本文選題：腦缺血再灌注損傷 + 甲狀腺激素��； 參考：《鄭州大學》2017年碩士論文

【摘要】：研究背景缺血性腦血管病是一類以缺血性損傷為主要臨床表現(xiàn)的疾病,多見于中老年人,其致殘率和死亡率較高,是威脅人類健康的主要疾病之一。因而找到一種可以改善缺血性損傷的治療方法或者藥物顯得尤為重要。神經(jīng)生長因子(NGF)和腦源性神經(jīng)營養(yǎng)因子(BDNF)屬于神經(jīng)營養(yǎng)因子家族成員,對神經(jīng)元生長發(fā)育、存活、分化均有重要作用;當腦缺血時,多種神經(jīng)營養(yǎng)因子被激活,進一步催化下游信號分子,促進神經(jīng)元再生及修復�？沟蛲龅鞍譈cl-2和促凋亡蛋白Bax是兩個重要的凋亡調(diào)控基因。在腦缺血后,凋亡相關基因被激活,Bax表達量增加,而Bcl-2表達量下降,同時激活大量細胞因子,引發(fā)神經(jīng)元凋亡。甲狀腺激素(T3)是在神經(jīng)系統(tǒng)發(fā)育成熟過程中至關重要,同時其在成年及老年人的神經(jīng)系統(tǒng)中亦至關重要,且可維持神經(jīng)系統(tǒng)的興奮性。研究發(fā)現(xiàn),腦缺血后,甲狀腺激素水平低下可能損害認知功能。但是其是否能夠促進缺血性腦血管病的神經(jīng)功能恢復及機制并無報道。因此,本研究通過建立大鼠大腦中動脈栓塞模型,觀察甲狀腺激素對缺血后NGF及BDNF,以及凋亡相關蛋白Bcl-2及Bax的影響,探討甲狀腺激素的對腦缺血再灌注損傷的作用及可能機制。目的觀察甲狀腺激素(T3)對大鼠大腦中動脈栓塞(MCAO)缺血再灌注損傷后NGF及BDNF的影響,并觀察其對凋亡相關分子Bax、Bcl-2的影響,探討甲狀腺激素的神經(jīng)保護作用及機制。方法1.SD大鼠隨機分為4組,每組8只,分別為假手術組(Sham 1);假手術+T3組(Sham 2);缺血再灌注組(IR);缺血再灌注+T3組(T3)。應用線栓法制備大鼠大腦中動脈栓塞(MCAO)缺血再灌注模型,于缺血后1小時及再灌注后6小時給予腹腔注射甲狀腺激素(T3)10μg/100g,生理鹽水為安慰劑對照。術后24 h觀察不同組別大鼠的神經(jīng)功能評分情況,并進行TTC染色觀察其梗死體積變化情況。2.收集不同組別大鼠缺血側大腦皮質,通過RT-PCR檢測腦源性神經(jīng)營養(yǎng)因子NGF及BDNF的mRNA水平變化。3.收集不同組別大鼠缺血側大腦皮質,通過Western Blot檢測腦源性神經(jīng)營養(yǎng)因子NGF及BDNF的蛋白表達變化。4.收集不同組別大鼠缺血側大腦皮質,通過Western Blot檢測抗凋亡蛋白Bcl-2及促凋亡蛋白Bax的蛋白表達變化。結果1.T3對腦缺血再灌注大鼠神經(jīng)功能評分的影響缺血再灌注后24小時,Longa評分可見,假手術組及假手術+T3組均為0分,無神經(jīng)功能缺損;而缺血再灌注組大鼠Longa評分(2.71±0.56),明顯高于假手術及假手術+T3組(n=8,P0.05),且大鼠可見明顯偏癱樣改變,軀體向偏癱側傾斜;給予T3處理的大鼠,其Longa評分(1.63±0.58),較缺血再灌注組減低(n=8,P0.05),同時偏癱樣改變亦有所緩解。2.T3對腦缺血再灌注大鼠腦組織梗死體積的影響缺血再灌注后24小時,計算梗死體積百分比可見,假手術組及假手術+T3組未見梗死灶,梗死體積百分比為0;而缺血再灌注組大鼠可見明顯梗死灶,梗死體積百分比(45.12±2.32),明顯高于假手術及假手術+T3組(n=8,P0.05);給予T3處理的大鼠,其梗死體積百分比(26.36±1.04),較缺血再灌注組減低(n=8,P0.05)。3.T3對腦缺血再灌注大鼠腦組織中NGF和BDNF mRNA水平的影響缺血再灌注后24小時,通過RT-PCR檢測缺血側大腦皮質NGF和BDNF mRNA的水平變化,可見,與假手術組及假手術+T3組相比,缺血再灌注組大鼠的NGF和BDNF mRNA水平均升高(n=8,P0.05);給予T3處理的大鼠,其NGF和BDNF mRNA水平較缺血再灌注大鼠進一步升高(n=8,P0.05)。4.T3對腦缺血再灌注大鼠腦組織中NGF和BDNF蛋白表達水平的影響缺血再灌注后24小時,通過Western Blot檢測缺血側大腦皮質NGF和BDNF蛋白水平變化,可見,與假手術組及假手術+T3組相比,缺血再灌注組大鼠的NGF和BDNF蛋白水平均升高(n=8,P0.05);給予T3處理的大鼠,其NGF和BDNF mRNA水平較缺血再灌注大鼠進一步升高(n=8,P0.05)。5.T3對腦缺血再灌注大鼠腦組織中Bcl-2和Bax表達水平的影響缺血再灌注后24小時,通過Western Blot檢測缺血側大腦皮質抗凋亡蛋白Bcl-2和促凋亡蛋白Bax水平變化,可見,與假手術組及假手術+T3組相比,缺血再灌注組大鼠的Bcl-2蛋白水平明顯降低(n=8,P0.05);給予T3處理的大鼠,其Bcl-2水平較缺血再灌注大鼠升高(n=8,P0.05)。同時,與假手術組及假手術+T3組相比,缺血再灌注組大鼠的Bax蛋白水平明顯升高(n=8,P0.05);給予T3處理的大鼠,其Bax水平較缺血再灌注大鼠降低(n=8,P0.05)。結論1.甲狀腺激素T3能明顯改善缺血再灌注損傷大鼠的神經(jīng)功能障礙,且減少其梗死體積百分比,說明T3對腦缺血再灌注損傷大鼠具有保護及修復作用。2.甲狀腺激素T3可使缺血再灌注損傷大鼠的NGF和BDNF的mRNA和蛋白水平均升高,促進神經(jīng)元再生和修復。3.甲狀腺激素T3可增加缺血再灌注損傷大鼠的抗凋亡蛋白Bcl-2,并降低促凋亡蛋白Bax的表達水平,從而抑制神經(jīng)元的凋亡,促進神經(jīng)功能的恢復。
[Abstract]:Background ischemic cerebrovascular disease (ischaemic cerebrovascular disease) is a kind of disease with ischemic injury as the main clinical manifestation. It is often seen in middle-aged and elderly people. The rate of disability and mortality is high, and it is one of the major diseases that threaten human health. Therefore, it is particularly important to find a therapeutic method or drug that can improve the ischemic injury. NG F) and brain derived neurotrophic factor (BDNF) are members of the neurotrophic factor family, and have important roles in the growth, development, survival and differentiation of neurons. When cerebral ischemia, a variety of neurotrophic factors are activated to further catalyze the downstream signal molecules and promote the rebirth and repair of neurons. Anti apoptotic protein Bcl-2 and apoptotic protein Bax are