【作者】 黄卫东；

【导师】 费舟； 贺晓生；

【作者基本信息】 第四军医大学 ， 外科学， 2005， 博士

【摘要】 谷氨酸是中枢神经系统内广泛存在、含量最高的氨基酸,对大脑皮层具有普遍而强烈的兴奋作用。颅脑损伤时,脑内谷氨酸蓄积,可继发神经细胞的多种急、慢性病理损害。弥漫性脑损伤后mGluRla变化最为明显,是加重神经元损害的关键因素。Homer蛋白对于第Ⅰ组mGluRs受体信号转导过程具有重要的调控作用,神经元损伤后Homer蛋白的表达变化与意义以及Homer分子调控mGluRla受体信号转导的具体机制尚不清楚。 实验一 体外培养大鼠脑皮层神经元机械性损伤模型的建立 目的 在细胞水平建立一种可靠的、简便易行的神经元机械性损伤模型,为进一步深入进行神经系统损伤的分子病理学研究奠定良好的基础。方法大鼠脑皮层神经元体外培养7d,通过光镜、电镜和免疫组织化学法鉴定培养的神经元。以微量移液器塑料枪头划割细胞,造成神经元机械性损伤。研究神经元机械性损伤后细胞形态学变化。检测细胞存活率和培养液乳酸脱氢酶活性变化,从病理生理学角度阐明神经元机械性损伤的效果。结果 神经元体外培养7d,光镜下见细胞核大而清晰、透亮,圆形或卵圆形,核仁明显,核膜清晰可见。胞体呈多形性,胞质透亮,更多还原

