高温联合辐射对胚胎神经发育毒性及脑热应激蛋白的影响
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摘要
背景 胚胎的中枢神经系统(特别是大脑)的发育对外界刺激极为敏感,受到外界不良环境的作用最易出现形态和行为畸形。脑蛋白质含量和HSPs的变化能准确反映脑分化和损伤程度,目前高温联合辐射对胚胎中枢神经系统发育,特别是脑HSPs及蛋白质合成的影响资料甚少。目的 探讨高温联合辐射致胚胎神经发育毒性与早期胚胎脑蛋白质和HSPs表达的关系，从蛋白质水平对高温联合环境毒物致畸机理进行研究，在实践上为人类的优生以及神经系统发育异常的预防提供了可靠思路。方法 孕鼠在妊娠第8、9天分别接受60Co (-射线全身照射各一次，两次照射剂量为1.0 GY,于孕第10天将孕鼠置各温度温箱中使其肛温保持37(0.5℃、41(0.5℃、42(0.5℃，并持续2、3、4、5分钟，孕鼠移出温箱2小时后，每组随机取5只，脱颈法处死，利用Western dot blot 和lowry方法定量分析胚胎脑HSP70和蛋白质含量；孕18天时，每组随机取10只，脱颈处死，观察胚胎神经系统畸形；其余10只，自然分娩，观察仔鼠体格发育、生理发育、神经反射、活动能力及学习记忆等神经行为指标。结果 高温联合辐射对胚胎神经毒性明显增加，并与高温强度及作用时间呈正相关；孕8天鼠接受高温联合辐射处理，其仔鼠的生理发育、早期反射、感觉功能、活动能力、学习记忆显著滞后于对照组（P<0.01），持续较长时间高温（>分钟）与辐射具有复合作用，加强了对胚胎神经系统发育的不良影响；高温联合辐射作用于胚胎早期对胎脑蛋白质降低有复合作用（P<0.01），并可诱导脑HSP70合成，但与单独高温或单独辐射比较HSP70的合成水平没有显著变化。结论 高温与辐射联合致神经发育毒性与胚胎分子水平上脑蛋白质合成及热应激蛋白的诱导有一定的相关性，胎脑蛋白质含量的下降和诱导HSPs的合成的变化规律与高温温度、高温持续时间、辐射以及二因素联合所致的神经发育毒性、神经行为毒性结果基本一致。
Background The central nervous system development ( brain above all ) is sensitive especially to outside stimulation. The abnormality of conformation and behavior will appear very easily when suffering outside bad environment. The content of brain proteins and change of HSPs can reflect exactly the degree of brain differentiation and damage. At present, the data is short about the effect of hyperthermia united with radiation on the central nervous system development, especially brain HSPs and other proteins. Objective To study the relation between the toxicity of embryonic nerve development made by hyperthermia united with radiation and expression of brain proteins of earlier period embryo and HSPs. To reveal the mechanism of the abnormality caused by hyperthermia united with environment poison from protein level. And to offer a reliable idea for human prepotency and prevention of the abnormality of nerve system development. Methods the rats who are pregnant for 8 or 9 days received respectively total body irradiation for one time by 60Co (-ray, and the dosage of irradiation for two times is 1.0 Gy. When the rats are pregnant for 10 days the temperature of rat anus is respectively kept at 37(0.5℃、 41(0.5℃、 42(0.5℃, in warming boxes. and lasted for 2、 3、 4、 5 minutes. After the pregnant rats are left warming boxes for two hours, 5 rats selected randomly from each team are executed by cervical vertebra luxation. The content of brain proteins and HSP70 of embryo is analyzed with western dot blot and Lowry method. When the rats are pregnant for 18 days, 10 rats selected randomly from each team are executed by cervical vertebra luxation, and to observe the abnormality of embryonic nerve system. And let another 10 of each team to born young rats naturally, and observe their body development, physio-development, nerve reflect, study and memory and other nerve behavior. Results The toxicity is increased markedly when hyperthermia combined with irradiation, and hyperthermia intensity and action time shows positive relativity. The rats who are pregnant for 8 days were treated by hyperthermia with irradiation, and physio-development, forepart reflect, sensory function, activity ability, study and memory of their son is more lower notably than control team (P<0.01). If hyperthermia lasted longer (>minutes), hyperthermia and irradiation have complex action, and the harmful effects on nerve system development of embryo are strengthened. At embryonic earlier period, hyperthermia with irradiation can depress the content of brain proteins (P<0.01), and induce synthesis of HSP70, and there is no significant difference between the combined effect group and single high temperature group and single irradiation group. Conclusion There is certain relativity to synthesis of brain proteins and
induction of HSP70 when hyperthermia combined with irradiation to produce toxicity to nerve development at molecular level. The change rule of the content drop of embryo brain proteins and the synthesis induction of HSP70 is basically consistent with hyperthermia temperature, lasting time of hyperthermia, irradiation and hyperthermia with irradiation to nerve development and nerve behavior.
