TUNEL法检测Cu~(2+)胁迫诱导的中国树花共生藻细胞的死亡
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摘要
为了探讨铜离子胁迫对中国树花共生藻细胞毒害作用的机制及细胞死亡的原因,通过徒手切片,采用两种TUNEL检测法(Promega和Roche试剂盒)对2mmol/L和4mmol/L Cu~(2+)胁迫24h的中国树花地衣体共生藻细胞凋亡情况进行观察。结果表明:(1)伊文思蓝染色法检测结果显示,中国树花共生藻细胞活力在2和4mmol/L Cu~(2+)胁迫下显著降低,其细胞的死亡率随着Cu~(2+)浓度的提高和处理时间的延长而明显增加;(2)在2和4mmol/L Cu~(2+)胁迫条件下,中国树花共生藻细胞的TUNEL阳性细胞率在Promega试剂盒中检测结果分别为50.30%和31.21%,而在Roche试剂盒中共生藻细胞TUNEL阳性标记核分别为53.17%和36.88%,两种TUNEL检测法结果类似。(3)与伊文思蓝染色法检测的Cu~(2+)胁迫中国树花共生藻细胞活力相比,发现较低浓度的Cu~(2+)(2mmol/L)胁迫对地衣共生藻细胞的毒害作用可诱导启动细胞凋亡程序,而较高浓度的Cu~(2+)(4mmol/L)胁迫对地衣共生藻产生较严重的毒害则导致大部分细胞的坏死,只有极少数细胞出现细胞凋亡。(4)两种试剂盒均可用于较低浓度Cu~(2+)胁迫引起的地衣体共生藻细胞凋亡的检测;直接将徒手切片材料用于TUNEL细胞凋亡原位检测中也得到了较理想的结果,避免了制备石蜡切片的繁琐步骤,缩短实验时间,简化了实验流程。
In order to understand the mechanism of toxic effects of Cu~(2+)stress on the phycobionts of Ramalina sinensis,we studied the cell death of symbiont alga in the hand-sliced sections of the lichen thallus which were treated with 2and 4mmol/L CuSO4 for 24hours respectively by using two kinds of TUNEL reaction kit(Promega DeadEndTM Colorimetric TUNEL System and Roche in situ Cell Death Detection Kit).The results of the study showed:(1)the phycobiont cell vitality decreased significantly under Cu~(2+)stress detected by Evan's Blue staining method.Moreover,there was a positive correlation between the cell vitality and the Cu~(2+)stress concentration and time.(2)When Promega DeadEndTM Colorimetric TUNEL System was used,the TUNEL positive phycobiont cells were 50.30% and 31.21%,respectivelyunder 2and 4mmol/L Cu~(2+)stress conditions.The TUNEL positive cells of phycobionts were 53.17%and36.88%,respectively when Roche in situ Cell Death Detection Kit was used under the same Cu~(2+)stress conditions,the results of both TUNEL kits was similar.(3)Considering the results of phycobionts cell vitality which was detected by Evan's Blue staining method under Cu~(2+)stress,it can be concluded that apoptosis(programmed cell death)of R.sinensis phycobiont were induced under lower Cu~(2+)concentration(2mmol/L)treatment,but the cell death of phycobionts were mainly caused by the necrosis and only few programmed cell death of the phycobionts was occured while the lichen was treated with higher Cu~(2+)concentration(4mmol/L).(4)Both kinds of TUNEL detection kit can be used for the study of apotopsis of lichen phycobionts under heavy metal Cu~(2+)stress conditions.In addition,the hand-sliced sections of lichen thalli were used in this study instead of the paraffin sections.The experiment procedure was simplified by avoiding the complicated experimental steps.This research will be an important scientific reference for the further understandings of lichen responses to the various metal stresses.
In order to understand the mechanism of toxic effects of Cu~(2+)stress on the phycobionts of Ramalina sinensis,we studied the cell death of symbiont alga in the hand-sliced sections of the lichen thallus which were treated with 2and 4mmol/L CuSO4 for 24hours respectively by using two kinds of TUNEL reaction kit(Promega DeadEndTM Colorimetric TUNEL System and Roche in situ Cell Death Detection Kit).The results of the study showed:(1)the phycobiont cell vitality decreased significantly under Cu~(2+)stress detected by Evan's Blue staining method.Moreover,there was a positive correlation between the cell vitality and the Cu~(2+)stress concentration and time.(2)When Promega DeadEndTM Colorimetric TUNEL System was used,the TUNEL positive phycobiont cells were 50.30% and 31.21%,respectivelyunder 2and 4mmol/L Cu~(2+)stress conditions.The TUNEL positive cells of phycobionts were 53.17%and36.88%,respectively when Roche in situ Cell Death Detection Kit was used under the same Cu~(2+)stress conditions,the results of both TUNEL kits was similar.(3)Considering the results of phycobionts cell vitality which was detected by Evan's Blue staining method under Cu~(2+)stress,it can be concluded that apoptosis(programmed cell death)of R.sinensis phycobiont were induced under lower Cu~(2+)concentration(2mmol/L)treatment,but the cell death of phycobionts were mainly caused by the necrosis and only few programmed cell death of the phycobionts was occured while the lichen was treated with higher Cu~(2+)concentration(4mmol/L).(4)Both kinds of TUNEL detection kit can be used for the study of apotopsis of lichen phycobionts under heavy metal Cu~(2+)stress conditions.In addition,the hand-sliced sections of lichen thalli were used in this study instead of the paraffin sections.The experiment procedure was simplified by avoiding the complicated experimental steps.This research will be an important scientific reference for the further understandings of lichen responses to the various metal stresses.
