细胞深低温冷冻损伤机制的实验研究
摘要
研究背景:近年来,深低温保存技术的应用越来越广泛。深低温可以降低细胞的呼吸代谢作用,延缓其功能的丧失,临床上将生物组织或器官采用特殊方法冷却至深低温,并长期保存;待需要时,再将其按特殊方法复温,以便获得活的生物体。
2002至2006年,我们先后将2例患者的断指在冷冻保护剂二甲亚砜(DMSO)的作用下,通过程序降温在-196℃液氮中保存后再植成功。但术后长期随访发现,患指均有不同程度萎缩。据冷冻损伤的两因素假说指出,造成细胞损伤有两个独立因素:胞内冰损伤和溶质损伤。一是胞内冰的形成,由过快冷却所产生的;冷却速率越快,此损伤越大。另一是“溶液损伤”,由过慢冷却所产生,使细胞在高浓度溶液中暴露时间过长而遭损伤,冷却越慢,此损伤越大。但是,目前对低温损伤两因素引起细胞的死亡方式没有进行阐明。
为阐明低温损伤两因素引起细胞的死亡方式,我们选取人肝癌细胞株BEL-7402经荧光染色后流式细胞仪检测细胞凋亡、坏死、和细胞碎片的变化。研究深低温冻存导致细胞死亡的方式,并观察对细胞周期的影响。
目的研究深低温冷冻损伤导致细胞死亡的机制。
方法在不同的电解质与冷冻保护剂浓度下:1倍离子浓度下50%甘油、0%、10%、50%DMSO及20倍离子浓度下50%DMSO,对人肝癌株细胞株(BEL-7402)程序降温至深低温后复苏。采用Annexin-V/PI荧光标记双染色、DNA-Prep stain荧光标记染色流式细胞仪分析冻存前后凋亡、坏死、存活、细胞碎片及BEL-7402细胞周期变化。
结果无冷冻保护剂时,冷冻后细胞坏死、碎片明显(P＜0.05)。随着离子浓度和DMSO浓度的增加,冷冻后BEL-7402细胞凋亡愈趋明显,但细胞坏死、碎片均不明显(P＞0.05);冷冻前后细胞周期变化不显著(P＞0.05)。
结论在冷冻过程中,造成细胞损伤有两个独立因素:胞内冰损伤和溶质损伤。胞内冰损伤引起细胞死亡的方式是坏死,而溶质损伤(包括电解质与DMSO)导致细胞凋亡。冷冻导致细胞死亡无周期特异性。
Background During the past ten years,biologists had found the method to cryopreserve many biomaterials,such as sperms,skin,parathyroid gland,leukocytes, bone marrow cells and antibiotics.Report showed that a cryopreserved Cyprinus Carpio embryo was still alive after thawing.In 2002,we succeeded in reimplanting a disjunct finger which was thermal-control cryopreserved in liquid nitrogen for 81 days.This success provided us a perspective in the cryopreservation of organs,or even the whole body of patients suffering from cancers.To investigate influence of cryodamage on cell viability,a study on mechanism of cryodamage in human hepatoma carcinoma cell was carried on.
Objective To investigate influence of cryodamage on cell viability.
Methods Human hepatoma carcinoma cell line BEL-7402 were cryopreserved under different concentrations of electrolytes and cryoprotective agents:1×50% glycerine,and 0%,10%or 50%DMSO;or 20×50%DMSO.Then apoptosis,cell necrosis,cryosurvival and debris rates of the cells,and cell cycle of BEL-7402 before and after freezing were analyzed by flow cytometry.
Results Without cryoprotective agents,cell necrosis and debris were obviously observed(P＜0.05)after freezing.When the concentrations of electrolytes and DMSO were increased,cell necrosis and debris got diminished(P＞0.05),while apoptosis of BEL-7402 turned to be significant gradually;There were no significant variation of cell cyle after freezing(P＞0.05).
Conclusion There are two independent kinds of cryodamage factors:one is endocelluar cryodamage,which can bring about cellular necrosis,and the other is solute-related damage,which may induce apoptosis.Both of these two kinds of cryodamage have no cyclic specificity.
