针刺对脑出血模型大鼠神经细胞内钙离子稳态调节及脑组织SODmRNA表达影响的实验研究
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摘要
目的：通过针刺对脑出血模型大鼠的脑细胞内Ca~(2+)浓度及SODmRNA表达的变化，来探讨脑出血病的发生、发展过程中，针刺对神经细胞内Ca~(2+)浓度的变化、Ca~(2+)颗粒的分布的改变，以及对SODmRNA表达水平的影响。进一步阐述脑出血后Ca~(2+)与SOD之间相互影响的关系，从细胞生物学、分子生物学方面来探讨针剌对脑出血的作用机理。为临床提供更为新的更有价值的、可靠的理论依据。
方法：采用脑内注入VII型(?)原酶诱导脑出血模型，同时设立正常组、假手术组、针刺组、脑出血组，其中脑出血组、针刺组按不同时相分别在5min，15min，30min，60min，180min观察脑组织细胞内Ca~(2+)变化及针刺对其的影响和3h，6h，24h，3d，7d观察脑组织SODmRNA表达变化及针刺对其的影响。对脑组织细胞内Ca~(2+)变化检测采用荧光指示剂Fara-2测定细胞Ca~(2+)原理和Ca~(2+)电镜细胞化学技术；利用原位杂交技术结合计算机图像分析，观察脑组织SODmRNA表达变化及针刺对其的影响。
结果：脑出血5min时，内质网的Ca~(2+)含量略减少，而细胞核、细胞浆、线粒体的Ca~(2+)含量则略增加，此时，未引起细胞超微结构的变化，随着Ca~(2+)内流增多，引起内质网、细胞核内的Ca~(2+)紊乱和线粒体、细胞浆内Ca~(2+)超载，线粒体数量减少，结构受到破坏，内质网扩张，线粒体肿胀。脑出血30min—60min，由于Ca~(2+)大量内流，细胞内各细胞器均发生变化，内质网内Ca~(2+)严重丢失，细胞核、细胞浆、线粒体内Ca~(2+)严重超载，导致细胞结构严重破坏，核固缩，胞质空化，细胞器减少等，180min，虽然Ca~(2+)超载略减轻，但细胞结构损伤依然很重，细胞膜结构不完整，内质网呈扩张状态等，针刺可良性调节各时相细胞器Ca~(2+)含量，减轻细胞内Ca~(2+)紊乱、超载，保护脑组织。
实验性脑出血可诱导Cu-ZnSOD基因在脑出血周围区(皮质)、海马、纹状体的异常表达，脑出血3h可见Cu-ZnSODmRNA表达上调，并随时间延长而增
中丈摘要
强，到24h达到高峰，然后，各脑区CoZns0DmRNA的表达逐渐减少，并与
7d趋于对照组水平，针刺组各时程Cu一ZnSODmRNA的表达均低于同时程脑出
血组，但仍高于对照组水平.提示针#.J可使脑出血后各脑区组织的
Cu一ZnsODmRNA表达下调，对脑出血造成的神经元损伤有保护作用.
结论:(l)脑出血后，导致脑血肿、脑水肿，引起脑出血周围组织缺血缺
氧，神经细胞内Ca2+超载;同时神经细胞亚细胞内的c矛十时间和空间分布发生
异常改变，神经细胞结构破坏;以60min最为显著.针刺可抑制神经细胞c扩十
内流，减轻ca2+超载，保护脑组织。(2)脑出血后，脑组织内cu一zns0Dm洲A
表达3h开始上调，24h达到峰值，针刺可使脑组织的Cu一ZnSODmRNA表达下
调，减轻脑组织损伤.
Obective: To study the mechanisms of acupuncture following intracerebral hemorrhage(ICH) and alterations of brain cell calcium inos regulation, actions of intracellular Ca2+ concentration in isolated rat
brain cell effects of SODmRNA expression in rat brain cell and its relationship with cell calcium and SOD, It provides scientific basis for clinical from the areas of cytobiology and molecular biology. Methods: Rat model of cerebral hemorrhage was made with collagenase, The rats were divided into 4 groups: the normal group, the sham experiments group, the intracerebral hemorrhage(ICH) group, the acupuncture group. According to the time of the intracerebral hemorrhage(ICH) formation, the intracerebral hemorrhage(ICH) group and acupuncture group were divided into 5 groups respectively, the groups of 5^ 15>3(K6(K 180 min are studied the influence of cell calcium ions and intervention of acupuncture ,the groups of 3 hour % 6 hour > 24 hour> 3 day > 7 day are studied the effects of SODmRNA and intervention of acupuncture.
