【作者】 陆新颜；

【导师】 洪光祥；

【作者基本信息】 华中科技大学 ， 外科学， 2006， 博士

【摘要】 周围神经损伤后轴突的再生是实现功能恢复的前提,如何维持神经轴突的生长,影响因素很多,其中,与损伤有关的神经细胞体活性的变化、坏死程度、坏死后的再生以及再生后的神经细胞是否具有正常细胞的功能是决定轴突生长、功能恢复的先决条件,神经细胞的凋亡与神经轴突的再生和修复有关。因此,认清神经元在轴突损伤后的病理变化过程具有重要意义。研究发现一系列的神经营养因子(Neurotrophic factors,NTF)、神神经递质和激素等在神经生长、发育、细胞增殖、凋亡、再生等过程中起重要作用。细胞外ATP类物质不仅可以作为神经递质及调质参与神经系统的功能,还可对胶质细胞及神经元产生营养作用,广泛参与细胞分化及凋亡、细胞线粒体形态变化、刺激细胞因子及神经生长因子的合成及释放等过程,对神经系统的发育及可塑性变化产生重要的影响。脊髓神经元和雪旺细胞表面P2Y受体亚型的存在及其介导的神经细胞的电生理变化和细胞增殖,证明细胞外ATP可以保护受损的神经元,这种作用是通过P2Y受体介导的。细胞的损伤修复启动时,发现MAPK系统激活,细胞外信号调节因子(ERK1/2)被激活,而这种信号途径也与P2Y受体有关。近来发现:Na+-K+-ATPase不仅具有离子泵的功能,还具有信号转导分子的作用。细胞内Ca2+离子作为一种重要的信号分子,参与细胞内多种生理生化反应。肌浆网丰富的Ca2+-ATPase对细胞内Ca2+平衡起着重要作用,ATPase能通过与细胞内其它蛋白质和因子的相互作用起传递细胞外信号的作用,调节细胞的功能。糖原合成酶激酶3(Glycogen synthasekinase3,GSK3)是信号传递系统中一个重要的高度保守的激酶,在许多输入信号通路中发挥作用,而其亚型GSK-3β是凋亡信号通路中一个重要的因子。那么,为了了解周围神经损伤后,神经元会主要发生什么样的变化,细胞外ATP对这种变化的作用如何,ATPase和P2Y受体在这个过程中的变化和作用如何这些问题,我们设计了这个实验。本实验主要分为以下三部分。第一部分细胞外ATP对坐骨神经损伤后脊髓前角运动神经元和靶肌肉ATPase活性影响的研究目的了解细胞外ATP对坐骨神经损伤后腓肠肌和相应脊髓节段ATP aase活性的影响。方法成年SD大鼠105只,随机分成三组,A组为损伤组:右侧坐骨神经损伤后靶肌肉局部注射生理盐水组,B组为对照组:右侧坐骨神经损伤后立即修复并靶肌肉局部注射生理盐水组;C组为实验组:右侧坐骨神经损伤后立即修复并靶肌肉局部注射0.5mgATP组。术后分别于12h、1d、3d、7d、14d、28d、56d测定腓肠肌和L4-6水平脊髓前角运动神经元Na+-K+-ATPase及Ca2+-ATPase活性和相应时期的肌湿重变化情况。结果细胞外ATP与修复神经后能显着改善ATPase的活性,这种改变与肌湿重的变化相一致,与损伤组和对照组对比有显着差异(P<0.05)。结论细胞外ATP可以通过对ATPase的影响对失神经骨骼肌和脊髓前角运动神经元具有一定的保护和促进功能恢复作用。第二部分坐骨神经损伤后脊髓前角运动神经元的变化及细胞外ATP对其作用的实验研究目的探讨大鼠坐骨神经损伤后,对应神经元的变化转归以及局部注射ATP后对脊髓神经元这种变化的影响及其方式。方法成年SD大鼠210只,随机分成三组,A组为损伤组:选择右坐骨神经损伤后靶肌肉局部注射生理盐水组,B组为对照组:右侧坐骨神经损伤后立即修复并靶肌肉局部注射生理盐水组;C组为实验组:右侧坐骨神经损伤后立即修复并靶肌肉局部注射0.5mgATP组。术后分别于12h、1d、3d、7d、14d、28d、56d通过免疫组织化学和Western Blot技术检测神经元结构及p-GSK-3β(ser9)表达变化情况,评价神经损伤后对应脊髓神经元的变化及ATP对这种变化的影响作用。结果坐骨神经损伤后对应脊髓神经元发生以凋亡为主的变化,细胞外ATP与修复神经后能显着改善凋亡的发生并能对GSK-3β的活性产生影响。结论周围神经损伤后脊髓神经元发生凋亡,局部注射ATP可以起到保护神经元的作用,这种作用与GSK-3 p有关。第三部分细胞外ATP对体外培养的机械损伤的脊髓前角运动神经元作用机制的实验研究目的研究细胞外ATP对体外培养的机械损伤的脊髓前角运动神经元的作用机制。方法取新生大鼠脊髓,通过常规的原代细胞培养程序,采用差速贴壁法分离出大鼠脊髓前角运动神经元,培养成熟后制作神经元机械损伤体外模型,分为A组:对照组、B组:100μMATP组、C组:100gMATP+20μg/ml Suramin组和D组:100μM ATP+IOμM Ouabain+10μg/ml Thapsigargin组,对各组机械损伤的运动神经元进行培养,1天后分别进行运动神经元计数、MTT比色实验观察运动神经元的存活及活性、流式细胞仪分析机械损伤的脊髓前角运动神经元凋亡百分率和Werstern Blot技术检测p-GSK-3β(ser9)的表达。结果A组机械损伤的神经元在培养1天后,运动神经元计数和神经元存活及活性值最低,与D组相比没有显著差异,但是与A组和C组差异显著;其凋亡细胞百分率最高,与其它三组相比有显著差异。B组运动神经元计数及MTT比色实验结果最高,与C组相比差异并不显著,但是与A组和D组有明显差异;其神经元凋亡百分率最低,与其它三组相比其结果有显著差异性;C组凋亡细胞百分率与其它三组有明显差异,但其结果优于A组差于其它两组;其活细胞数与B组差异不大,明显优于A组和D组。D组神经元凋亡百分率明显低于A组,与B组和C组相比没有显著差异,但是其存活细胞数与B组和C组存在显著差异,与A组差异不大。A组中可见p-GSK-3β(ser9)阳性产物低表达,B组和D组见较多量p-GSK-3p(ser9)阳性产物表达,表达显著,c组中可见中量的p-GSK-3β(ser9)阳性产物表达。结论机械损伤的脊髓前角运动神经元会发生坏死,这种坏死改变以凋亡为主,p-GSK-3β参与到这种凋亡信号的传递中。细胞外ATP对损伤的细胞能起到保护作用,减少凋亡的发生,这种作用可以通过P2Y受体介导,ATPase也能在这个信号传递系统中发挥作用,保护细胞,避免损伤加重。更多还原

【Abstract】 When the peripheral nerve injured, the precondition of recovery of function is the regeneration of axon. There are many factors can influence the growth of axon. The change of activity, the extent of necrosis, the regeneration after necrosis and the function after the regeneration of neuron connected with peripheral nerve are the prerequisite of the axon growth and recovery of function. The neuron apoptosis is connected with axon regeneration and repair. So, it has an important meaning to identify clearly the pathological change of the axon after injury. The research found that some neurotrophic factors, neurotransmitters and hormones have an important effect on the nerve growth, development, cell proliferation, apoptosis and regeneration. The species of extracellular ATP can participate the effect of nerve system not only as the neurotransmitter and modulator but also has a trophic action to glial cell and neuron. It participates in many courses, e.g. cell differentiation and