2002至2006年,我们先后将2例患者的断指在冷冻保护剂二甲亚砜(DMSO)的作用下,通过程序降温在-196℃液氮中保存后再植成功。但术后长期随访发现,患指均有不同程度萎缩。据冷冻损伤的两因素假说指出,造成细胞损伤有两个独立因素:胞内冰损伤和溶质损伤。一是胞内冰的形成,由过快冷却所产生的;冷却速率越快,此损伤越大。另一是“溶液损伤”,由过慢冷却所产生,使细胞在高浓度溶液中暴露时间过长而遭损伤,冷却越慢,此损伤越大。但是,目前对低温损伤两因素引起细胞的死亡方式没有进行阐明。
为阐明低温损伤两因素引起细胞的死亡方式,我们选取人肝癌细胞株BEL-7402经荧光染色后流式细胞仪检测细胞凋亡、坏死、和细胞碎片的变化。研究深低温冻存导致细胞死亡的方式,并观察对细胞周期的影响。
目的研究深低温冷冻损伤导致细胞死亡的机制。
方法在不同的电解质与冷冻保护剂浓度下:1倍离子浓度下50%甘油、0%、10%、50%DMSO及20倍离子浓度下50%DMSO,对人肝癌株细胞株(BEL-7402)程序降温至深低温后复苏。采用Annexin-V/PI荧光标记双染色、DNA-Prep stain荧光标记染色流式细胞仪分析冻存前后凋亡、坏死、存活、细胞碎片及BEL-7402细胞周期变化。
结果无冷冻保护剂时,冷冻后细胞坏死、碎片明显(P＜0.05)。随着离子浓度和DMSO浓度的增加,冷冻后BEL-7402细胞凋亡愈趋明显,但细胞坏死、碎片均不明显(P＞0.05);冷冻前后细胞周期变化不显著(P＞0.05)。
结论在冷冻过程中,造成细胞损伤有两个独立因素:胞内冰损伤和溶质损伤。胞内冰损伤引起细胞死亡的方式是坏死,而溶质损伤(包括电解质与DMSO)导致细胞凋亡。冷冻导致细胞死亡无周期特异性。
Background During the past ten years,biologists had found the method to cryopreserve many biomaterials,such as sperms,skin,parathyroid gland,leukocytes, bone marrow cells and antibiotics.Report showed that a cryopreserved Cyprinus Carpio embryo was still alive after thawing.In 2002,we succeeded in reimplanting a disjunct finger which was thermal-control cryopreserved in liquid nitrogen for 81 days.This success provided us a perspective in the cryopreservation of organs,or even the whole body of patients suffering from cancers.To investigate influence of cryodamage on cell viability,a study on mechanism of cryodamage in human hepatoma carcinoma cell was carried on.
Objective To investigate influence of cryodamage on cell viability.
Methods Human hepatoma carcinoma cell line BEL-7402 were cryopreserved under different concentrations of electrolytes and cryoprotective agents:1×50% glycerine,and 0%,10%or 50%DMSO;or 20×50%DMSO.Then apoptosis,cell necrosis,cryosurvival and debris rates of the cells,and cell cycle of BEL-7402 before and after freezing were analyzed by flow cytometry.
Results Without cryoprotective agents,cell necrosis and debris were obviously observed(P＜0.05)after freezing.When the concentrations of electrolytes and DMSO were increased,cell necrosis and debris got diminished(P＞0.05),while apoptosis of BEL-7402 turned to be significant gradually;There were no significant variation of cell cyle after freezing(P＞0.05).
Conclusion There are two independent kinds of cryodamage factors:one is endocelluar cryodamage,which can bring about cellular necrosis,and the other is solute-related damage,which may induce apoptosis.Both of these two kinds of cryodamage have no cyclic specificity.
引文
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[4]Bolognesi MP,Chen LF,Seaber AV,et al.Protective effect of hypothermia on contractile force in skeletal muscle.J Orthop Res,1996;14(3):390
[5]潘达德,程国良,林宗礼,等.冷藏对离体缺血组织的保护作用:组织化学观察.中华显微外科杂志,1994;17(1):31
[6]Kihira M,Miura T,Ishiguro N.Preservation of skeletal muscle in tissue transfers using rat hindlimbs.Plast Reconstr Surg,1991;88(2):275
[7]Turk JB,Zhang WX,Babajanian M,et al.The use of a new perfusate in experimental microvascular flaps:A threshold increase in ischemic tolerance.J Reconstr Microsurg,1991;4(4):305
[8]Norden MA,Rao VK,Southard JH.Imrpoved preservation of rat hindlimbs with the University of Wisconsin solution and butanedione monoxime.Plast Reconstr Surg,1997;100(9):957
[9]Yokoyama K,Kimura M,Itoman M.rat whole-limb viability after cold immersion using University of Wisconsin and Euro-Collins solutions.Transplantation,1996;62(7):884
[10]Gordon L,Levinsoha DG,Boronsky CD,et al.Improved preservation of skeletal muscle in amputated limbs using pulsatil hypothermic perfusion with University of Wisconsin solution.J Bone Joint Surg(Am),1992;74(6):1358
[11]Kour Y,Phone MH,Chia J,et al.A preliminary report of tissue preservation
with University of Wisconsin cold storage solution in major limb replantation.Ann A cad Med Singapore,1995;24(4 suppl):37.