【Abstract】 Glutamic acid is the most important amino acid in the central nervous system, and has extensive and intense exciting effects on neurons of cortex. Accumulation of glutamic acid in brain after traumatic injury is related to various acute or chronic pathological insults on neurons. Change of metabotrobic glutamate receptor 1a (mGluR1a) is the most prominent in the families of mGluRs after diffuse brain injury and plays an important role in aggravating neuronal damages. Homer takes part in modulation of group I mGluRs’ signal transduction. Expression and significance of Homer and the detailed mechanism of signal transduction of mGluR1a after neuronal injury remain unclear. Part I. Mechanically injured model of cortical neurons of rat in vitroObjective Traumaticlly injured model of neurons in vitro can establish a foundation for molecular pathological mechanism research of central nervous system injury. In this study, we aim to form a novel, reliable and easy-conducted mechanically injured model of cortical neurons of rat in vitro.Methods Cortical neurons were identified with microscope, electric microscope and immunohistochemistry after 7 days in vitro cultivation. Mechanical injury was manipulated with a plastic tip crossing onto the neuronal culture, which resulted in three kinds of injury according to the degree of insults. After that, morphological changes were observed. In addition, cellular viability and lactate dehydrogenase (LDH) concentration were detected to elucidate the mechanical injury effects from the pathphysiological aspect. Results The neuronal nucleus was large and clear with round or elliptical outline. The nucleolus and the membrane of nucleus were easily found. The figures of neurons were various, and the cytoplasm was bright with many dendrites and axons striking out from the cellular body, which connected each other to form a complicated reticular formation. The chromatin was mainly euchromatin. Plenty of organelle including mitochondria and Golgi complex, and neuronal specific structure neurofilament and dendrite were also found. The percent of neurofilament 200 (NF200) positive neurons was more than 90%. Phenomena of necrosis and apoptosis were discovered 6 hours after mechanical injury and regeneration of injured neurons 72 hours after injury. The cellular viability decreased and the LDH concentration increased after mechanical injury of neurons in vitro. Conclusion In our study, cortical neurons were successfully cultured. It was confirmed that tip-caused injury could actually produce neuronal insults, and the degree of damage depended on the extension of tip-caused injury. This simple and effective mechanically injured model of neurons can simulate the pathological mechanism of traumatic brain injury, and is convenient for the study of morphology, molecular pathology and therapeutics after traumatic brain injury.Part II. Expression of metabotropic glutamate receptor la and neuroprotective effects of its antagonist AID A on the mechanically injured cortical neurons in vitroObjective It is aimed to study the expression and significance of mGluRla after mechanical injury in neurons in vitro and the effects of 1-aminoindan-l, 5-dicarboxylic acid (AIDA), a selective antagonist for mGluRla, on the pathphysiological process of traumatic neuronal injury. Methods Based on the mechanically injured model of cortical neurons previously established, expression of mGluRla was detected with the method of immunohistochemistry, and neuroprotective effects of AID A on injured neurons were analyzed by the measurement of LDH concentration as well as inner-cellular calcium level. Results There was weak expression of mGluRla in neurons in the control group, with the positive particles distributing in the neuronal cytoplasm, cyto-membrane and dendrites. But no mGluRla was found in the nucleus. While in the injury group, there were wide spread expression of mGluRla positive neurons from 10 minutes to 24 hours after injury and distribution of the positive particles were mainly in the cytoplasm around the nucleus 10 minutes after injury, and in the dendrites 24 hours after injury. Alive neurons were rare 72 hours after injury, with weak mGluRla expression. Although there was no significantly statistical differences in LDH concentration between group with AIDA treatment and group without any interference from 30 minutes to 6 hours after injury (P > 0.05), the concentration of LDH decreased significantly 12-72 hours after injury in the AIDA treated group compared with that in the injured group alone (P<0.05). Calcium fluorescence intensity unit was 63.6 + 7.3 in the neurons of control group, but the intensity unit decreased to 24.8 + 7.7 thirty minutes afterapplication of AIDA in the injured neurons. Conclusion Intense expression and consistent activity of mGluRla was found after mechanical injury of neurons, and increased synthesis of mGluRla in cytoplasm and transportation from cytoplasm to dendrites were also observed after neuronal injury. AIDA could significantly decrease neuronal calcium concentration, which may be the mechanism of its neuroprotective effects. It was illuminated that AIDA could antagonize the excessive excitement of mGluRla which was due to accumulation of glutamic acid as a result of mechanical insults of neurons. In this study, the pattern of mGluRla expression after neuronal injury in vitro and the treatment time window of AIDA were clarified. It was proved that mGluRla played a critical role in the pothphysiological process of traumatic injury of neurons, and a theoretical foundation was formed for the development of new neuroprotective agents and the improvement of prognosis for patients with traumatic and degenerative disease in central nervous system. Part III. Expression and significance of Homer after mechanical injury of cortical neurons in vitroObjective The expression patterns of Homer-1 after neuronal injury and its effcets on the function of mGluRla were discussed. Methods Based on the mechanically injured model of cortical neurons, expression patterns of Homer-1 were detected with the method of immunohistochemistry, immunoblotting and RT-PCR. Results It was indicated by nondenaturing agarose gels electrophoresis that no Homer-la specific DNA fragment was found in the control group, but it was found 10 minutes after neuronal injury, and increased significantly 30 minutes to 72 hours after injury. There was Homer-lb/c expression in control group, but no change of Homer-lb/c expression was observed after injury. Meanwhile, the expression patterns ofHomer-1 protein were similar to that of Homer-1 mRNA. Immunohistochemical analysis showed that there was a weak expression of Homer-la in control group. And Homer-la was increased significantly from 10 minutes to 72 hours after mechanical injury. The positive particles were distributed in the neuronal cytoplasm, cyto-membrane and dendrites. No change of Homer-lb/c expression was found before and after injury of neurons. Conclusion It was verified for the first time that traumatic injury could induce Homer-la gene expression, which may be due to the excitement of neurons as a result of glutamic acid release after injury. Expression of Homer-lb/c was not modulated by the neuronal exciting activity. Dynamically expressed Homer-la and constitutively expressed Homer-lb/c might further modulate the distribution and function of mGluRla. Potential mechanism might be that Homer-la, which competes with Homer-lb/c to couple to mGluRla, regulates the distribution of receptor at synapse and the efficiency of signal transduction, prevents neurons from excess excitement. Homer-la functions as a natural dominant negative to selectively regulate association ofmGluRla with Homer-lb/c after neuronal excitement increased.Part IV. Homer induced mGluRla signal transduction after mechanicalinjury of neurons in vitroObjective Constitutive expression of Homer-lb/c in neurons, indicated in our previous results, implies that it participates in the physiological activity of mGluRla. How does Homer-lb/c affect the distribution of mGluRla in neurons? How does Homer-lb/c regulate signal transduction of mGluRla? If Homer-lb/c plays a critical role in the pathphysiological process of neuronal injury, whether changes of its expression have neuroprotective effects on injured neurons? The answers were uncertain, and the questions were also thepurpose of this study. Methods RNA interference technology was performed to knock down Homer-lb/c gene, and to inhibit Homer-lb/c expression in neurons. Immunohistochemistry, immunoblotting and RT-PCR were used to identify Homer- lb/c expression after siRNA transfection. Calcium concentration was measured with laser scanning confocal microscopy 36 hours after Homer-lb/c siRNA transfection in neurons. Analysis of neuroprotective effects of Homer-lb/c gene knocking down on injured neurons was performed by detection of the LDH level in the medium. Results Expression of Homer-lb/c was inhibited and mGluRla appeared only in cytoplasm 36 hours after siRNA transfection. Calcium fluorescence intensity unit was 51.8 + 7.5 in neurons of control group, but was decreased to 23.4+ 2.8 in neurons after siRNA transfection with statistically significant difference between the two groups (P<0.05). LDH concentrations were (78 + 19.5) U/L, (76+18.1) U/L and (81+21.3) U/L in control group, transfection without siRNA group and siRNA transfected group respectively before the mechanical injury with insignificantly statistical difference among these groups (P>0.05). LDH concentrations were increased significantly in control [ (158+23.0) U/L ] and transfection without siRNA groups [ (145+25.1) U/L ] compared with that in siRNA transfected group [ (93+20.1) U/L ] 24 hours after traumatic neuronal injury (P<0.05). Conclusion Homer-lb/c gene knocking down was achieved with 36 hours siRNA transfection in neurons. Homer-lb/c plays an important role in mGluRla transportation from cytoplasm to dendrites and targeting at the synapse. Retention of mGluRla in cytoplasm is related to insufficient Homer-lb/c synthesis, but it does not influence the total volume of mGluRla expression. Homer-lb/c participates in cellular calcium releasing and is an important regulating molecule in the mGluRla-IP3R-Ca2+更多还原