two. After cerebral ischemia, the apoptosis related genes are activated, the expression of Bax is increased, and the expression of Bcl-2 is decreased, and a large number of cytokines are activated and the neuronal apoptosis is activated. Thyroid hormone (T3) is crucial in the process of maturation of the nervous system, and it is also critical in the nervous system of adult and elderly people. The study found that hypothyroidism may damage cognitive function after cerebral ischemia. However, it is not reported that it can promote the recovery of neurologic function and the mechanism of ischemic cerebrovascular disease. Therefore, this study has established a rat model of middle cerebral artery embolism to observe the deficiency of thyroid hormone. The effect of NGF and BDNF after blood, as well as the effect of apoptosis related protein Bcl-2 and Bax, to explore the effect of thyroid hormone on cerebral ischemia reperfusion injury and its possible mechanism. Objective To observe the effect of thyroid hormone (T3) on NGF and BDNF after cerebral artery embolism (MCAO) ischemia reperfusion injury in rats and to observe the effect of thyroid hormone on Bax and Bcl-2 of apoptosis related molecules. To explore the neuroprotective effect and mechanism of thyroid hormone. Methods 1.SD rats were randomly divided into 4 groups, 8 rats in each group, including sham operation group (Sham 1), sham operation +T3 group (Sham 2), ischemia reperfusion group (IR) and ischemia reperfusion +T3 group (T3). The ischemia reperfusion model of middle cerebral artery embolism (MCAO) in rats was prepared by means of thread thrombus, 1 hours after ischemia. And 6 hours after reperfusion, the intraperitoneal injection of thyroid hormone (T3) was 10 g/100g, and the saline was placebo control. The neurological function score of different groups of rats was observed after 24 h, and the changes of infarct volume were observed by TTC staining and.2. was used to collect the cerebral cortex of the ischemic side of different groups of rats, and the brain derived operation was detected by RT-PCR. The mRNA level changes of nutrient factors NGF and BDNF collects the ischemic cerebral cortex of different groups of rats and the changes of the protein expression of NGF and BDNF of the brain derived neurotrophic factor by Western Blot to collect the ischemic cerebral cortex in different groups of rats, and detect the protein expression of anti apoptotic protein Bcl-2 and apoptotic protein through Western Blot. Results the effect of 1.T3 on the neurological function score of cerebral ischemia reperfusion rats was 24 hours after ischemia-reperfusion. The Longa score showed that the sham operation group and the sham operation +T3 group were 0 points and no nerve function defect, while the Longa score of the ischemia reperfusion group was (2.71 + 0.56), obviously higher than the sham operation and the sham operation +T3 group (n=8, P0.05), and the rats were visible. The change of hemiplegia, the body inclined to the hemiplegia side, the Longa score of the rats treated with T3 (1.63 + 0.58) was lower than that of the ischemia reperfusion group (n=8, P0.05), and the changes of hemiplegia also alleviated the effect of.2.T3 on the infarct volume of cerebral tissue in the cerebral ischemia reperfusion rats, 24 hours after the blood