脑创伤后儿茶酚胺递质对神经细胞损害的线粒体调控机制的实验研究
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摘要
创伤性脑损伤后的脑组织损害是由于直接机械性损伤和继发性损害所引起。继发性脑损害是一系列细胞、分子事件作用的结果。当前，颅脑损伤的研究主要是围绕颅脑损伤后继发性损伤机制展开的。大量资料表明创伤性脑损伤可导致细胞的死亡，而在生理和绝大多数病理条件下，细胞死亡都呈现为凋亡的典型特征。目前我们已认识到脑创伤可引起大量的神经细胞凋亡，因此研究引起细胞凋亡的机制可能为脑创伤的救治提供新的前景。
近年的研究表明有多种脑内的生化物质涉及脑创伤后继发损害的病理过程。而作为内源性的儿茶酚胺类中枢性神经递质是神经递质中一个大类型，脑组织中儿茶酚胺递质的改变是反映颅内神经代谢的敏感指标。儿茶酚胺递质包括多巴胺、去甲肾上腺素、肾上腺素，它们不但参与脑的正常神经生理生化功能，还与脑的继发性损害有密切关系。最近几年的研究表明，在中枢性退行性病变及颅脑损伤后的继发性损害中，儿茶酚胺类神经递质皆发生紊乱。我们的体外实验研究显示：过量的儿茶酚胺类神经递质对神经细胞具有细胞毒性作用，可直接引起细胞死亡，且具有时间和剂量效应。但在在体内是否如此仍未确定。
最近有研究表明，线粒体除了合成ATP外还有多种重要生理功能，包括产生超氧阴离子等活性氧，调节氧化还原电势和细胞的氧化还原信号转导，调控细胞凋亡和基因表达等，此外，还发现它在生物生长，发育，衰老，疾病，死亡等方面都有重要意义，近来研究细胞的生化途径时发现，在生理条件下线粒体生物能力学因素具有导致细胞死亡和细胞凋亡的能力。而已往研究创伤性脑损伤导致的神经细胞凋亡基本都集中于细胞核的形态与核的信号改变上，对线粒体在脑创伤后神经细胞凋亡中所起的作用很少涉及。
因此，本研究首先建立脑损伤模型，研究线粒体在脑创伤后儿茶酚
胺类递质诱导神经细胞凋亡中的作用及机制。
第一部分脑创伤后儿茶酚胺类递质的变化及其对神经细胞的影响
目的:
明确脑创伤后儿茶酚胺类递质的变化规律及其对神经细胞的影响。
方法:
1、sD大鼠，用自由落体撞击法制作左顶叶脑挫裂伤模型，随机分
为假手术组和损伤后lh、6h、24h、48h、72h及168h组。
2、Tunel法及Hochest3334荧光染色检测各组损伤侧皮质、海马的
神经细胞死亡形式。
3、免疫荧光组织化学方法检测酪氨酸经化酶(TH)在各组损伤侧
皮质、海马，脑干的表达，并定量分析。
4、2‘7‘一二氯氢化荧光乙酞乙酸盐荧光素标记活体脑片中神经细
胞的活性氧物质的含量，并定量分析。
5、2‘7‘一二氯氢化荧光乙酞乙酸盐与碘化毗咤两种荧光素双标记
活体脑片，观察活性氧物质对神经细胞的影响。
6、用SPSS10.0统计软件，非配对两样本均数比较的t检验或多样
本均数比较的方差分析对实验数据进行统计学处理，p<0.05表示有显著
性差异。
结果:
1、发现脑外伤组6h起有较多凋亡的阳性细胞，并逐渐增多，24h
后凋亡细胞无明显继续增加，假手术组仅见少量的阳性细胞。
2、脑创伤后的早期(lh)，在损伤侧皮层、海马TH表达增加，6h
时达到高峰，之后开始下降，72h时已有较明显的下降，而在脑干TH表
达无明显的改变。免疫荧光染色后，利用共聚焦显微镜测荧光强度创伤
组/假手术组比率半定量分析示:脑外伤后皮质一小时起TH升高，为假
手术组的2.23倍(p(0.01)，6h达到高峰，是假手术组的3.19倍(p(0.01)，
之后开始下降，至72h时是对照组及假手术组的1.35倍(p<0.05)，168h
时低于假手术组，为0.89倍。海马lh为对照组及假手术组的2.13倍
(p<0.01)，6h达到高峰，是假手术组的2二89倍(p<0 .01)，之后开始下
降，至72h时是假手术组的1.21倍(p<0.05)，168h时低于假手术组，
为0 .93倍。
3、脑损伤后2‘7‘一二氯氢化荧光乙酞乙酸盐标记，皮层与海马的
荧光强度增强，皮层1h组为假手术组的3.13倍(p<。.01)，24h达到高
峰，是假手术组的4.87倍(p<0 .01)，之后一直维持在这一水平，至72h
时稍有下降，是假手术组的2.26倍(p<0.01)。海马的荧光强度较皮层
稍低。比较TH变化与过氧化物水平的关系，发现:过氧化物与TH在6h
内均随时间而增高，24h后TH明显降低，而过氧化物24h降低不明显。
4、荧光探针2‘7‘一二氯氢化荧光乙酞乙酸盐与PI双标记发现，在
2‘7‘一二氯氢化荧光乙酸乙酸盐其荧光强度增强区域都有PI荧光着色。
结论:
脑创伤后急性期儿茶酚胺类递质过度分泌，并且递质可以经上行纤
维的传导分布到皮层和海马，在创伤后缺血缺氧情况下儿茶酚胺递质可