[12]顾玉东,李继峰,袁伟,等.离断肢体血管与肌肉保护的实验研究,中华创伤杂志,1996;12(1):25
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[22]胡成栋.玻璃化深低温保存生物组织与器官[J].空军总医院学报,2004,20(4):219-222.
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[27]Hua TC,Chang ZH,Chen Y.Cryobiological engineering[M].Shanghai Institute of Mechanical Engineering,1985.
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[29]Bianchi MVA,et al.Thermal analysis of a cryomicoscope:Effect of heat spreading and contact resistance[J].Intema -tional Journal of Heat and Mass Transfer,1998,41(23):3873.
[30]Wildt DE.Genome resource banking for wildlife research,management,and conservation[J].ILAR,2000,41:228-234.
[31]Jackson TH,Ungan A,Critser JK,et al.Novel microwave technology for cryopreservation of biomaterials by suppression of apparent ice formation[J].Cryobiology,1997,34:363-372.
[32]胡军祥,应华波,周国英,等.微波复温对玻璃化冻存大鼠胚脑细胞活性的影响[J].浙江大学报,2005,31(2):190-194.
[33]骆晓东,何立群,高大勇.低温保存后生物组织微波复温过程的数值分析[J].北京生物医学工程,2006,25(1):20-25.
[34]Karlsson JO,Toner M.Long-term storage of tissues by cryopreservation:critical issues[J].Biomaterials,1996,17:243-256.
[35]Hagedom M,Hsu EW,Pilatus U,et al.Magnetic resonance microscopy and spectroscopy reveal kinetics of cryoprotectant permeation in a multicompartmental biological system[J].Proc Natl Acad Sci U S A.1996,93:7454-7459.
[36]Zhang Lu,Zhang Lin,Jiang Yong,et al.Role of p38 protein kinase in heat-induced Raw cells apoptosis.Chin Med J 2000;113(4):354-357
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[2]Ono H,Nakagawa Y,Mizumoto S,et al.Evaluation of vascular compliance and vasoconstractive reaction in amputated hind-limbs of rats.J Orthop Res,1995;13(3):375
[3]Ono H,Nakagawa Y,Mizumoto S,et al.Vascular compliance and vasconstractive reactions in rat hindlimbs comparison between storage temperature of -1℃ and 4℃.J Reconstr Microsurg,1997;13(6):409
[4]Bolognesi MP,Chen LF,Seaber AV,et al.Protective effect of hypothermia on contractile force in skeletal muscle.J Orthop Res,1996;14(3):390
[5]潘达德,程国良,林宗礼,等.冷藏对离体缺血组织的保护作用:组织化学观察.中华显微外科杂志,1994;17(1):31
[6]Kihira M,Miura T,Ishiguro N.Preservation of skeletal muscle in tissue transfers using rat hindlimbs.Plast Reconstr Surg,1991;88(2):275
[7]Turk JB,Zhang WX,Babajanian M,et al.The use of a new perfusate in experimental microvascular flaps:A threshold increase in ischemic tolerance.J Reconstr Microsurg,1991;4(4):305
[8]Norden MA,Rao VK,Southard JH.Imrpoved preservation of rat hindlimbs with the University of Wisconsin solution and butanedione monoxime.Plast Reconstr Surg,1997;100(9):957
[9]Yokoyama K,Kimura M,Itoman M.rat whole-limb viability after cold immersion using University of Wisconsin and Euro-Collins solutions.Transplantation,1996;62(7):884
[10]Gordon L,Levinsoha DG,Boronsky CD,et al.Improved preservation of skeletal muscle in amputated limbs using pulsatil hypothermic perfusion with University of Wisconsin solution.J Bone Joint Surg(Am),1992;74(6):1358
[11]Kour Y,Phone MH,Chia J,et al.A preliminary report of tissue preservation
with University of Wisconsin cold storage solution in major limb replantation.Ann A cad Med Singapore,1995;24(4 suppl):37.
