【作者】 刘媛；

【导师】 王正国；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 近年来,创伤性脑损伤(Traumatic brain injury,TBI)的发生率逐年上升,无论是在发达国家还是在发展中国家,已成为年轻人,特别是35岁以下年轻人死亡的主要原因之一。因此,有关TBI的发病机制及其治疗措施的研究一直受到国内外临床医生和神经科学研究者的高度关注。脑创伤后无论是机械性的直接损伤,还是随后出现的缺血、缺氧、离子失衡以及兴奋性氨基酸的毒性作用均可引起神经元损害和死亡,并导致继发性脑水肿和脑功能障碍。TBI时海马是重要的作用靶点,对损伤高度敏感。海马的功能与学习记忆密切相关,它在空间信息的获得、检索、巩固和存储过程中发挥重要作用。脑创伤后由于海马受损所导致的学习、记忆功能障碍目前尚无有效的治疗措施,它是造成TBI救治效果差、伤残率高的重要原因之一。因此,研究TBI后海马神经元损伤的机制及防治成为近年来国内外关注的热点和难点。大量研究表明:谷氨酸是海马突触环路里最主要的一种神经递质,而谷氨酸及其受体的作用也一直是TBI分子机制研究的重要内容。近年来针对谷氨酸(glutamate, Glu)受体研制了多种拮抗药物,但其效果均不尽人意,而且存在明显的副作用。其原因是由于这些药物的特异性不强,在阻断钙离子的同时,也阻断了正常的突触内信息传递。因此,深入研究海马细胞Glu受体亚单位在TBI后的表达变化、转运调控过程,不仅对于深化认识TBI的发病机制具有重要的理论意义,而且对于设计出特异性更强、且副作用更小的谷氨酸受体阻断药物具有重要的临床价值。谷氨酸受体包括代谢型(G蛋白耦联的受体)和离子通道型两类受体。其中离子通道型Glu受体分为N-甲基-D-天门冬氨酸(NMDA)受体,α-氨基羟甲基恶唑丙酸(AMPA)受体和海人藻酸(KA)受体。研究表明海马CA1区NMDA和AMPA受体的含量均高于其它脑区,正常情况下该区神经细胞膜上的NMDA受体对钙离子有较高通透性,而AMPA受体对钙离子不通透。AMPA受体介导神经元钙离子通透性的作用主要与GluR2亚单位有关,其中含有GluR2亚单位的AMPA受体不通透钙离子,不含GluR2亚基的AMPA受体则对钙离子有很高的通透性。有关NMDA受体在TBI中的作用已经进行了广泛的研究。通过阻断NMDA受体可对TBI后海马神经元细胞具有一定的保护作用,但仍然未达到预期的效果。目前有关AMPA受体在TBI后的生物学作用却引起了广泛的关注。最近研究报道大鼠脑缺血损伤可导致海马神经元突触后膜表面含有GluR2的AMPA受体数量显著减少,而不含GluR2的AMPA受体在突触后膜形成增多,导致钙离子内流增加及伤后神经元死亡数量明显增多,但其具体的机制仍然不清楚。AMPA受体是一种膜受体,神经细胞膜受体的功能虽然受细胞内外多种过程的调节,但磷酸化作用对膜受体功能的影响最重要。PTEN基因,即第10号染色体同源丢失性磷酸酶张力蛋白基因(Phosphatase and tensin homology deleted on chromosome ten, PTEN),是1997年克隆出的一种新型肿瘤抑制基因,PTEN可使(PI3、4、5P3 )脱磷酸,从而阻断PI3K信号通路,抑制细胞生长。此外,PTEN可阻断细胞周期,诱导细胞凋亡,抑制细胞的转移、扩散、聚集和粘附。最近的研究提示PTEN参与了脑缺血损伤的过程,并与兴奋性氨酸受体NMDAR的生物学功能具有一定的联系。而有关创伤性脑损伤所导致海马损伤过程中,PTEN的表达变化及其与兴奋性谷氨酸AMPA受体相互作用的关系目前尚未见报道。鉴于此,我们首先通过Impactor II重物打击装置制备了中度脑创伤模型,并利用细胞培养、神经病理、行为学以及分子生物学等技术方法,通过离体和在体实验观察PTEN与TBI后海马神经元形态和功能变化的关系及其对AMPA受体的调控作用,并初步分析PTEN在TBI后海马神经元损伤中的调控作用,探讨其作用机制。主要技术方法:1.采用Wise Young改良的Impactor-II重物打击装置,通过对大鼠右侧大脑皮层进行打击,制备创伤性脑损伤模型。2.用50g.cm的打击强度制备中度脑创伤模型,实验动物分为对照组、单纯损伤组和PTEN抑制剂bpv侧脑室注射治疗组,利用免疫组织化学、RT-PCR、Western Blot方法检测脑创伤后不同时间点PTEN mRNA及蛋白表达的变化,采用TUNEL和NF-200免疫荧光染色检测了损伤不同时间海马神经元的凋亡和神经元的存活情况。3.利用体外培养的海马神经元,制备了牵张损伤模型,实验分为正常对照组、单纯损伤组和bpv处理组,利用免疫荧光染色观察了各组神经元损伤后GluR2表达的变化。同时通过PI染色检测了损伤后神经元的死亡情况。4.利用Western blot和RT-PCR检测了培养神经元牵张损伤后神经元细胞PTEN和磷酸化PTEN以及GluR2表达情况,并通过Fura-3标记神经元细胞内游离钙离子,利用激光共聚焦显微镜实时扫描检测了损伤情况下细胞内钙离子浓度的变化。主要结果:1.采用Wise Young改良的Impactor-II重物打击装置,通过对大鼠右侧大脑皮层进行20g.cm、50g.cm、100g.cm强度的打击后,制备了轻、中、重度的创伤性脑损伤模型。2.中度TBI后损伤侧海马CA1区凋亡神经元数量显著增加,在1天时达到高峰,并维持到第3天,7天后显著减少,提示TBI造成海马损害具有区域选择性,CA1区神经元对损伤最敏感。TBI后伤侧海马神经元PTENmRNA的表达升高,12小时到达高峰;同时PTEN蛋白在伤后24小时增加最为显著,表明PTEN参与了TBI的病理生理变化过程。通过侧脑室注射PTEN蛋白的抑制剂bpv后,可减少TBI后海马神经元的凋亡和丢失。3.培养海马神经元牵张损伤后,PTENmRNA和蛋白的表达均增加,同时死亡神经元的数量也明显增加,在给予100 nM、200 nM和500 nM PTEN抑制剂bpv预处理后,可减少神经元的死亡,其中200nM bpv对神经元的保护作用明显好于100nM,但与500 nM组无显著差别,提示PTEN抑制剂对损伤神经元的保护作用具有一定的剂量依赖性。4.通过Western blot检测首次观察到,培养海马神经元牵张损伤后12、24和72小时,损伤组和bpv组PTEN和p-PTEN的水平均明显高于正常对照组,各时相点损伤组和bpv治疗组间PTEN的表达却无显著差异。但bpv治疗组中p-PTEN的表达水平在各时相点均低于损伤组,表明bpv对损伤海马神经元细胞PTEN的磷酸化水平具有一定的抑制作用。5.培养海马神经元牵张损伤后,免疫荧光染色提示细胞膜上GluR2蛋白的表达减少并对钙离子的通透性增加,在给予PTEN抑制剂bpv的条件下,可有效抑制膜表面GluR2的减少及钙离子的内流。通过Western blot检测观察到细胞总GluR2蛋白的表达无显著变化,提示PTEN抑制剂bpv通过抑制PTEN的磷酸酶活性,阻止神经元细胞膜上AMPA受体中GluR2亚单位的减少,减轻细胞外钙离子内流,这可能PTEN抑制剂对AMPA受体进行调控并对神经元产生保护作用的重要机制。6.通过激光共聚焦显微镜实时扫描Fura-3荧光强度变化观察到:阻断培养神经元NMDA受体,可部分阻止谷氨酸刺激所引起的钙离子内流;同时给予bpv和NMDA受体拮抗剂(MK-801)可明显减少、但不能完全阻止谷氨酸刺激所引起的钙离子内流;在给予bpv+ MK801+LY294002预处理的神经元后,由谷氨酸刺激所引起的钙离子明显高于bpv+ MK801预处理组,表明PTEN对损伤神经元AMPA受体的调控是通过PI3K-Akt通路发挥作用。结论:1. TBI后脑组织的损害具有选择性,海马CA1区神经元对损伤因素最敏感。本研究首次观察到PTEN参与了TBI后海马神经元的继发性损伤过程,并与神经元的凋亡具有一定的关系。2.体内、外实验证实PTEN抑制剂bpv对损伤海马神经元具有保护作用。3.本课题首次观察到PTEN抑制剂bpv是通过抑制PTEN的磷酸酶活性,阻止神经元细胞膜上AMPA受体中GluR2亚单位的减少,减轻细胞外钙离子内流而实现对损伤神经元的保护作用。4. PTEN对GluR2的调控,可通过PI3K-Akt途径来实现。更多还原