apoptosis, the morphologic change of chondriosome, to stimulate the synthesis and release of cytokine and nerve growth factor. It has a great influence to neurological growth and plasticity. The found of receptor P2Y on the neuron and schwann cell and the change of it mediated on electrophysiology variation and cell proliferation, these improved that the extracellular ATP can protect the injured neuron, and this effect has been mediated with receptor P2Y. When the reparation start after injury, we found the MAPK system been activated, the ERK1/2 been activated, and the course concerned with receptor P2Y. In recent years, people found that Na-K-ATPase can not only as a ion pump, but also have the feature of as a signal transduction molecule. The ion Ca in cell is an important signal molecule in many physiological and biochemical event of cell living. The abundant Ca- ATPase in arcoplasmic reticulum can balance the ion Ca in the cell. The ATPase through the interaction with other proteins in the cell can transfer the extracellular signal into the cell and regulate the cell functions. The Glycogen synthase kinase3（GSK3） is a conservative kinase in the signal transmission system, it can produce a marked effect in many input signal path, and its subtype GSK—3β is an important factor of apoptosis. In order to understand the change of neuron following peripheral injury, what the effect of extracellular ATP and what’s the effect of ATPase and receptor P2Y in the course, we design the experiment. The experiment is divided into three parts as following.Part 1Effects of extracellular ATP on the ATPase activity of denervated skeletal muscle and motor neurons of anterior spinal cordObjective To investigate the effect of extracellular ATP on ATPase activity of denervated gastrocnemius and motor neurons of anterior spinal cord following sciatic nerve injury in SD rat. Method Sprague-Dawley rats were used, randomly divided into three groups: The right side sciatic nerves were transected but not sutured group （injury group）, the right side sciatic nerves were transected and sutured immediately, and the ATP （experimental group） and normal sodium （control group） were injected into the right side gastrocnemius with same volume. After 12hour、 1day、 3day、 7day、 14day、 28day and 56day of the operation, measure the muscles wet weight and Na⁺-K⁺-ATPase and Ca²⁺ -ATPase activities with both of the two tissues. Result Compared with the three groups, The ATPase bioactivity in experimental and control groups were superior to that in the injury group at some times, and this change was the same with the change of the muscle wet weight. Statistically significant significance was found. Conclusions The effect of extracellular ATP can improve the ATPase activity of denervated muscles and motor neurons of anterior spinal cord at SD rat, which suggested it has certain protective and promote regeneration effects on denervated muscle and motor neurons of anterior spinal cord, and these effects may produce through ATPase.Part 2Experimental Study of Change of motor neurons of anterior spinal cord following sciatic nerve injury and the effect of extracellular ATPObjective To explor the Change of motor neurons of anterior spinal cord following sciatic nerve injury and the effect of extracellular ATP on the motor neurons SD rat. Method Sprague-Dawley rats were used, randomly divided into three groups: The right side sciatic nerves were transected but not sutured group （injury group）, the right side sciatic nerves were transected and sutured immediately, and the ATP （experimental group） and normal sodium （control group） were injected into