自氧化及被氧化酶分解。产生大量的活性氧物质，引起神经细胞凋亡，
儿茶酚胺类递质过度分泌早于细胞出现凋亡形态。创伤急性期过后，由
于负反馈作用，儿茶酚胺递质分泌受抑制，低于正常水平。
第二部分儿茶酚胺递质诱导神经细胞凋亡的线粒体调控机制
目的:
探讨线粒体在脑创伤后儿茶酚胺类递质诱导神经细胞凋亡中的作
用及机制。
方法:
1、动物模型的制作及实验分组同实验第一部分。
2、梯度离心方法分离及提取游离的线粒体和细胞质。
3、
The brain tissue damage resulted from both direet meehanical injury and
seeondary brain imPairment.They were related and cooPerated in a series of
events of eells and moleeules.The researeh of brain damage reeently
cmPhasis on evolution of secondary brain imPairment after trau们natie brain
injury(TBI).Inereasing evidenees showed that TBI might induee eell death.
However， under eircumstanee of normal Physiologieal and in many
Pathologieal Proeesses，eell death emerged tyPieal eharaeteristies of apoPtosis.
TBI indueing apoPtosis of a large number of neurons had been肋own.
Therefore further studies on the meehhasms of neuron apoPtosis might giVe
a new insighton the treatnlent ofTBI.
A series of studies Provided convineing evidence that Pathologieal
Proeesses of secondary brain ilnPairment involved in a eascade of
bioehemieal ehanges of TBI.Serve as endogenous neurotransmitter in the
nervous system，the ehanges Catecholamines(CAs)reflected a sensitive
marker of metabolism.CAs，sueh as DA，NE andE，Played an imPortant role
in normal neuroPhysiobiologieal Proeesses of eentral nervous system，as well
as involved in Pathologieal Proeesses of seeondary brain imPairment.Reeent
studies de们以onstrated that CAs was disturbed following seeondal，y
illlPairment of neurodegenerative disorders and TBI.Our in vitro indicated
that overdose CAs were toxieally to neurons and might induee aPoPtosis of
them direetly，几吐hermore，there time eourse and dose一response·However，
whether this sitL讯tione劝sted in vivo remained unelear.
Reeent studies rePresented that mitoeholidria(Mt) eatried out many
imPortant fullction exeePt ATP synthesis:generating reaetive oxygen sPeeies
(ROS)，regulating oxidoreduction Potential alld eellular oxidoreduction signal
transduetion，as well as the apoPtosis of neurons and the exPression of genes.