Background The central nervous system development ( brain above all ) is sensitive especially to outside stimulation. The abnormality of conformation and behavior will appear very easily when suffering outside bad environment. The content of brain proteins and change of HSPs can reflect exactly the degree of brain differentiation and damage. At present, the data is short about the effect of hyperthermia united with radiation on the central nervous system development, especially brain HSPs and other proteins. Objective To study the relation between the toxicity of embryonic nerve development made by hyperthermia united with radiation and expression of brain proteins of earlier period embryo and HSPs. To reveal the mechanism of the abnormality caused by hyperthermia united with environment poison from protein level. And to offer a reliable idea for human prepotency and prevention of the abnormality of nerve system development. Methods the rats who are pregnant for 8 or 9 days received respectively total body irradiation for one time by 60Co (-ray, and the dosage of irradiation for two times is 1.0 Gy. When the rats are pregnant for 10 days the temperature of rat anus is respectively kept at 37(0.5℃、 41(0.5℃、 42(0.5℃, in warming boxes. and lasted for 2、 3、 4、 5 minutes. After the pregnant rats are left warming boxes for two hours, 5 rats selected randomly from each team are executed by cervical vertebra luxation. The content of brain proteins and HSP70 of embryo is analyzed with western dot blot and Lowry method. When the rats are pregnant for 18 days, 10 rats selected randomly from each team are executed by cervical vertebra luxation, and to observe the abnormality of embryonic nerve system. And let another 10 of each team to born young rats naturally, and observe their body development, physio-development, nerve reflect, study and memory and other nerve behavior. Results The toxicity is increased markedly when hyperthermia combined with irradiation, and hyperthermia intensity and action time shows positive relativity. The rats who are pregnant for 8 days were treated by hyperthermia with irradiation, and physio-development, forepart reflect, sensory function, activity ability, study and memory of their son is more lower notably than control team (P<0.01). If hyperthermia lasted longer (>minutes), hyperthermia and irradiation have complex action, and the harmful effects on nerve system development of embryo are strengthened. At embryonic earlier period, hyperthermia with irradiation can depress the content of brain proteins (P<0.01), and induce synthesis of HSP70, and there is no significant difference between the combined effect group and single high temperature group and single irradiation group. Conclusion There is certain relativity to synthesis of brain proteins and
induction of HSP70 when hyperthermia combined with irradiation to produce toxicity to nerve development at molecular level. The change rule of the content drop of embryo brain proteins and the synthesis induction of HSP70 is basically consistent with hyperthermia temperature, lasting time of hyperthermia, irradiation and hyperthermia with irradiation to nerve development and nerve behavior.
引文
Tilson HA．Mitchlo Ceurobehavioral techniques to access the effects of Chemical on the nervous system.Ann Revpharmmacel Toxical,1984;24
梁友信，陈自中．行为毒理学方法的回顾与展望，中华劳动卫生职业病杂志，1999；17（2）
庄志雄．分子毒理学研究的现状与展望．中华劳动卫生职业病杂志，2000；18（1）
Dimbeeg Y,Tottmar opberg A,et al.Effects of Low dose X—irradiation on mouse—brain aggregation cultures.Int J Radiat Boit,1992；16(3)
Cheng Y, Liu Y F,Liang J. Protective effect of Zinc: a potent heat shock protein inducer in cold preservation of rat liver. Hepatobiliary Pancreat Dis Int.2002 May;1(2)258-61