the right side gastrocnemius with same volume. After 12hour、 1day、 3day、 7day、 14day、 28day and 56day of the operation, observe the motor neuron’s change on immunohistochemistry and measure the change of protein of p—GSK—3β（ser9）. Result After the injury of sciatic nerve, the main change of motor neurons of anterior spinal cord is apoptosis. Use extracellular ATP and ripair sciatic nerve can decrease neuron apoptosis and influence the activity of protein GSK—3β. Conclusion The main change of motor neuron of anterior spinal cord following sciatic nerve injury is apoptosis, after inject with ATP, it can protect the motor neuron and this effection may concern with protein GSK—3β.Part 3Experimental Study of the Mechanism of Action of Extracellular ATP on Motor Neurons of Anterior Spinal Cord in Vitro Following Machinery Injury Objective To explore the mechanism of action of extracellular ATP on motor neurons in vitro following machinery injury. Method Get the neonate Sprague-Dawley rat’s medulla spinalis, through the conventional primary cell culture procedure, we use the method of difference adherence time and get the motor neurons of anterior spinal cord. When the neuron is in maturity, we make the mechanical injury model in vitro. All the models were divided into four groups: group A is control group; group B is 100μM ATP group; group C is 100μM ATP+20μg/ml suramin group and group D is 100μM ATP+10μM ouabain+10μg/ml Thapsigargin group. Culture the four groups neurons, after one day, we count the motor neuron, observe the survival and activity of neurons through MTT shade selection experiment, use flow cytometry to analyze the percentage of apoptosis of motor neurons of anterior spinal cord and detect the expression of protein p-GSK-3β （ser9） through Western-Blot technology. Result After we cultured the neuron one day, we found the counting of motor neuron and the survival and activity of neurons of group A was the lowest. They had no significant deviation controlling with group D, and had significant deviation controlling with group A and C. The percentage of apoptosis of motor neurons in group A was the highest, it had significant deviation controlling with others. The counting of motor neuron and the survival and activity of neurons of group B was the highest. They had no significant deviation controlling with group C, and had significant deviation controlling with group A and D. The percentage of apoptosis of motor neurons in group B was the lowest, it had significant deviation controlling with others. The percentage of apoptosis of neurons of group C was better than group A and worth than the others, the counting of living cells was no significant deviation controlling with group B, but better than group A and D. The percentage of apoptosis of neurons of group D was lower than group A, but had no significant deviation with others. The counting of living cells had significant deviation controlling with group B and C, and had no significant deviation controlling with group A in it. In group A, we can see the lower expression of p-GSK-3β （ser9） , and in group B and D, we only can see a amounts of it, it was significant. In the group C had a few expression of p-GSK-3β （ser9） . Conclusion The cultured machinery injured motor neuron of anterior spinal cord will apoptosis. In the apoptosis, the GSK-3β may in the course of signal conduction. Extracellular ATP can protect the injured neurons, decrease the count of apoptosis and this effect may mediate with receptor P2Y, and ATPase can influence the signal conduction, produce a marked effect of protecting the neuron and lightening the damage.更多还原