创伤性脑损伤后大鼠脑组织钙结合蛋白S100A6的表达及意义
发布时间：2018-11-03 15:07
【摘要】：创伤性脑损伤（traumatic brain injury TBI）在各类创伤性损伤中最为常见，多与其他各种创伤合并存在，以高死亡率、高致残率而位居各类创伤之首。TBI后患者轻者出现一过性意识障碍、轻度认知功能障碍等神经系统症状，重者常出现昏迷、瘫痪、严重认知功能障碍、癫痫等神经系统症状，严重影响患者的生存及生活质量。对于TBI后神经系统的病理生理改变，主要的热点集中在各种损伤因素导致的最终共同通路：神经细胞内钙离子爆发引发的细胞坏死。随着对创伤性脑损伤的研究的不断深入，人们认识到TBI后神经细胞不仅存在坏死，而且存在凋亡。而后关于TBI后钙爆发引起的细胞凋亡的机制成为新的研究方向。目前关于TBI后神经细胞内生化改变还缺乏全面深入的认识，因此临床上对TBI后患者的治疗时机、方法、疗效至今没有突破性的进展和定论。有学者从钙爆发后与下游分子结合而引发的细胞凋亡与抗凋亡通路的角度进行了深入研究。有研究发现钙结合蛋白S100家族蛋白可与钙离子结合，进一步可与钙周期素结合蛋白CacyBp结合进一步转导信号，从而提出TBI后可能存在的调控细胞凋亡的细胞内信号转导通路：Ca~(2+)/S100/CacyBp/β-catenin（β-tubulin）。目前国内外尚无TBI后S100A6量变及与神经元损伤后认知功能的改变之间关系的报道。作者设计本实验，通过建立大鼠TBI模型初步探讨TBI后大鼠脑组织中S100A6分子表达变化的规律。 目的： 探寻创伤性脑损伤的神经损伤机制，建立中度脑损伤大鼠模型，通过测定TBI后不同时间点大鼠脑组织中S100A6分子的表达，分析其规律及与TBI的关系，旨在从蛋白表达变化的角度认识TBI后神经细胞损伤发展的分子机制，以期通过动物实验为临床诊治创伤性脑损伤提供有价值的理论依据及新的干预点以提高临床疗效。 方法： 健康雄性SD大鼠72只，清洁等级，体重（220±20g），按照随机数字表法分为TBI组（64只）和正常对照组（8只）；TBI模型的制备使用瞬间头颅侧向旋转致伤法制作，按伤后时间分为：1h，3h，6h，12h，24h，72h，7d，14d共8个亚组，每组8只，各时间点处死大鼠。在各时间点处死大鼠并进行甲醛灌注固定，采集脑组织制作石蜡切片，应用免疫组化法检测S100A6蛋白在大鼠脑组织海马及皮层中的分布情况。S100A6蛋白阳性结果的判断标准：以细胞浆内出现棕黄染色颗粒为阳性，反之为阴性。将各时间点脑组织切片高倍镜图片用图像处理软件计算阳性细胞的灰度值，实验数据用均数±标准差（x±SD）表示，采用在各损伤时间组与正常对照组间分别行两独立大样本均数间的u检验，P0.05为差异有统计学意义。 结果： 免疫组化结果显示大鼠脑组织中S100A6蛋白在皮层与海马组织细胞的核膜、胞质、细胞膜中均有表达，正常对照组的海马及皮层神经元细胞核膜、胞质中表现为棕色染色。与正常对照组相比：损伤后1h组皮层与海马细胞表达均最低（P0.01），表现为棕黄色淡染，随后表达量逐渐恢复，至损伤后7d、14d其表达量与阴性对照组比较差异无统计学意义（P0.05）。 结论： 在创伤性脑损伤后，大鼠海马、皮层组织中S100A6蛋白明显下降至最低值低，随着创伤后时间推移大鼠海马、皮层中S100A6蛋白表达逐渐回升，到伤后一周时与正常对照组比较差异无统计学意义。推测S100A6蛋白在TBI后与大量爆发的Ca2+结合而大量消耗，并共同与CacyBp结合进行信号转导，造成免疫组化表现为TBI后迅速降低。而后随着细胞凋亡及抗凋亡机制的启动，Ca2+爆发的恢复，以及S100A6蛋白的新的合成，S100A6表现为表达随时间回升。这样的先降低而后回升的规律，与临床上TBI患者意识深而后恢复，，认知功能降低而后回升的规律一致，提示脑组织中S100A6的变化与神经细胞的损伤与恢复有一定关系。为进一步深入研究S100A6的变化及其与TBI后细胞信号转导上下游分子的相互作用提供依据。也为临床上治疗TBI后Ca2+引起的神经细胞损伤提供新的干预位点，从而为进一步研究提供思路。
[Abstract]:Traumatic brain injury (TBI) is the most common in all kinds of traumatic injuries. After TBI, mild cognitive dysfunction, mild cognitive dysfunction and other neurological symptoms occurred in the patients with mild cognitive dysfunction. The patients frequently experienced neurological symptoms such as coma, paralysis, severe cognitive dysfunction and epilepsy, which seriously affected the survival and quality of life of the patients. For the pathophysiological changes of the nervous system after TBI, the main focus is on the final common pathway caused by various damage factors: cell necrosis induced by calcium ion burst in nerve cells. With the development of traumatic brain injury, it is recognized that nerve cells after TBI have not only necrosis but also apoptosis. Then the mechanism of apoptosis induced by calcium eruption after TBI became the new research direction. At present, there is still a lack of thorough understanding of the biochemical changes in nerve cells after TBI, so there is no breakthrough in the treatment time and method of patients with TBI. Some scholars studied the apoptosis and anti-apoptotic pathway of apoptosis induced by the combination of calcium and downstream molecules. It has been found that the S100 family protein of calcium-binding protein can be combined with calcium ions to further transduce signals with cyclin-binding protein CacyBp, thus suggesting possible intracellular signal transduction pathways that regulate apoptosis of cells after TBI: Ca ~ (2 +)/ S100/ CacyBp/ Thr-catenin. At present, there are no reports of the relationship between the amount of TBI and the change of cognitive function