【Abstract】 Recently, the incidence rate of traumatic brain injury (TBI)has increased year by year. Both in developed and developing countries, TBI has become one of the main causes of death for young people, especially those under 35 years. Therefore, clinicians and neuroscientists closely pay attention to the research on mechanism and treating measures of TBI.After traumatic brain injury, whether mechanical injury or ischemia, hypoxia, ionic disequilibrium and toxic effects of excitory amino acids appeared subsequently all could casuse neurons damage and death, further lead to secondary edema and dysfunction of brain. Hippocampus is an important target and highly sensitive to injury when traumatic brain injury occurrs. The function of hippocampus is closely related to learning and memory, which plays main roles in the process of acquiring, indexing, consolidating and storing spatial information. There are no effective measures at present to cure dysfunctions of learning and memeory because of hippocampus damage, which has become one of the important reasons resulting in badly treating effect and high physical disability after TBI. Therefore, the injured mechanism and treatment of hippocampal neurons after TBI have evoked considerable interests all over the world in recent years.It is indicated that glutamate is one of the primary neurotransmitters in synapse circuit of hippocampus. At the same time, the effects of glutamate and its receptors have been the main research content about molecular mechanism of TBI. Though many antagonists against glutamate receptor have been developed recent years, the results are not satisfying because of evident side effects. The reason of which is that low specificity of antagonists when the antagonist blocks calcium ions ,it also obstructs normal information transmission in synapses. So, it is very necessary to study the expression of Glu subunit of hippocampal neurons after TBI and the regulating process of transport deeply. It has not only theoretical significance to recognize the pathogenetic mechanism of TBI, but also clinical value to design more specific and less side effect drug against glutamate receptor.The Glutamate receptors (GluRs) are categorized into two classes, metabotropic ( namely G-proteins coupled receptor) and ionotropic receptors. The ionotropic receptors (iGluRs) are further subdivided into three groups: N-methyl-D-aspartate (NMDA) receptor,a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate(AMPA) and kainate receptors. It is demonstrated that the content of NMDA and AMPA receptors in CA1 region of hippocampus are more than those in other regions. In general, NMDA receptors located on membrane of CA1 neurons are characterized by a high permeability to Ca2+, while AMPA receptors show no permeability to Ca2+.The permeability to Ca2+ of AMPA receptors are related to GluR2 subunit, among which AMPA receptors with GluR2 subunit may prevent the channels from cellular influx of Ca2+ whereas AMPA receptors without GluR2 subunit show significant permeability to Ca2+. Extensive researches have been carried out about effects of NMDA receptor on TBI. Although blockage of NMDA receptor can protect hippocampal neurons from TBI, there was a distance to prospective effects. The biological effects of AMPA receptor in TBI attracted wide attention at present.It has been reported that in global ischemia of rat AMPA receptors with GluR2 located on postsynaptic