引文
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[7]阿孜古丽·玉苏普,托合提·克依木,阿布都拉·阿巴斯,等.利用地衣微核评价乌鲁木齐南郊空气质量[J].新疆农业科学,2010,47(8):1 637-1 642.AZIGULI Y,TOHITI K,ABDULLA A,et al.Using lichens micronucleus southern suburbs of Urumqi air quality[J].Xinjiang Agricultural Science Evaluation 2010,47(8):1 637-1 642.
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[9]热依拉·热合曼,帕提古丽·依明,阿布都拉·阿巴斯,等.Cu2+胁迫对两种地衣细胞结构的影响[J].西北植物学报,2015,35(1):1-8.REYILA R,PATIGU I,PAZILAITIB,et al.Effects of Cu2+stress on the cell structure of two species of lichen[J].Acta Botanica Boreali-Occidentalia Sinica,2015,35(1):1-8.
[10]PASTIKKA E,KAIRAVUO M,SERSEN F,et al.Excess copper predisposes photosystemⅡto photo inhibition in vivo by outcompeting iron and causing decrease in leaf chlorophyll[J].Plant Physiology,2002,129:1 359-1 367.
[11]DEMIDCHIK V,SOKOLIK A,YURIN V.The effect of Cu2+on ion transport systems of the plant cell plasmalemma[J].Plant Physiology,1997,114:1 313-1 325.
[12]JOHNSON FB,SINCLAIR DA,GUARENTE L.Molecular biology of aging[J].Cell.1999,96:291-302.
[13]张贵又,田瑞华,戴尧仁.植物细胞凋亡的研究进展[J].生物工程进展,2001,21(6):22-26.ZHANG G Y,TIAN R H,DAI Y R.Research Progress on plant cell apoptosis[J].Progress in Biological Engineering,2001,21(6):22-26.
[14]高丰,金天明,姜宁.细胞凋亡的研究现状与发展趋势[J].动物医学进展,2003,24(6):22-26.GAO F,JIN T Y,MEI N.Research status and development trend of apoptosis in[J].Animal Medicine,2003,24(6):22-26.
[15]李向军,刘玉峰.细胞凋亡及检测方法[J].动物科技,2004,21(12):30-34.LI S J,LIU Y F.Cell apoptosis and detection methods[J].Animal Science and Technology,2004,21(12):30-34.
[16]徐昌杰,陈昆松,Hong Ding.植物程序性死亡检测技术[J].遗传,2004,26(1):127-130.XU CJ,CHEN KS,HONG D.Plant programmed death detection technique[J].Inheritance,2004,26(1):127-130.
[17]LIU P P,BHATTACHRJEE S,KLESSIG D F,et al.Systemic acquired resistance is induced by gene-mediated responses independent of cell death[J].Mol Pathol,2010,11(1):155-160.
[18]MITTLER R,SMON L,LAM E,et al,Pathogen-induced programmed cell death in tobacco[J].J.Cell Sci,1997,110:1 333-1 344.
[19]BRUSSAARD C P D,NOORDELOOS A A M,RIEGMAN R.Autolysis kinetics of the marine diatom Ditylum brightwellii(Bacillariophyceae)under nitrogen and phosphorus limitation and starvation[J].Journal of Phycology,1997,33(6):980-987.
[20]NOVAETIS,PHARMA A G.Cell apoptosis[J].Toxicol Pathology,2002,30:1 137-1 143
[21]LEE D Y,RHEE G Y.Kinetics of cell death in the cyanobacterium Anabaena flos-aquae and the production of dissolved organic carbon[J].Phycol,1997,(33):991-998.
[22]NING S B,GUO H L,WANG L,et al.Salt stress induces programmed cell death in prokaryotic organism Anabaena[J].Appl.Microbiol,2002,(93):15-28.
[23]SEGOVIA M,HARAMATY L,BERGES J A,et al,Cell death in the unicellular chlorophyte Dunaliella tertiolecta.A hypothesis on the evolution of apoptosis in higher plants and metazoans[J].Plant Physiol,2003,(132):99-105.