after neuronal damage at home and abroad. By establishing the rat TBI model, the authors designed the rat TBI model to investigate the changes of CYP1A6 molecule expression in brain tissues of rats after TBI. Objective: To explore the mechanism of nerve injury in traumatic brain injury and to establish a rat model of moderate brain injury. The aim of BI is to recognize the molecular mechanism of nerve cell damage development after TBI from the perspective of protein expression change, with a view to providing valuable theoretical basis and new intervention point for clinical diagnosis and treatment of traumatic brain injury through animal experiment. high clinical Methods: 72 healthy male SD rats were divided into TBI group (64 only) and normal control group (8 only) according to the random number table method. The following time is divided into: 1h, 3h, 6h, 12h, 24h, 72h, 7d, 14d in total of 8 subgroups, each group 8 rats were killed at each time point. Rats were killed at each time point and formalin fixed, paraffin sections were collected in brain tissue, and immunohistochemical method was applied to detect the brain tissue of rat brain tissue. Distribution in hippocampus and cortex. Criteria for determination of positive results of CYP1A6 protein: brown-yellow staining in cytoplasm The gray values of positive cells were calculated by image processing software for each time point brain tissue slice at high magnification, and the experimental data were expressed by standard deviation (x/ SD). u-test between the number of large samples, po. 05 is Results: The results showed that the expression of cyclin A6 protein in the cortex and hippocampal cells, the cytoplasm and the cell membrane were all expressed in the cortex and hippocampus of rats, and the hippocampus and cortex of the normal control group were observed. Compared with the normal control group, the expression of the cortex and the hippocampal cells was lowest (P0.01). The expression of the cell nucleus membrane and the hippocampal cells was lowest (P0.01). The expression level was gradually restored, and the expression level and the negative control group were observed after the injury. comparative difference Conclusion: In traumatic brain injury, the expression of cyclin A6 protein in hippocampus and cortex of rats was obviously reduced to the lowest value. It was not statistically significant to compare with the normal control group at one week after injury. The result was that the AA6 protein was consumed by the combination of Ca2 + with a large number of bursts after TBI, and was combined with CacyBp. With the initiation of cell apoptosis and anti-apoptosis mechanism, the recovery of Ca 2 + burst, and the egg yolk A6 egg. The new synthesis of white, the expression of cyclin A6 showed up with time. In this way, the regularity of the back-up was decreased and then the consciousness of clinical TBI patients was deep and then recovered, the cognitive function decreased and then the rule of recovery was consistent, suggesting that S1 in brain tissue There is a certain relationship between the change of 00A6 and the damage and recovery of nerve cells. In order to further study the changes of A6A6 and its relationship with T The interaction between the upstream and downstream molecules of the BI post-cell signal transduction provides a basis for clinical treatment of the nerve cells induced by Ca2 + after TBI.