Brain cell were isolated from rat brain with Fura2-AM measurements of intracellular Ca2+ concentration([Ca2+]j). The distribution of Ca2+ and changes of ultrastural in brain neuron were determined by using K-pyroantimonate cytochemistry method, in the mean time, the energy-dispersive X-ray microanalysis method was used to measure the intracellular Ca2+ concentration. The SODmRNA expression in brain tissue were observed with In situ hybridization method.
Resuts: The Ca2+ concentration and distribution of the endoplasmic reticulum in brain cell was obvious at 5 min after intracerebral hemorrhage(ICH), descended rpidly at 30 min, peaked at 60min, then risen gradually. The Ca2+ concentration and distribution of the cytoblast, cytoplasm and mitochondria in brain cell was obvious at 5min after intracerebral hemorrhage(ICH), rose rpidly at 30 min, peaked at 60 min, then descended gradually. The changes of ultrastructral in neuron was obvious at 5 min after intracerebral hemorrhage(ICH), peaked at 60min and alleviated gradually. Calcium ions fluxes from the extracellular fuid into the intracellular space may trigger irreversible injury that is the endoplasmic reticulum expind, mitochondria swelling at 5 min, karyopyknsis, cytoplasm cavi tion and organell decrease at 60 min and cytoarchitecture destroy and endoplasmic reticulum expand at 180 min. Acupuncture can two-sided regulate Ca2+ concentration and distribution of organell, de ;reased Ca2+ verloading, the alleviating the injury of neurons.
The abnormal expressioi of SOD mRNA in rat brain tissue(cortex, hippocampus, striatum) was obvious at 3 hour after intracerebral hemorrhage(ICH), rose rapic y at 6 hour, peaked at 24 hour, then descended gradually at 3 da . Acupuncture can down-regulating the experimental of SODmRNA andd alleviating the injury of neurons. Conclusion: (i) Brain hem torn, brain edema formation following intracerebral tv morrhage(ICI I) lead to ischemia and hypoxia, Ca2+ overloading of ,he brain cell. The Ca2+ distribution of ultrastructural in the brain neuroh changed in time and pace, cytoarchitecture destroy and the peaked at 6) min. Acupuncture can play a role in inhibiting fluxed from the extracellular fluid into the intracellular space and descended
Ca2+ overloading of brain cell and protected the neuronal injury induce
y intracerebral hemorrhage. (2) The expression of SODmRNA is up-regulated in rat brain tissue that it was obvious at 3 hour after intracerebral hemorrhage(ICH), rose rapidly at 6 hour, peaked at 24 hour. Acupuncture can down-regulated the experimental of SODmRNA and alleviating the injury of neurons after intracerebral hemorrhage.
方法：采用脑内注入VII型(?)原酶诱导脑出血模型，同时设立正常组、假手术组、针刺组、脑出血组，其中脑出血组、针刺组按不同时相分别在5min，15min，30min，60min，180min观察脑组织细胞内Ca~(2+)变化及针刺对其的影响和3h，6h，24h，3d，7d观察脑组织SODmRNA表达变化及针刺对其的影响。对脑组织细胞内Ca~(2+)变化检测采用荧光指示剂Fara-2测定细胞Ca~(2+)原理和Ca~(2+)电镜细胞化学技术；利用原位杂交技术结合计算机图像分析，观察脑组织SODmRNA表达变化及针刺对其的影响。
结果：脑出血5min时，内质网的Ca~(2+)含量略减少，而细胞核、细胞浆、线粒体的Ca~(2+)含量则略增加，此时，未引起细胞超微结构的变化，随着Ca~(2+)内流增多，引起内质网、细胞核内的Ca~(2+)紊乱和线粒体、细胞浆内Ca~(2+)超载，线粒体数量减少，结构受到破坏，内质网扩张，线粒体肿胀。脑出血30min—60min，由于Ca~(2+)大量内流，细胞内各细胞器均发生变化，内质网内Ca~(2+)严重丢失，细胞核、细胞浆、线粒体内Ca~(2+)严重超载，导致细胞结构严重破坏，核固缩，胞质空化，细胞器减少等，180min，虽然Ca~(2+)超载略减轻，但细胞结构损伤依然很重，细胞膜结构不完整，内质网呈扩张状态等，针刺可良性调节各时相细胞器Ca~(2+)含量，减轻细胞内Ca~(2+)紊乱、超载，保护脑组织。
实验性脑出血可诱导Cu-ZnSOD基因在脑出血周围区(皮质)、海马、纹状体的异常表达，脑出血3h可见Cu-ZnSODmRNA表达上调，并随时间延长而增
中丈摘要
强，到24h达到高峰，然后，各脑区CoZns0DmRNA的表达逐渐减少，并与
7d趋于对照组水平，针刺组各时程Cu一ZnSODmRNA的表达均低于同时程脑出
血组，但仍高于对照组水平.提示针#.J可使脑出血后各脑区组织的
Cu一ZnsODmRNA表达下调，对脑出血造成的神经元损伤有保护作用.