[24]ZUPPINI A,GEROTTO C,BALDAN B.Programmed cell death and adaptation:two different types of abiotic stress response in a unicellular chlorophyte[J].Plant Cell Physiol,2010,(51):884-895.
[25]BIDLE K D,FALKOWSKI P G,Cell death in planktonic,photosynthetic microorganisms[J].Nat.Rev.Microbiol,2004,(2):643-655.
[26]MOHARIKAR S,DSOUZA J S,KULKARNI A B,et al,Apoptotic-like cell death pathway is induced in unicellular chlorophyte Chlamydomonas reinhardtii(Chlorophyceae)cells following UV irradiation:detection and functional analyses[J].Phycol,2006,(42):423–433.
[27]SIRISHA V L,SINHA M,DSOUZA J S.Menadione-induced caspase-dependent programmed cell death in the green chlorophyte Chlamydomonas reinhardtii[J].Phycol,2014,(50):587-601.
[28]DILEK UNAL,YIGIT UYANIKGIL.UV-Binduces cell death in the lichen Physcia semipinnata(J.E.Gmel)[J].Turk J Biol,2011,(35):137-144
[29]孙英丽,赵允,刘春香.细胞色素C能诱导植物细胞编程性死亡[J].植物学报,1999,41:87SUN Y L,ZHAO Y,LIU C X.Cytochrome C can induce programmed cell death in plant[J].Acta Botanica Sinica,1999,41:87.
[30]Labat-Moleur F,Guillermet C,Lofimier.TUNEL apoptotic cell detection in tissue sections:critical evaluation and improvement[J].Histochem Cytochern,1998,46(3):327-334.
[31]张晶,黄贵祥,赵恭华.影响TUNEL法阳性结果的因素探讨[J].中华病理学杂志,2000,29(6):467-468ZHANG J,HUANG G X,ZHAO G H.A study on the factors of positive results of method in TUNEL[J].Chinese Journal of Pathology,2000,29(6):467-468.
[2]魏江春.中国地衣综览[M].中国北京:万国学术出版社,1991.
[3]KIRK PM,CANNON PF,DAVID JC,et al.Dictionary of The Fungi.[M].9th Edition CAB International,Wallingford,Oxon,.2001:1-655.
[4]MASON E H.The Biology of Lichens[M].London:Edward Arnold Publishers Ltd,1983.
[5]帕孜来提·拜合提,祁要鹏,阿孜古丽·玉苏普,等.铜胁迫对两种地衣植物生理生化特征的影响[J].生态环境学报,2010,19(11):2 708-2 712.PAZILAITI B,QI Y P,AZIGULI Y,et al.Copper stress on physiological and biochemical characteristics of two different lichens species[J].Journal of Ecological Environment,2010,19(11):2 708-2 712
[6]帕孜来提·拜合提,阿孜古丽·玉苏普,居玛古丽,等.地衣对乌鲁木齐市空气污染生物指示的研究[J].生物技术,2010,20:79-82.PAZILAITI B,QI Y P,AZIGULI Y,et al.Study on the biological indicator of air pollution in Urumqi City by lichen[J],Biotechnology,2010,20:79-82.
[7]阿孜古丽·玉苏普,托合提·克依木,阿布都拉·阿巴斯,等.利用地衣微核评价乌鲁木齐南郊空气质量[J].新疆农业科学,2010,47(8):1 637-1 642.AZIGULI Y,TOHITI K,ABDULLA A,et al.Using lichens micronucleus southern suburbs of Urumqi air quality[J].Xinjiang Agricultural Science Evaluation 2010,47(8):1 637-1 642.
[8]艾尼瓦尔·吐米尔,阿地里江·阿布都拉,阿布都拉·阿巴斯.利用地衣移植法对乌鲁木齐市大气质量的评价[J].环境污染与防治,2011,33(23):1-9.AINIWAR T,ADILIJIANG A,ABDULLA A.Evaluation of air quality in Urumqi City by using the method of lichen transplant[J]Environmental Pollution and Control,2011,33(23):1-9.
[9]热依拉·热合曼,帕提古丽·依明,阿布都拉·阿巴斯,等.Cu2+胁迫对两种地衣细胞结构的影响[J].西北植物学报,2015,35(1):1-8.REYILA R,PATIGU I,PAZILAITIB,et al.Effects of Cu2+stress on the cell structure of two species of lichen[J].Acta Botanica Boreali-Occidentalia Sinica,2015,35(1):1-8.
[10]PASTIKKA E,KAIRAVUO M,SERSEN F,et al.Excess copper predisposes photosystemⅡto photo inhibition in vivo by outcompeting iron and causing decrease in leaf chlorophyll[J].Plant Physiology,2002,129:1 359-1 367.
