178-185.[Tang Y H,Wang J,Li J G,Xie H C,Li T,He J P.2013.Comparision of antioxidase and ATPase activity in heart between Myospalax cansus and Rattus norvegicus under acute hypoxia[J].Acta Theriologica Sinica,33(2):178-185.]
王剑.2010.急性低氧下甘肃鼢鼠心脏抗氧化酶及ATP酶活性研究[D].西安:陕西师范大学.[Wang J.2010.Study on antioxidant enzymes and ATPase activities in heart of Gansu zokor in acute hypoxia[J].Xi’an:Shaanxi Normal University.]
韦日明,高雅,张可锋,李迎迎.2016.杠板归总黄酮对小鼠酒精性肝损伤的保护作用[J].南方农业学报,47(4):664-669.[Wei R M,Gao Y,Zhang K F,Li Y Y.2016.Protective effect of total flavonoids extracted from Polygonum knotweed L.on mice with alcoholic liver injury[J].Journal of Southern Agriculture,47(4):664-669.]
毋亚男,宋丽华,郭春花,来丽娜,张晓一,张晓京.2017.NOS/NO在白藜芦醇抗大鼠心肌缺血再灌注损伤中的作用[J].长治医学院学报,31(5):321-323.[Wu Y N,Song L H,Guo C H,Lai L N,Zhang X Y,Zhang X J.2017.The role of NOS/NO on myocardial in the protective effect of resveratrol against myocardial ischemia-reperfusion injury in rats[J].Journal of Changzhi Medical College,31(5):321-323.]
吴志昊,尤锋,王英芳,文爱韵,马得友,徐永立,张培军.2011.低氧和高氧对大菱鲆幼鱼红细胞核异常及氧化抗氧化平衡的影响[J].上海海洋大学学报,20(6):808-813.[Wu Z H,You F,Wang Y F,Wen A Y,Ma D Y,Xu Y L,Zhang P J.2011.The effects of hypoxia and hyperoxia on nucleus anomaly,SOD,CAT activities and MDA content in juvenile turbot Scophthalmus maximus[J].Journal of Shanghai Ocean University,20(6):808-813.]
谢惠春.2016.三种小型哺乳动物低氧适应生理生化机制研究[D].西安:陕西师范大学.[Xie H C.2016.Three kinds of small mammals to adapt to the physiological and biochemical mechanisms of hypoxia[D].Xi’an:Shaanxi Normal University.]
谢印芝,杨曦明,马子敏,尹昭云.2000.低氧大鼠脑、肺组织NO、NOS变化及其机理研究[J].高原医学杂志,10(1):4-6.[Xie Y Z,Yang X M,Ma Z M,Yin Z Y.2000.Changes of nitric oxide and nitric oxide synthase in rats during acuta and intermittent hypoxia[J].Journal of High Altitude Medicine,10(1):4-6.]
杨明阳.2017.低氧和高氧对斑马鱼胚胎心血管发育的影响[D].重庆:西南大学.[Yang M Y.2017.The influence of hypoxia and hyperoxia on cardiovascular development in zebrafish embryo[D].Chongqing:Southwest University.]
杨文君,钟梅芳,张宁,陈昉里,吴秀凤,周兆年,陈红.2010.不同强度短期间歇性减压低氧对血液及心血管组织抗氧化物质的影响[J].上海交通大学学报(医学版),30(6):656-659.[Yang W J,Zhong M F,Zhang N,Chen FL,Wu X F,Zhou Z N,Chen H.2010.Effects of different levels of short-term intermittent hypobaric hypoxia on antioxidants of blood and cardiovascular tissues in rats[J].Journal of Shanghai Jiaotong University(Medical Edition),30(6):656-659.]
赵燕静,狄桂兰,孔祥会,张杰,李莉,聂国兴,李学军.2016.低氧环境下poly I:C刺激对淇河鲫肝胰脏抗氧化防护的影响[J].水产学报,40(12):1830-1841.[Zhao Y J,Di G L,Kong X H,Zhang J,Li L,Nie G X,Li X J.2016.Effect on antioxidant defense in hepatopancreas of Carassius auratus after poly I:C injection under hypoxia[J].Journal of Fisheries of China,40(12):1830-1841.]
Galvin D J,Watson R W G,O’Neill A,Coffey R N T,Taylor C,Gillespie J I,Fitzpatrick J M.2005.Hypoxia inhibits human bladder smooth muscle cell proliferation:A potential mechanism of bladder dysfunction[J].Neurourology and Urodynamics,23(4):342-348.
Genbacev O,Krtolica A,Kaelin W,Fisher S J.2001.Human cytotrophoblast expression of the von hippel-lindau protein is downregulated during uterine invasion in situ and upregulated by hypoxia in vitro[J].Developmental Biology,233(2):526-536.
Ivanovic Z.2009.Hypoxia or in situ normoxia:The stem cell paradigm[J].Journal of Cellular Physiology,219(2):271-275.
Min J,Jin Y M,Moon J S,Sung M S,Jo S A,Jo I.2006.Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells[J].Hypertension,47(6):1189-1196.
Morrison S J.2001.Neuronal potential and lineage determination by neural stem cells[J].Current Opinion in Cell Biology,13(6):666-672.
Tzeng Y W,Lee L Y,Chao P L,Lee H C,Wu R T,Lin A M.2010.Role of autophagy in protection afforded by hypoxic preconditioning against MPP+-induced neurotoxicity in SH-SY5Y cells[J].Free Radical Biology&Medicine,49(5):839-846.