membrane of hippocampal neurons diminished notably, whereas AMPA receptors without GluR2 increased, which obviously induced Ca2+ influx into cytoplasm and neuronal death. The mechanism of that remains unclear.AMPA receptors are located on the plasma membrane of neural cell. Although the function of receptors are regulated by many procedures outside or inside the cell, phosphorylation is the most important factor influencing the function of plasma receptors. Phosphatase and tensin homology deleted on chromosome ten (PTEN) was cloned as a novel tumor suppressor gene in 1997. PTEN can antagonize PI3K signaling pathway and inhibit the growth of cells by dephosphorylation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3).In addition, PTEN can interrupt cell cycle and induce apoptosis,further, suppresses migration, diffusion, aggregation and trafficking of cells. Recent studies implicated PTEN took part in the process of cerebral ischemia injury and was associated with biological function of excitory amino acid NMDA receptors. However the changes of PTEN expression and interaction with AMPA receptors in hippocampus damage by TBI has not been reported . Thus, we firstly established moderate traumatic brain injury model of rats by Impactor II weight-drop equipment; Secondly, the relationship between PTEN and changes in morphology and function of hippocampal neurons after TBI as well as the regulation on AMPA receptor were observed; Finally, the regulated effect of PTEN on hippocampal neurons injury after TBI was initially analyzed and the mechanism of which was discussed.Main techniques and methods1. Models of traumatic brain injury were established through strength inducing to temporal right side of the rat’s cortex with Impactor-II weight-drop equipment modified by Wise Young.2. The model of moderate traumatic brain injury was produced by 50g.cm forece’s strike. The animals were divided into three groups: normal controlled group, injured group; group treated with PTEN inhibitor bpv through intracerebroventricular injection. The expression of PTEN mRNA and protein at different intervals post injury was detected by immunohistochemistry, RT-PCR and western blot. The apoptosis and survival of neurons in hippocampus at different intervals post injury were observed by TUNEL and NF-200 immunofluorescent staining respectively.3. A stretch injury model was established with primary cultured hippocampal neurons in vitro. In this experiment , the cells were divided into normal controlled group, injured group and bpv treated group. Expression of GluR2 in neurons post-injury was detected by immunofluorescent staining. At the same time, the death of injured neurons was examined by PI staining.4. The expression of PTEN, p-PTEN and GluR2 in neurons after loaded stretch injury was estimated by western Blot and RT-PCR. The free Ca2+ in cytoplasm of neurons was labeled with Fura-3 and the changes of Ca2+ concentration were scanned with laser confocal microscope.Main Results:1. Mild,moderate,severe traumatic brain injury models were established through 20g.cm、50g.cm、100g.cm force’s strike inducing to temporal right side of the rat’s cortex respectively, with Impactor-II weight-drop equipment modified by Wise Young.2. Apoptotic neurons of hippocampus in injured side increased notably after moderate TBI, reaching peak on the 1st day, maintaining on the 3rd day and decreasing significantly on 7th day, which indicated that hippocampus damage caused by