【摘要】：創(chuàng)傷性腦損傷（traumatic brain injury TBI）在各類創(chuàng)傷性損傷中最為常見，多與其他各種創(chuàng)傷合并存在，以高死亡率、高致殘率而位居各類創(chuàng)傷之首。TBI后患者輕者出現(xiàn)一過性意識障礙、輕度認知功能障礙等神經(jīng)系統(tǒng)癥狀，重者常出現(xiàn)昏迷、癱瘓、嚴重認知功能障礙、癲癇等神經(jīng)系統(tǒng)癥狀，嚴重影響患者的生存及生活質(zhì)量。對于TBI后神經(jīng)系統(tǒng)的病理生理改變，主要的熱點集中在各種損傷因素導致的最終共同通路：神經(jīng)細胞內(nèi)鈣離子爆發(fā)引發(fā)的細胞壞死。隨著對創(chuàng)傷性腦損傷的研究的不斷深入，人們認識到TBI后神經(jīng)細胞不僅存在壞死，而且存在凋亡。而后關(guān)于TBI后鈣爆發(fā)引起的細胞凋亡的機制成為新的研究方向。目前關(guān)于TBI后神經(jīng)細胞內(nèi)生化改變還缺乏全面深入的認識，因此臨床上對TBI后患者的治療時機、方法、療效至今沒有突破性的進展和定論。有學者從鈣爆發(fā)后與下游分子結(jié)合而引發(fā)的細胞凋亡與抗凋亡通路的角度進行了深入研究。有研究發(fā)現(xiàn)鈣結(jié)合蛋白S100家族蛋白可與鈣離子結(jié)合，進一步可與鈣周期素結(jié)合蛋白CacyBp結(jié)合進一步轉(zhuǎn)導信號，從而提出TBI后可能存在的調(diào)控細胞凋亡的細胞內(nèi)信號轉(zhuǎn)導通路：Ca~(2+)/S100/CacyBp/β-catenin（β-tubulin）。目前國內(nèi)外尚無TBI后S100A6量變及與神經(jīng)元損傷后認知功能的改變之間關(guān)系的報道。作者設(shè)計本實驗，通過建立大鼠TBI模型初步探討TBI后大鼠腦組織中S100A6分子表達變化的規(guī)律。 目的： 探尋創(chuàng)傷性腦損傷的神經(jīng)損傷機制，建立中度腦損傷大鼠模型，通過測定TBI后不同時間點大鼠腦組織中S100A6分子的表達，分析其規(guī)律及與TBI的關(guān)系，旨在從蛋白表達變化的角度認識TBI后神經(jīng)細胞損傷發(fā)展的分子機制，以期通過動物實驗為臨床診治創(chuàng)傷性腦損傷提供有價值的理論依據(jù)及新的干預(yù)點以提高臨床療效。 方法： 健康雄性SD大鼠72只，清潔等級，體重（220±20g），按照隨機數(shù)字表法分為TBI組（64只）和正常對照組（8只）；TBI模型的制備使用瞬間頭顱側(cè)向旋轉(zhuǎn)致傷法制作，按傷后時間分為：1h，3h，6h，12h，24h，72h，7d，14d共8個亞組，每組8只，各時間點處死大鼠。在各時間點處死大鼠并進行甲醛灌注固定，采集腦組織制作石蠟切片，應(yīng)用免疫組化法檢測S100A6蛋白在大鼠腦組織海馬及皮層中的分布情況。S100A6蛋白陽性結(jié)果的判斷標準：以細胞漿內(nèi)出現(xiàn)棕黃染色顆粒為陽性，反之為陰性。將各時間點腦組織切片高倍鏡圖片用圖像處理軟件計算陽性細胞的灰度值，實驗數(shù)據(jù)用均數(shù)±標準差（x±SD）表示，采用在各損傷時間組與正常對照組間分別行兩獨立大樣本均數(shù)間的u檢驗，P0.05為差異有統(tǒng)計學意義。 結(jié)果： 免疫組化結(jié)果顯示大鼠腦組織中S100A6蛋白在皮層與海馬組織細胞的核膜、胞質(zhì)、細胞膜中均有表達，正常對照組的海馬及皮層神經(jīng)元細胞核膜、胞質(zhì)中表現(xiàn)為棕色染色。與正常對照組相比：損傷后1h組皮層與海馬細胞表達均最低（P0.01），表現(xiàn)為棕黃色淡染，隨后表達量逐漸恢復(fù)，至損傷后7d、14d其表達量與陰性對照組比較差異無統(tǒng)計學意義（P0.05）。 結(jié)論： 在創(chuàng)傷性腦損傷后，大鼠海馬、皮層組織中S100A6蛋白明顯下降至最低值低，隨著創(chuàng)傷后時間推移大鼠海馬、皮層中S100A6蛋白表達逐漸回升，到傷后一周時與正常對照組比較差異無統(tǒng)計學意義。推測S100A6蛋白在TBI后與大量爆發(fā)的Ca2+結(jié)合而大量消耗，并共同與CacyBp結(jié)合進行信號轉(zhuǎn)導，造成免疫組化表現(xiàn)為TBI后迅速降低。而后隨著細胞凋亡及抗凋亡機制的啟動，Ca2+爆發(fā)的恢復(fù)，以及S100A6蛋白的新的合成，S100A6表現(xiàn)為表達隨時間回升。這樣的先降低而后回升的規(guī)律，與臨床上TBI患者意識深而后恢復(fù)，，認知功能降低而后回升的規(guī)律一致，提示腦組織中S100A6的變化與神經(jīng)細胞的損傷與恢復(fù)有一定關(guān)系。為進一步深入研究S100A6的變化及其與TBI后細胞信號轉(zhuǎn)導上下游分子的相互作用提供依據(jù)。也為臨床上治療TBI后Ca2+引起的神經(jīng)細胞損傷提供新的干預(yù)位點，從而為進一步研究提供思路。
[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.
【學位授予單位】：延安大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R651.15

【參考文獻】

相關(guān)期刊論文 前2條

1 賀曉生,易聲禹,章翔,費舟,張劍寧,梁景文,楊利孫;腦彌漫性軸索損傷軸索內(nèi)鈣超載及鈣拮抗劑的治療作用[J];中華神經(jīng)外科雜志;2000年01期

2 賀曉生,易聲禹,章翔,費舟,張劍寧,梁景文,楊利孫;大鼠頭顱瞬間旋轉(zhuǎn)致腦彌漫軸索損傷的形態(tài)學觀察與機理探討[J];中華神經(jīng)外科雜志;1998年01期

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