In addition，Mt also Played an lmPortant role in living thing，5 grov改h，
develoPment，aging，disease and death.Reeent bioehemieal studies revealed
another asPeet of Mt funetion:indueing aPoPtosis and eell death by triggering
sPecifie bioehemical reaetion.The Previous researches on TBI indueing
neuron apoPtosis focused on stUdying the morphologieal and signalling
Pathways ehanges of nueleus.There were relatiVely few rePorts on Mt
Playing role in neuronal eell apoPtosis after TBI，
Therefore，these studles iultially established damage model，and
investigated the meehanisms and the exPanding role of Mt on CAs inducing
neuronal eell apoPtosis after TBI.
Part 1 Studies on the ehanges of CAs and its effeet involved in neuronal
eells after TBI
PurPose:To identify the eharaeteristies of the ehanges of CAs and its effeets
involved in neu『onal eells
Methods:
1 .Healthy male SPrague一Dawley(SD)rats were randomized into the
sham一injury grouP andl，6，24，48， 72，168h grouP after traumatie injured.
The eontUsion model on brain was made use ffee一falling body.
2 .The dead form of neuronal eells in injured eortex and hiPPocamPus
were deteeted using Tunel and Hoehest 33342 fluoreseenee staining.
3 .The exPression of TH in injured eortex，hiPPoeamPus and brain stem
were detected and semiquatitative measurelnents by fluoreseenee
histochemistry technique.
4 .The eontain of ROS of neuronal eells in living brain sliees were
labeled withZ’7’一diehlorodi勿drofluroresein diaeetate and semiquatitative
measllre幻 nellts.
l0
5 .ROS affecting neuronal eells were obs柳ed living brain sliees
double一labeled withZ’7’一diehlorodihydrofluroresein diacetate and
ProgPidium iodide.
Results:
1)APoPtotie eell emerged in injured eortex and hiPPoeamPus at lh，then
increased gradually，reaehed the Peak at 24h.There、vere a few neuronal eells
with weak and dll’f1卫se brown TIJNEL Positive staining in sham一injury grouP.
2)At early Period(lh) after TBI，the Positive exPression of TH in
injured eortex and hiPPoeamPus signifieantly increased eomPared with the
sham一injury grouP，and reaeh the Peak at 6h，then began to deereased，
apParently deerea
近年的研究表明有多种脑内的生化物质涉及脑创伤后继发损害的病理过程。而作为内源性的儿茶酚胺类中枢性神经递质是神经递质中一个大类型，脑组织中儿茶酚胺递质的改变是反映颅内神经代谢的敏感指标。儿茶酚胺递质包括多巴胺、去甲肾上腺素、肾上腺素，它们不但参与脑的正常神经生理生化功能，还与脑的继发性损害有密切关系。最近几年的研究表明，在中枢性退行性病变及颅脑损伤后的继发性损害中，儿茶酚胺类神经递质皆发生紊乱。我们的体外实验研究显示：过量的儿茶酚胺类神经递质对神经细胞具有细胞毒性作用，可直接引起细胞死亡，且具有时间和剂量效应。但在在体内是否如此仍未确定。