芦春林，Kimme CAKmel GL,等.高温致胚胎发育毒性机理的研究.北京医科大学学报，1993；25(6)
Sun XQ, Li JS, Wu XY. The express of heat shock protein 70 in rat brain after GZ exposure. J Gravit physio， 2002 Jul; 9(1):23-24
Carolyn MKB,Hales.Beat—Shock induced tolerance to the embryotoxic effedts of hyperthermia and cadmium in mouse embryos in vitro.Teratology,1991；43
Kimmel CA,Kimmel GL,Lu CL,et al． Stress protein systhesis as a potential biomaker for heat—induced development toxicity.Teratology,1991；43
Nakashima K,Kawamata A,Fujiki I,et al.The individual and combined effects of cigarette smoke and hyperthermia on early somite mouse embryos in culture.Teratology,1991；44
Fisher BR,Kimmel GL,Kimmel CA.et al.The association of heat—induced alteration in protein systhesis with somatic defects in rat embryos.Teratology,1991；43
Golub,M A.Maremal toxicity and the identifieation of inorganic arsenic as a development toxicant.Reprot Toxical， 1994；8(4)
Mirks,P E.Hyperthermia and the indution of neural tube defects in mice.Teratology,1994；49:135—142
Geman,J. Embryos stress hypothesis of teratogensis.Am J.Meh,1984；76:293
Cheng Y, Liu Y F,Liang J. Protective effect of Zinc: a potent heat shock protein inducer in cold preservation of rat liver. Hepatobiliary Pancreat Dis Int.2002 May;1(2)258-61
芦春林，Kimme CAKmel GL,等.高温致胚胎发育毒性机理的研究.北京医科大学学报，1993；25(6)
Sun XQ, Li JS, Wu XY. The express of heat shock protein 70 in rat brain after GZ exposure. J Gravit physio， 2002 Jul; 9(1):23-24
Carolyn MKB,Hales.Beat—Shock induced tolerance to the embryotoxic effedts of hyperthermia and cadmium in mouse embryos in vitro.Teratology,1991；43
Kimmel CA,Kimmel GL,Lu CL,et al． Stress protein systhesis as a potential biomaker for heat—induced development toxic
Fisher BR,Brown NT,Heredia,D J.et al.Occipotal encepalocele and early gestational hyperthemia. Teratology,1995；52:90
高卫民，周湘艳.出生前氚水照射对仔代大鼠生长发育及神经行为的影响.中华放射医学与防护杂志，1998；18（6）
谭涣然，杨磊，沈丽.射线照射对仔代大鼠生长发育及神经行为的影响.中华放射医学与防护杂志，1998；18（6）
蒋武成,袁厚积.生物物质常用化学分析法.北京.科学出版社.1991；93～97
何生,潘秋萍,邬堂春等． Western 斑点印迹法检测热应激蛋白．中华劳动卫生职业病杂志,1996；14(3)
Novey L, Scharrf KD. Heat stress proteins and transcription factors cell. Mol. Lifesci, 1997；53:80
Chin LK, David TV, Gary BT,et al．Compartive genotoxicity testing of mainstream whole smoke from cigarette which burn or heat
tobacco.Mutation Res,1990；8:89
Chen H, Liu Z, Zhou Z, Jiang M, Qian L, WuS.The regulatory effect of memantine on expression and synthesis of heat shock protein 70 gene in neonatal rat models with cerebral hypoxic ischemia. Chin Med J.2003 Apr;116(1):558-64
Kanpinga HH, Brinsting JF, Stege GJ,et al.Thermal protein denaturation and protein aggregation in cells made thermotolerant by various chemicals: role of heat shock proteins Exp Cell Res,1995;219
Pannen BH, Maeda K,Ayuse T, et al Hepatic heat shock and acte phase gene expression are induced simultaneously after cellotomy in the anesthetized pig. Anesthesiology. 1995；83
Mirkes PE, Hypothermia and the induction of neural tube defects in mices.Teratology,1994；49
Jensh P,Bent BL.studies concerning the effects of low level prenatal X—irradiation on postnatal growth and adult behavior in the wistar at. Int Tadiat Biol,1986；5(6):1069
尹武英,李衡华,彭力.咖啡因对小鼠的行为致畸学研究.卫生研究,1998；27(2):115
王宏，陈德清，高长文，等.低剂量60Co(-射线宫内持续照射对小鼠生后发育及反射行为的影响.中华放射医学与防护杂志，1991；11（5）323
cellular Ca, intracellular PH, and HSP70 Ann Thorac Cardiovasc Surg.2003 Oct;9(5):301-6
李勇. 热休克蛋白在应激环境中对胚胎生长发育的影响[J]. 国外医学.卫生分册，1997；24（1）：20
36． 张玲，王颖群，余绍祖．脑缺血诱导的基因表达．卒中与神经疾病，1999；6(3)：191
37. Shawlot W , et al. Nature.1995;374:425—430
梁友信，陈自中．行为毒理学方法的回顾与展望，中华劳动卫生职业病杂志，1999；17（2）
庄志雄．分子毒理学研究的现状与展望．中华劳动卫生职业病杂志，2000；18（1）
Dimbeeg Y,Tottmar opberg A,et al.Effects of Low dose X—irradiation on mouse—brain aggregation cultures.Int J Radiat Boit,1992；16(3)