reperfusion, the percentage of infarct volume was calculated and the false hand was calculated. There was no infarct in group and sham +T3 group, the percentage of infarct volume was 0, but the infarct size was obvious in the ischemia reperfusion group (45.12 + 2.32), which was significantly higher than that of sham operation and sham operation group +T3 (n=8, P0.05), and the percentage of death volume (26.36 + 1.04) of the rats treated with T3 was lower than that of the ischemia reperfusion group (n=8, P0.0). 5) the effect of.3.T3 on the level of NGF and BDNF mRNA in cerebral tissue of cerebral ischemia reperfusion rats was 24 hours after ischemia-reperfusion. The level of NGF and BDNF mRNA in the cerebral cortex of ischemic side was detected by RT-PCR, and the NGF and BDNF mRNA levels of the rats in the ischemia reperfusion group were higher than those in the sham operation group and the sham operation group. The level of NGF and BDNF mRNA in the rats treated with ischemia and reperfusion was further increased (n=8, P0.05).4.T3 on the expression of NGF and BDNF protein in the brain tissue of cerebral ischemia reperfusion rats, 24 hours after ischemia reperfusion, and the changes of NGF and BDNF protein levels in the cerebral cortex of ischemic side were detected by Western Blot, and it was seen with the sham operation group and the sham operation group. The levels of NGF and BDNF protein in the rats of the +T3 group were increased (n=8, P0.05), and the mRNA level of NGF and BDNF in the rats treated with T3 was higher than that of the ischemia-reperfusion rats (n=8, P0.05). The effect of.5.T3 on the expression level of the cerebral ischemia reperfusion rats was 24 hours after ischemia reperfusion. Western Blot detected the changes in the level of anti apoptotic protein Bcl-2 and apoptotic protein Bax in the cerebral cortex of the ischemic side. Compared with the sham operation group and the sham operation +T3 group, the Bcl-2 protein level in the ischemia reperfusion group was significantly lower (n=8, P0.05), and the Bcl-2 level of the rats treated with T3 was higher than that of the ischemia-reperfusion rats (n=8, P0.05). Meanwhile, the level of Bcl-2 was higher than that of the ischemia reperfusion rats (n=8, P0.05). Compared with the sham operation group and the sham operation +T3 group, the Bax protein level in the ischemia-reperfusion group was significantly increased (n=8, P0.05), and the Bax level of the rats treated with T3 was lower than that of the ischemia reperfusion rats (n=8, P0.05). Conclusion 1. thyroid hormone T3 can obviously improve the nerve dysfunction in the rats with ischemia reperfusion injury and reduce the infarct volume. T3 has protective and repair effects on cerebral ischemia reperfusion injury in rats..2. thyroid hormone T3 can increase the level of mRNA and protein of NGF and BDNF in rats with ischemia-reperfusion injury, and the regeneration of neurons and the repair of.3. thyroid hormone T3 can increase the anti apoptotic protein Bcl-2 in rats with ischemia-reperfusion injury and decrease the apoptosis of the rats. The expression level of dead protein Bax inhibits neuronal apoptosis and promotes the recovery of neurological function.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743

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