结论:(l)脑出血后，导致脑血肿、脑水肿，引起脑出血周围组织缺血缺
氧，神经细胞内Ca2+超载;同时神经细胞亚细胞内的c矛十时间和空间分布发生
异常改变，神经细胞结构破坏;以60min最为显著.针刺可抑制神经细胞c扩十
内流，减轻ca2+超载，保护脑组织。(2)脑出血后，脑组织内cu一zns0Dm洲A
表达3h开始上调，24h达到峰值，针刺可使脑组织的Cu一ZnSODmRNA表达下
调，减轻脑组织损伤.
Obective: To study the mechanisms of acupuncture following intracerebral hemorrhage(ICH) and alterations of brain cell calcium inos regulation, actions of intracellular Ca2+ concentration in isolated rat
brain cell effects of SODmRNA expression in rat brain cell and its relationship with cell calcium and SOD, It provides scientific basis for clinical from the areas of cytobiology and molecular biology. Methods: Rat model of cerebral hemorrhage was made with collagenase, The rats were divided into 4 groups: the normal group, the sham experiments group, the intracerebral hemorrhage(ICH) group, the acupuncture group. According to the time of the intracerebral hemorrhage(ICH) formation, the intracerebral hemorrhage(ICH) group and acupuncture group were divided into 5 groups respectively, the groups of 5^ 15>3(K6(K 180 min are studied the influence of cell calcium ions and intervention of acupuncture ,the groups of 3 hour % 6 hour > 24 hour> 3 day > 7 day are studied the effects of SODmRNA and intervention of acupuncture.
Brain cell were isolated from rat brain with Fura2-AM measurements of intracellular Ca2+ concentration([Ca2+]j). The distribution of Ca2+ and changes of ultrastural in brain neuron were determined by using K-pyroantimonate cytochemistry method, in the mean time, the energy-dispersive X-ray microanalysis method was used to measure the intracellular Ca2+ concentration. The SODmRNA expression in brain tissue were observed with In situ hybridization method.
Resuts: The Ca2+ concentration and distribution of the endoplasmic reticulum in brain cell was obvious at 5 min after intracerebral hemorrhage(ICH), descended rpidly at 30 min, peaked at 60min, then risen gradually. The Ca2+ concentration and distribution of the cytoblast, cytoplasm and mitochondria in brain cell was obvious at 5min after intracerebral hemorrhage(ICH), rose rpidly at 30 min, peaked at 60 min, then descended gradually. The changes of ultrastructral in neuron was obvious at 5 min after intracerebral hemorrhage(ICH), peaked at 60min and alleviated gradually. Calcium ions fluxes from the extracellular fuid into the intracellular space may trigger irreversible injury that is the endoplasmic reticulum expind, mitochondria swelling at 5 min, karyopyknsis, cytoplasm cavi tion and organell decrease at 60 min and cytoarchitecture destroy and endoplasmic reticulum expand at 180 min. Acupuncture can two-sided regulate Ca2+ concentration and distribution of organell, de ;reased Ca2+ verloading, the alleviating the injury of neurons.
The abnormal expressioi of SOD mRNA in rat brain tissue(cortex, hippocampus, striatum) was obvious at 3 hour after intracerebral hemorrhage(ICH), rose rapic y at 6 hour, peaked at 24 hour, then descended gradually at 3 da . Acupuncture can down-regulating the experimental of SODmRNA andd alleviating the injury of neurons. Conclusion: (i) Brain hem torn, brain edema formation following intracerebral tv morrhage(ICI I) lead to ischemia and hypoxia, Ca2+ overloading of ,he brain cell. The Ca2+ distribution of ultrastructural in the brain neuroh changed in time and pace, cytoarchitecture destroy and the peaked at 6) min. Acupuncture can play a role in inhibiting fluxed from the extracellular fluid into the intracellular space and descended
Ca2+ overloading of brain cell and protected the neuronal injury induce
y intracerebral hemorrhage. (2) The expression of SODmRNA is up-regulated in rat brain tissue that it was obvious at 3 hour after intracerebral hemorrhage(ICH), rose rapidly at 6 hour, peaked at 24 hour. Acupuncture can down-regulated the experimental of SODmRNA and alleviating the injury of neurons after intracerebral hemorrhage.
引文
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