[11]DEMIDCHIK V,SOKOLIK A,YURIN V.The effect of Cu2+on ion transport systems of the plant cell plasmalemma[J].Plant Physiology,1997,114:1 313-1 325.
[12]JOHNSON FB,SINCLAIR DA,GUARENTE L.Molecular biology of aging[J].Cell.1999,96:291-302.
[13]张贵又,田瑞华,戴尧仁.植物细胞凋亡的研究进展[J].生物工程进展,2001,21(6):22-26.ZHANG G Y,TIAN R H,DAI Y R.Research Progress on plant cell apoptosis[J].Progress in Biological Engineering,2001,21(6):22-26.
[14]高丰,金天明,姜宁.细胞凋亡的研究现状与发展趋势[J].动物医学进展,2003,24(6):22-26.GAO F,JIN T Y,MEI N.Research status and development trend of apoptosis in[J].Animal Medicine,2003,24(6):22-26.
[15]李向军,刘玉峰.细胞凋亡及检测方法[J].动物科技,2004,21(12):30-34.LI S J,LIU Y F.Cell apoptosis and detection methods[J].Animal Science and Technology,2004,21(12):30-34.
[16]徐昌杰,陈昆松,Hong Ding.植物程序性死亡检测技术[J].遗传,2004,26(1):127-130.XU CJ,CHEN KS,HONG D.Plant programmed death detection technique[J].Inheritance,2004,26(1):127-130.
[17]LIU P P,BHATTACHRJEE S,KLESSIG D F,et al.Systemic acquired resistance is induced by gene-mediated responses independent of cell death[J].Mol Pathol,2010,11(1):155-160.
[18]MITTLER R,SMON L,LAM E,et al,Pathogen-induced programmed cell death in tobacco[J].J.Cell Sci,1997,110:1 333-1 344.
[19]BRUSSAARD C P D,NOORDELOOS A A M,RIEGMAN R.Autolysis kinetics of the marine diatom Ditylum brightwellii(Bacillariophyceae)under nitrogen and phosphorus limitation and starvation[J].Journal of Phycology,1997,33(6):980-987.
[20]NOVAETIS,PHARMA A G.Cell apoptosis[J].Toxicol Pathology,2002,30:1 137-1 143
[21]LEE D Y,RHEE G Y.Kinetics of cell death in the cyanobacterium Anabaena flos-aquae and the production of dissolved organic carbon[J].Phycol,1997,(33):991-998.
[22]NING S B,GUO H L,WANG L,et al.Salt stress induces programmed cell death in prokaryotic organism Anabaena[J].Appl.Microbiol,2002,(93):15-28.
[23]SEGOVIA M,HARAMATY L,BERGES J A,et al,Cell death in the unicellular chlorophyte Dunaliella tertiolecta.A hypothesis on the evolution of apoptosis in higher plants and metazoans[J].Plant Physiol,2003,(132):99-105.
[24]ZUPPINI A,GEROTTO C,BALDAN B.Programmed cell death and adaptation:two different types of abiotic stress response in a unicellular chlorophyte[J].Plant Cell Physiol,2010,(51):884-895.
[25]BIDLE K D,FALKOWSKI P G,Cell death in planktonic,photosynthetic microorganisms[J].Nat.Rev.Microbiol,2004,(2):643-655.
[26]MOHARIKAR S,DSOUZA J S,KULKARNI A B,et al,Apoptotic-like cell death pathway is induced in unicellular chlorophyte Chlamydomonas reinhardtii(Chlorophyceae)cells following UV irradiation:detection and functional analyses[J].Phycol,2006,(42):423–433.
[27]SIRISHA V L,SINHA M,DSOUZA J S.Menadione-induced caspase-dependent programmed cell death in the green chlorophyte Chlamydomonas reinhardtii[J].Phycol,2014,(50):587-601.
[28]DILEK UNAL,YIGIT UYANIKGIL.UV-Binduces cell death in the lichen Physcia semipinnata(J.E.Gmel)[J].Turk J Biol,2011,(35):137-144
[29]孙英丽,赵允,刘春香.细胞色素C能诱导植物细胞编程性死亡[J].植物学报,1999,41:87SUN Y L,ZHAO Y,LIU C X.Cytochrome C can induce programmed cell death in plant[J].Acta Botanica Sinica,1999,41:87.
[30]Labat-Moleur F,Guillermet C,Lofimier.TUNEL apoptotic cell detection in tissue sections:critical evaluation and improvement[J].Histochem Cytochern,1998,46(3):327-334.
[31]张晶,黄贵祥,赵恭华.影响TUNEL法阳性结果的因素探讨[J].中华病理学杂志,2000,29(6):467-468ZHANG J,HUANG G X,ZHAO G H.A study on the factors of positive results of method in TUNEL[J].Chinese Journal of Pathology,2000,29(6):467-468.