最近有研究表明，线粒体除了合成ATP外还有多种重要生理功能，包括产生超氧阴离子等活性氧，调节氧化还原电势和细胞的氧化还原信号转导，调控细胞凋亡和基因表达等，此外，还发现它在生物生长，发育，衰老，疾病，死亡等方面都有重要意义，近来研究细胞的生化途径时发现，在生理条件下线粒体生物能力学因素具有导致细胞死亡和细胞凋亡的能力。而已往研究创伤性脑损伤导致的神经细胞凋亡基本都集中于细胞核的形态与核的信号改变上，对线粒体在脑创伤后神经细胞凋亡中所起的作用很少涉及。
因此，本研究首先建立脑损伤模型，研究线粒体在脑创伤后儿茶酚
胺类递质诱导神经细胞凋亡中的作用及机制。
第一部分脑创伤后儿茶酚胺类递质的变化及其对神经细胞的影响
目的:
明确脑创伤后儿茶酚胺类递质的变化规律及其对神经细胞的影响。
方法:
1、sD大鼠，用自由落体撞击法制作左顶叶脑挫裂伤模型，随机分
为假手术组和损伤后lh、6h、24h、48h、72h及168h组。
2、Tunel法及Hochest3334荧光染色检测各组损伤侧皮质、海马的
神经细胞死亡形式。
3、免疫荧光组织化学方法检测酪氨酸经化酶(TH)在各组损伤侧
皮质、海马，脑干的表达，并定量分析。
4、2‘7‘一二氯氢化荧光乙酞乙酸盐荧光素标记活体脑片中神经细
胞的活性氧物质的含量，并定量分析。
5、2‘7‘一二氯氢化荧光乙酞乙酸盐与碘化毗咤两种荧光素双标记
活体脑片，观察活性氧物质对神经细胞的影响。
6、用SPSS10.0统计软件，非配对两样本均数比较的t检验或多样
本均数比较的方差分析对实验数据进行统计学处理，p<0.05表示有显著
性差异。
结果:
1、发现脑外伤组6h起有较多凋亡的阳性细胞，并逐渐增多，24h
后凋亡细胞无明显继续增加，假手术组仅见少量的阳性细胞。
2、脑创伤后的早期(lh)，在损伤侧皮层、海马TH表达增加，6h
时达到高峰，之后开始下降，72h时已有较明显的下降，而在脑干TH表
达无明显的改变。免疫荧光染色后，利用共聚焦显微镜测荧光强度创伤
组/假手术组比率半定量分析示:脑外伤后皮质一小时起TH升高，为假
手术组的2.23倍(p(0.01)，6h达到高峰，是假手术组的3.19倍(p(0.01)，
之后开始下降，至72h时是对照组及假手术组的1.35倍(p<0.05)，168h
时低于假手术组，为0.89倍。海马lh为对照组及假手术组的2.13倍
(p<0.01)，6h达到高峰，是假手术组的2二89倍(p<0 .01)，之后开始下
降，至72h时是假手术组的1.21倍(p<0.05)，168h时低于假手术组，
为0 .93倍。
3、脑损伤后2‘7‘一二氯氢化荧光乙酞乙酸盐标记，皮层与海马的
荧光强度增强，皮层1h组为假手术组的3.13倍(p<。.01)，24h达到高
峰，是假手术组的4.87倍(p<0 .01)，之后一直维持在这一水平，至72h
时稍有下降，是假手术组的2.26倍(p<0.01)。海马的荧光强度较皮层
稍低。比较TH变化与过氧化物水平的关系，发现:过氧化物与TH在6h
内均随时间而增高，24h后TH明显降低，而过氧化物24h降低不明显。
4、荧光探针2‘7‘一二氯氢化荧光乙酞乙酸盐与PI双标记发现，在
2‘7‘一二氯氢化荧光乙酸乙酸盐其荧光强度增强区域都有PI荧光着色。
结论:
脑创伤后急性期儿茶酚胺类递质过度分泌，并且递质可以经上行纤
维的传导分布到皮层和海马，在创伤后缺血缺氧情况下儿茶酚胺递质可
自氧化及被氧化酶分解。产生大量的活性氧物质，引起神经细胞凋亡，
儿茶酚胺类递质过度分泌早于细胞出现凋亡形态。创伤急性期过后，由
于负反馈作用，儿茶酚胺递质分泌受抑制，低于正常水平。
第二部分儿茶酚胺递质诱导神经细胞凋亡的线粒体调控机制
目的:
探讨线粒体在脑创伤后儿茶酚胺类递质诱导神经细胞凋亡中的作
用及机制。
方法:
1、动物模型的制作及实验分组同实验第一部分。
2、梯度离心方法分离及提取游离的线粒体和细胞质。
3、
The brain tissue damage resulted from both direet meehanical injury and
seeondary brain imPairment.They were related and cooPerated in a series of
events of eells and moleeules.The researeh of brain damage reeently
cmPhasis on evolution of secondary brain imPairment after trau们natie brain
injury(TBI).Inereasing evidenees showed that TBI might induee eell death.
However， under eircumstanee of normal Physiologieal and in many
Pathologieal Proeesses，eell death emerged tyPieal eharaeteristies of apoPtosis.
TBI indueing apoPtosis of a large number of neurons had been肋own.