TBI was of regional selectivity and neurons in CA1 region were most sensitive to injury. The expression of PTENmRNA in injured hippocampal neurons increased remarkably 12 hours post-injury, so as the PTEN protein,which was prominent at 24 hours post-injury. All of the results implyed PTEN participated the pathophysiological procedure of TBI. The apoptosis and loss of hippocampal neurons after TBI can be diminished through intracerebroventricular injection of PTEN inhibitor bpv.3. PTENmRNA and protein in cultured hippocampal neurons both increased after stretched injury, accompanied with added dead neurons. The number of dead neurons post-injury was decreased by 100nM, 200nM and 500nM PTEN inhibitor pre-treatment, among which the protective effect of 200nM bpv was better than that of 100nM, but had no difference compared with 500nM group. This data demonstrated that to some extent the protective effect of PTEN inhibitor was of dose-dependent.4. It was firstly clarified by western blot text that level of PTEN and p-PTEN in injured group or bpv treated group were both more than that in normal controlled group at 12、24 and 72 hours after neurons stretched injury, but there was no difference between the injured group and bpv treated group. However, level of p-PTEN in bpv treated group was lower than that in the injured group at all time post-injury. It was indicated that bpv antagonized the phosphorylation of PTEN in injured hippocampal neurons.5. It was observed that expression of GluR2 on membrane of hippocampal neurons decreased by immunofluorescent staining and the permeability to Ca2+ increased after stretched injury. PTEN inhibitor could suppress GluR2 diminishing and Ca2+ influx. However total GluR2 in neurons didn’t change post injury, which indicated PTEN inhibitor bpv antagonized phosphatase activity of PTEN, resulting in prevention GluR2-containing AMPA receptor located on membrane decreasing and relieving of neuron injury by Ca2+ influx, which may be the possible mechanism of PTEN inhibitor regulation of AMPA receptor and have protective effect on neurons.6. The changes of Fura-3 fluorescent intensity were detected by laser confocal microscope real-time scanning. It was observed that blockage of NMDA receptor partly prevented Ca2+ from influx into cytoplasm casused by Glu, at the same time , bpv combined with MK-801 could significantly decrease but not completely forbid Ca2+ influx stimulated by Glu. Ca2+ influx in bpv+ MK801+LY294002 pre-treated neurons stimualted by Glu was much more than those in bpv+ MK801 pre-treated group, which demonstrated that PTEN regulated AMPA receptor in the injured neurons through PI3K-Akt signaling pathway.Conclusions:1. The damage to brain tissue after TBI is of selectivity. Neurons in hippocampal CA1 region are most sensitive to injured factors. In this study we fisrtly observed that PTEN took part in the process of secondary injury to hippocampal neurons after TBI and was associated with apoptosis and survival of neurons.2. It was identified PTEN inhibitor bpv had protective effect on injured hippocampal neurons both in vivo and in vitro.3. It was firstly observed that PTEN inhibitor bpv antagonized phosphatase activity of PTEN, resulting in preventing GluR2-containing AMPA receptor located on membrane decreasing and relieving of neuron injury by Ca2+ influx.It may be the protective mechanism of bpv .4. It was well established that PTEN, via PI3K-Akt pathway, mediated GluR2 regulation.更多还原