[12]顾玉东,李继峰,袁伟,等.离断肢体血管与肌肉保护的实验研究,中华创伤杂志,1996;12(1):25
[13]朱兆明等.玻璃化速冻储存皮肤时应用液氮降温后豚鼠皮活力的变化[J].军医进修学院创伤中心烧伤资料汇编,1990,86.
[14]Wowk B,Darwin M,Harris SB,et al.Effect of solute methoxylation on glass-forming ability and stability of vitrification solutions[J].Cryobiology,1999,39(3):215-227.
[15]Hua YC,Chang ZH and Chen Y.Cryobiological engineering[M],Shanghai Institute of Mechanical Engineering,1985,98-99.
[16]陈鸿书.低温技术在医学上的应用[M].北京:中国科学技术出版社,1991,35-36.
[17]Mazur P,Leibo SP,Chu EHY.A two factor hypothesis of freezing injery-evidence from Chinese hamster tissue culture cells[J].Exp Cell Res,1972,71:345-355.
[18]Fahy GM.Vitrification,in "Low Temperature Biotech -nology"[J].ASME,1988,113-146.
[19]李广武,郑成义,唐兵.低温生物学[M].湖南:湖南科学技术出版社,1997.69-71,101-105.
[20]Vannereau H,Novakoviteh G,Carin M.Cryobiology of complex tissues[J].Contracept Fertil Sex,1998,26:573-577.
[21]Baudot A,Cacela C,Duarte ML,et al.Thermal study of simple amino-alcohol solutions[J].Cryobiology,2002,44:150-160.
[22]胡成栋.玻璃化深低温保存生物组织与器官[J].空军总医院学报,2004,20(4):219-222.
[23]Zhang XS,Zhao L,Hua TC.A Study on the cryopreservation of common carp cyprinvs carpioembryo[J].Cryo-Letteres,1989,10;271-278.
[24]Hua TC,Thermal control of cryopreservation of biomaterial[J],in:Cryogenics and refrigeration-proceedings of international conference.Intern Acadedic Publishers, 1989,280-285.
[25]Mackenzie AP.Death of frozen yeast in the course of slow warming[J].Cryobiology,1973,10:157-170.
[26]Lovelock JE.The haemolysis of human red blood cells by freezing and thawing[J].Biophysics Acta,1953,10:414-426.
[27]Hua TC,Chang ZH,Chen Y.Cryobiological engineering[M].Shanghai Institute of Mechanical Engineering,1985.
[28]Diller KR,Cravalho EG.A cryomicrocope for the study of freezing and thawing processes in biological cells[J],Cryobilogy,1971,7:191-199.
[29]Bianchi MVA,et al.Thermal analysis of a cryomicoscope:Effect of heat spreading and contact resistance[J].Intema -tional Journal of Heat and Mass Transfer,1998,41(23):3873.
[30]Wildt DE.Genome resource banking for wildlife research,management,and conservation[J].ILAR,2000,41:228-234.
[31]Jackson TH,Ungan A,Critser JK,et al.Novel microwave technology for cryopreservation of biomaterials by suppression of apparent ice formation[J].Cryobiology,1997,34:363-372.
[32]胡军祥,应华波,周国英,等.微波复温对玻璃化冻存大鼠胚脑细胞活性的影响[J].浙江大学报,2005,31(2):190-194.
[33]骆晓东,何立群,高大勇.低温保存后生物组织微波复温过程的数值分析[J].北京生物医学工程,2006,25(1):20-25.
[34]Karlsson JO,Toner M.Long-term storage of tissues by cryopreservation:critical issues[J].Biomaterials,1996,17:243-256.
[35]Hagedom M,Hsu EW,Pilatus U,et al.Magnetic resonance microscopy and spectroscopy reveal kinetics of cryoprotectant permeation in a multicompartmental biological system[J].Proc Natl Acad Sci U S A.1996,93:7454-7459.
[36]Zhang Lu,Zhang Lin,Jiang Yong,et al.Role of p38 protein kinase in heat-induced Raw cells apoptosis.Chin Med J 2000;113(4):354-357
[37]Teodorczyk-Injeyan JA,Chembrzynsk NM,Lalani S,et al.Immune deficiency following thermal trauma is associated with apoptosis cell death.J Clin Immunol 1995; 15:318-328
[38]Chitnis D, Dickerson C, Munster AM, et al. Inhibition of apoptosis in polymorphonuclear neutrophils from burn patients. J of Leukoc Biol 1996; 59: 835 -839
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