Cheng Y, Liu Y F,Liang J. Protective effect of Zinc: a potent heat shock protein inducer in cold preservation of rat liver. Hepatobiliary Pancreat Dis Int.2002 May;1(2)258-61
芦春林，Kimme CAKmel GL,等.高温致胚胎发育毒性机理的研究.北京医科大学学报，1993；25(6)
Sun XQ, Li JS, Wu XY. The express of heat shock protein 70 in rat brain after GZ exposure. J Gravit physio， 2002 Jul; 9(1):23-24
Carolyn MKB,Hales.Beat—Shock induced tolerance to the embryotoxic effedts of hyperthermia and cadmium in mouse embryos in vitro.Teratology,1991；43
Kimmel CA,Kimmel GL,Lu CL,et al． Stress protein systhesis as a potential biomaker for heat—induced development toxicity.Teratology,1991；43
Nakashima K,Kawamata A,Fujiki I,et al.The individual and combined effects of cigarette smoke and hyperthermia on early somite mouse embryos in culture.Teratology,1991；44
Fisher BR,Kimmel GL,Kimmel CA.et al.The association of heat—induced alteration in protein systhesis with somatic defects in rat embryos.Teratology,1991；43
Golub,M A.Maremal toxicity and the identifieation of inorganic arsenic as a development toxicant.Reprot Toxical， 1994；8(4)
Mirks,P E.Hyperthermia and the indution of neural tube defects in mice.Teratology,1994；49:135—142
Geman,J. Embryos stress hypothesis of teratogensis.Am J.Meh,1984；76:293
Cheng Y, Liu Y F,Liang J. Protective effect of Zinc: a potent heat shock protein inducer in cold preservation of rat liver. Hepatobiliary Pancreat Dis Int.2002 May;1(2)258-61
芦春林，Kimme CAKmel GL,等.高温致胚胎发育毒性机理的研究.北京医科大学学报，1993；25(6)
Sun XQ, Li JS, Wu XY. The express of heat shock protein 70 in rat brain after GZ exposure. J Gravit physio， 2002 Jul; 9(1):23-24
Carolyn MKB,Hales.Beat—Shock induced tolerance to the embryotoxic effedts of hyperthermia and cadmium in mouse embryos in vitro.Teratology,1991；43
Kimmel CA,Kimmel GL,Lu CL,et al． Stress protein systhesis as a potential biomaker for heat—induced development toxic
Fisher BR,Brown NT,Heredia,D J.et al.Occipotal encepalocele and early gestational hyperthemia. Teratology,1995；52:90
高卫民，周湘艳.出生前氚水照射对仔代大鼠生长发育及神经行为的影响.中华放射医学与防护杂志，1998；18（6）
谭涣然，杨磊，沈丽.射线照射对仔代大鼠生长发育及神经行为的影响.中华放射医学与防护杂志，1998；18（6）
蒋武成,袁厚积.生物物质常用化学分析法.北京.科学出版社.1991；93～97
何生,潘秋萍,邬堂春等． Western 斑点印迹法检测热应激蛋白．中华劳动卫生职业病杂志,1996；14(3)
Novey L, Scharrf KD. Heat stress proteins and transcription factors cell. Mol. Lifesci, 1997；53:80
Chin LK, David TV, Gary BT,et al．Compartive genotoxicity testing of mainstream whole smoke from cigarette which burn or heat
tobacco.Mutation Res,1990；8:89
Chen H, Liu Z, Zhou Z, Jiang M, Qian L, WuS.The regulatory effect of memantine on expression and synthesis of heat shock protein 70 gene in neonatal rat models with cerebral hypoxic ischemia. Chin Med J.2003 Apr;116(1):558-64
Kanpinga HH, Brinsting JF, Stege GJ,et al.Thermal protein denaturation and protein aggregation in cells made thermotolerant by various chemicals: role of heat shock proteins Exp Cell Res,1995;219
Pannen BH, Maeda K,Ayuse T, et al Hepatic heat shock and acte phase gene expression are induced simultaneously after cellotomy in the anesthetized pig. Anesthesiology. 1995；83
Mirkes PE, Hypothermia and the induction of neural tube defects in mices.Teratology,1994；49
Jensh P,Bent BL.studies concerning the effects of low level prenatal X—irradiation on postnatal growth and adult behavior in the wistar at. Int Tadiat Biol,1986；5(6):1069
尹武英,李衡华,彭力.咖啡因对小鼠的行为致畸学研究.卫生研究,1998；27(2):115
王宏，陈德清，高长文，等.低剂量60Co(-射线宫内持续照射对小鼠生后发育及反射行为的影响.中华放射医学与防护杂志，1991；11（5）323
cellular Ca, intracellular PH, and HSP70 Ann Thorac Cardiovasc Surg.2003 Oct;9(5):301-6
李勇. 热休克蛋白在应激环境中对胚胎生长发育的影响[J]. 国外医学.卫生分册，1997；24（1）：20
36． 张玲，王颖群，余绍祖．脑缺血诱导的基因表达．卒中与神经疾病，1999；6(3)：191
37. Shawlot W , et al. Nature.1995;374:425—430