Therefore further studies on the meehhasms of neuron apoPtosis might giVe
a new insighton the treatnlent ofTBI.
A series of studies Provided convineing evidence that Pathologieal
Proeesses of secondary brain ilnPairment involved in a eascade of
bioehemieal ehanges of TBI.Serve as endogenous neurotransmitter in the
nervous system，the ehanges Catecholamines(CAs)reflected a sensitive
marker of metabolism.CAs，sueh as DA，NE andE，Played an imPortant role
in normal neuroPhysiobiologieal Proeesses of eentral nervous system，as well
as involved in Pathologieal Proeesses of seeondary brain imPairment.Reeent
studies de们以onstrated that CAs was disturbed following seeondal，y
illlPairment of neurodegenerative disorders and TBI.Our in vitro indicated
that overdose CAs were toxieally to neurons and might induee aPoPtosis of
them direetly，几吐hermore，there time eourse and dose一response·However，
whether this sitL讯tione劝sted in vivo remained unelear.
Reeent studies rePresented that mitoeholidria(Mt) eatried out many
imPortant fullction exeePt ATP synthesis:generating reaetive oxygen sPeeies
(ROS)，regulating oxidoreduction Potential alld eellular oxidoreduction signal
transduetion，as well as the apoPtosis of neurons and the exPression of genes.
In addition，Mt also Played an lmPortant role in living thing，5 grov改h，
develoPment，aging，disease and death.Reeent bioehemieal studies revealed
another asPeet of Mt funetion:indueing aPoPtosis and eell death by triggering
sPecifie bioehemical reaetion.The Previous researches on TBI indueing
neuron apoPtosis focused on stUdying the morphologieal and signalling
Pathways ehanges of nueleus.There were relatiVely few rePorts on Mt
Playing role in neuronal eell apoPtosis after TBI，
Therefore，these studles iultially established damage model，and
investigated the meehanisms and the exPanding role of Mt on CAs inducing
neuronal eell apoPtosis after TBI.
Part 1 Studies on the ehanges of CAs and its effeet involved in neuronal
eells after TBI
PurPose:To identify the eharaeteristies of the ehanges of CAs and its effeets
involved in neu『onal eells
Methods:
1 .Healthy male SPrague一Dawley(SD)rats were randomized into the
sham一injury grouP andl，6，24，48， 72，168h grouP after traumatie injured.
The eontUsion model on brain was made use ffee一falling body.
2 .The dead form of neuronal eells in injured eortex and hiPPocamPus
were deteeted using Tunel and Hoehest 33342 fluoreseenee staining.
3 .The exPression of TH in injured eortex，hiPPoeamPus and brain stem
were detected and semiquatitative measurelnents by fluoreseenee
histochemistry technique.
4 .The eontain of ROS of neuronal eells in living brain sliees were
labeled withZ’7’一diehlorodi勿drofluroresein diaeetate and semiquatitative
measllre幻 nellts.
l0
5 .ROS affecting neuronal eells were obs柳ed living brain sliees
double一labeled withZ’7’一diehlorodihydrofluroresein diacetate and
ProgPidium iodide.
Results:
1)APoPtotie eell emerged in injured eortex and hiPPoeamPus at lh，then
increased gradually，reaehed the Peak at 24h.There、vere a few neuronal eells
with weak and dll’f1卫se brown TIJNEL Positive staining in sham一injury grouP.
2)At early Period(lh) after TBI，the Positive exPression of TH in
injured eortex and hiPPoeamPus signifieantly increased eomPared with the
sham一injury grouP，and reaeh the Peak at 6h，then began to deereased，
apParently deerea
引文
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2、章翔。加强对弥漫性脑损伤的基础与应用研究。中华创伤杂志，2003，19，135—139。
3、Vink R, Nimmo AJ, Cemak Ⅰ. An overview of new and novel phamaco therapies for use in traumatic brain injury. Clin Exp Phzrmacol Physiol, 2001,28,919-921.