【学位授予单位】：延安大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R651.15
本文编号：2308148
[Abstract]:Traumatic brain injury (TBI) is the most common in all kinds of traumatic injuries. After TBI, mild cognitive dysfunction, mild cognitive dysfunction and other neurological symptoms occurred in the patients with mild cognitive dysfunction. The patients frequently experienced neurological symptoms such as coma, paralysis, severe cognitive dysfunction and epilepsy, which seriously affected the survival and quality of life of the patients. For the pathophysiological changes of the nervous system after TBI, the main focus is on the final common pathway caused by various damage factors: cell necrosis induced by calcium ion burst in nerve cells. With the development of traumatic brain injury, it is recognized that nerve cells after TBI have not only necrosis but also apoptosis. Then the mechanism of apoptosis induced by calcium eruption after TBI became the new research direction. At present, there is still a lack of thorough understanding of the biochemical changes in nerve cells after TBI, so there is no breakthrough in the treatment time and method of patients with TBI. Some scholars studied the apoptosis and anti-apoptotic pathway of apoptosis induced by the combination of calcium and downstream molecules. It has been found that the S100 family protein of calcium-binding protein can be combined with calcium ions to further transduce signals with cyclin-binding protein CacyBp, thus suggesting possible intracellular signal transduction pathways that regulate apoptosis of cells after TBI: Ca ~ (2 +)/ S100/ CacyBp/ Thr-catenin. At present, there are no reports of the relationship between the amount of TBI and the change of cognitive function after neuronal damage at home and abroad. By establishing the rat TBI model, the authors designed the rat TBI model to investigate the changes of CYP1A6 molecule expression in brain tissues of rats after TBI. Objective: To explore the mechanism of nerve injury in traumatic brain injury and to establish a rat model of moderate brain injury. The aim of BI is to recognize the molecular mechanism of nerve cell damage development after TBI from the perspective of protein expression change, with a view to providing valuable theoretical basis and new intervention point for clinical diagnosis and treatment of traumatic brain injury through animal experiment. high clinical Methods: 72 healthy male SD rats were divided into TBI group (64 only) and normal control group (8 only) according to the random number table method. The following time is divided into: 1h, 3h, 6h, 12h, 24h, 72h, 7d, 14d in total of 8 subgroups, each group 8 rats were killed at each time point. Rats were killed at each time point and formalin fixed, paraffin sections were collected in brain tissue, and immunohistochemical method was applied to detect the brain tissue of rat brain tissue. Distribution in hippocampus and cortex. Criteria for determination of positive results of CYP1A6 protein: brown-yellow staining in cytoplasm The gray values of positive cells were calculated by image processing software for each time point brain tissue slice at high magnification, and the experimental data were expressed by standard deviation (x/ SD). u-test between the number of large samples, po. 05 is Results: The results showed that the expression of cyclin A6 protein in the cortex and hippocampal cells, the cytoplasm and the cell membrane were all expressed in the cortex and hippocampus of rats, and the hippocampus and cortex of the normal control group were observed. Compared with the normal control group, the expression of the cortex and the hippocampal cells was lowest (P0.01). The expression of the cell nucleus membrane and the hippocampal cells was lowest (P0.01). The expression level was gradually restored, and the expression level and the negative control group were observed after the injury. comparative difference Conclusion: In traumatic brain injury, the expression of cyclin A6 protein in hippocampus and cortex of rats was obviously reduced to the lowest value. It was not statistically significant to compare with the normal control group at one week after injury. The result was that the AA6 protein was consumed by the combination of Ca2 + with a large number of bursts after TBI, and was combined with CacyBp. With the initiation of cell apoptosis and anti-apoptosis mechanism, the recovery of Ca 2 + burst, and the egg yolk A6 egg. The new synthesis of white, the expression of cyclin A6 showed up with time. In this way, the regularity of the back-up was decreased and then the consciousness of clinical TBI patients was deep and then recovered, the cognitive function decreased and then the rule of recovery was consistent, suggesting that S1 in brain tissue There is a certain relationship between the change of 00A6 and the damage and recovery of nerve cells. In order to further study the changes of A6A6 and its relationship with T The interaction between the upstream and downstream molecules of the BI post-cell signal transduction provides a basis for clinical treatment of the nerve cells induced by Ca2 + after TBI.
【学位授予单位】：延安大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R651.15
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2 贺晓生,易声禹,章翔,费舟,张剑宁,梁景文,杨利孙;大鼠头颅瞬间旋转致脑弥漫轴索损伤的形态学观察与机理探讨[J];中华神经外科杂志;1998年01期
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