【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(shí)障礙、輕度認(rèn)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(yán)重認(rèn)知功能障礙、癲癇等神經(jīng)系統(tǒng)癥狀，嚴(yán)重影響患者的生存及生活質(zhì)量。對(duì)于TBI后神經(jīng)系統(tǒng)的病理生理改變，主要的熱點(diǎn)集中在各種損傷因素導(dǎo)致的最終共同通路：神經(jīng)細(xì)胞內(nèi)鈣離子爆發(fā)引發(fā)的細(xì)胞壞死。隨著對(duì)創(chuàng)傷性腦損傷的研究的不斷深入，人們認(rèn)識(shí)到TBI后神經(jīng)細(xì)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(xì)胞凋亡的機(jī)制成為新的研究方向。目前關(guān)于TBI后神經(jīng)細(xì)胞內(nèi)生化改變還缺乏全面深入的認(rèn)識(shí)，因此臨床上對(duì)TBI后患者的治療時(shí)機(jī)、方法、療效至今沒有突破性的進(jìn)展和定論。有學(xué)者從鈣爆發(fā)后與下游分子結(jié)合而引發(fā)的細(xì)胞凋亡與抗凋亡通路的角度進(jìn)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(jìn)一步可與鈣周期素結(jié)合蛋白CacyBp結(jié)合進(jìn)一步轉(zhuǎn)導(dǎo)信號(hào)，從而提出TBI后可能存在的調(diào)控細(xì)胞凋亡的細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)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(rèn)知功能的改變之間關(guān)系的報(bào)道。作者設(shè)計(jì)本實(shí)驗(yàn)，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(dá)變化的規(guī)律。 目的： 探尋創(chuàng)傷性腦損傷的神經(jīng)損傷機(jī)制，建立中度腦損傷大鼠模型，通過測定TBI后不同時(shí)間點(diǎn)大鼠腦組織中S100A6分子的表達(dá)，分析其規(guī)律及與TBI的關(guān)系，旨在從蛋白表達(dá)變化的角度認(rèn)識(shí)TBI后神經(jīng)細(xì)胞損傷發(fā)展的分子機(jī)制，以期通過動(dòng)物實(shí)驗(yàn)為臨床診治創(chuàng)傷性腦損傷提供有價(jià)值的理論依據(jù)及新的干預(yù)點(diǎn)以提高臨床療效。 方法： 健康雄性SD大鼠72只，清潔等級(jí)，體重（220±20g），按照隨機(jī)數(shù)字表法分為TBI組（64只）和正常對(duì)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(shí)間分為：1h，3h，6h，12h，24h，72h，7d，14d共8個(gè)亞組，每組8只，各時(shí)間點(diǎn)處死大鼠。在各時(shí)間點(diǎn)處死大鼠并進(jìn)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(biāo)準(zhǔn)：以細(xì)胞漿內(nèi)出現(xiàn)棕黃染色顆粒為陽性，反之為陰性。將各時(shí)間點(diǎn)腦組織切片高倍鏡圖片用圖像處理軟件計(jì)算陽性細(xì)胞的灰度值，實(shí)驗(yàn)數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差（x±SD）表示，采用在各損傷時(shí)間組與正常對(duì)照組間分別行兩獨(dú)立大樣本均數(shù)間的u檢驗(yàn)，P0.05為差異有統(tǒng)計(jì)學(xué)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(xì)胞的核膜、胞質(zhì)、細(xì)胞膜中均有表達(dá)，正常對(duì)照組的海馬及皮層神經(jīng)元細(xì)胞核膜、胞質(zhì)中表現(xiàn)為棕色染色。與正常對(duì)照組相比：損傷后1h組皮層與海馬細(xì)胞表達(dá)均最低（P0.01），表現(xiàn)為棕黃色淡染，隨后表達(dá)量逐漸恢復(fù)，至損傷后7d、14d其表達(dá)量與陰性對(duì)照組比較差異無統(tǒng)計(jì)學(xué)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(shí)間推移大鼠海馬、皮層中S100A6蛋白表達(dá)逐漸回升，到傷后一周時(shí)與正常對(duì)照組比較差異無統(tǒng)計(jì)學(xué)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(jìn)行信號(hào)轉(zhuǎn)導(dǎo)，造成免疫組化表現(xiàn)為TBI后迅速降低。而后隨著細(xì)胞凋亡及抗凋亡機(jī)制的啟動(dòng)，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(dá)隨時(shí)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(shí)深而后恢復(fù)，，認(rèn)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(xì)胞的損傷與恢復(fù)有一定關(guān)系。為進(jìn)一步深入研究S100A6的變化及其與TBI后細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)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(xì)胞損傷提供新的干預(yù)位點(diǎn)，從而為進(jìn)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(xué)位授予單位】：延安大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R651.15

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本文編號(hào)：2308148