4、瞿文军，徐如祥。单胺类递质对神经细胞的影响。解放军医学，2003，28，76—78。
5、瞿文军，徐如祥。多巴胺对体外培养神经细胞的影响。中华老年医学杂志，2000，19，368—372。
6、Oppenheim RW. Cell death during deleveloment of the nervous system. Annu Rer. Neurosci, 1991,14,453-501.
7、Johnson EM Jr, Greenlund LJ, Akins PT, et al. Neuronal apoptosis: Current understanding of molecular mechanisms and potential role in ischemic brain injury. J Neurotrauma, 1995,12,843-852.
8、Bernardi P, Scorrano L, Colonna R, et al. Mitochondria and cell death. Eur J Biochem, 1999,264,687-701.
9、Fiskum G. Mitochondrial participation in ischemic and traumatic neural cell death. J Neurotrauma, 2000, 17, 843-855.
10、Xiaodong Wang. The expanding role of mitochondria in apoptosis. Genes Der, 2001,15,2922-2933.
11、Liu, X., Kim, C.N., Yang, J., et al. Induction ofapoptotic program in cell-free extracts: Requirement for dATP and cytochrome c. Ce11,1996, 86,147-157.
12、Jiang, X. and Wang, X. Cytochrome c promotes caspase-9
activation by inducing nucleotide binding to Apaf-1. J. Biol. Chem, 2000, 275, 31199-31203.
13、Crompton M. The mitochondrial permeability transition pore and its role in cell death. J Biochem.,1999,341,233-249.
14、Feeney DM, Boyeson MG, Linn RT. Response to cortical injury:Ⅰ. Methodogy and local effects of contusions in the rat. Brain Res, 1981,211,67-77.
15、方敏，王晓东。细胞凋亡的线粒体通路。北京大学学报(医学版)，2002，34，1—10。
16、Paakkari L, Lindsbergs P. Nitric oxide in the central nervous system. Ann Med, 1995,27,369-377.
17、王晓英，邢虹，何其华，等。利用共聚焦扫描显微镜研究大鼠脑片缺血缺氧损伤。科学通报，1999，44，
18、韩济生。神经科学原理，第二版，北京：北京医科大学出版社，1999，455—481。
19、Joseph M, Masserano, Li gong et al. Dopamine induces apoptotic cell death of a catecholaminergic cell line derived from the central nervous system. Meolecular Pharmacology 1996, 50: 130901—5.
20、韩济生，关新民，主编。医用神经生物学，湖北：武汉出版社，1995，第一版
21、Jakel RJ, Maragos WF. Neuronal cell death in Huntington's disease: a potential role for dopamine. Trends Neurosci. 2000, 23(6):239-245.
22、Levin BE, Brown KL, Pawar G, Dunn-Meynell A. Widespread and lateralization effects of acute traumatic brain injury on norepinephrine turnover in the rat brain. Brain Res. 1995, 20;674(2):307-13.
23、Clifton GL, Ziegler MG, Grossman RG. Circulating catecholamines and sympathetic activity after head injury. Neurosurgery. 1981, 8(1):10-4.
24、Solomon RA, McCormack BM, Lovitz RN, et al. Elevation of brain norepinephrine concentration after experimental
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25、Yan HQ, Kline AE, Ma X, et al. Tyrosine hydroxylase, but not dopamine beta-hydroxylase, is increased in rat frontal cortex after traumatic brain injury. Neuroreport. 2001 Aug 8;12(11):2323-7.
26、丘永明，张保中，费智敏，等。损伤性犬大脑皮质运动区单胺类递质的变化。临床神经病学杂志，2000，13(6)：323—325。
27、Jai Sung Noh,* Eun Young Kim,. Jang Sook Kang, et al. Neurotoxic and Neuroprotective Actions of Catecholamines in Cortical Neurons. Exp Nrurology, 1999, 159, 217—224.
28、Huang W, Chen Y, Shohami E, et al. Neuroprotective effect of rasgiline, a selective moniamine oxidase-B inhibitor, against closed head injury in the mouse. Eur J Pharmacol, 1999, 366(2-3): 127—35.
29、Wahl M, Mediators of vascular and parenchymal mechanisms in secondary damage. Acta Neurochirurgica, 1993, 57(Suppl): 64-69.
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