甲状腺激素对大鼠脑缺血再灌注损伤后NGF和BDNF表达的影响

发布时间：2018-04-27 21:47

本文选题：脑缺血再灌注损伤 + 甲状腺激素　；参考：《郑州大学》2017年硕士论文

【摘要】：研究背景缺血性脑血管病是一类以缺血性损伤为主要临床表现的疾病,多见于中老年人,其致残率和死亡率较高,是威胁人类健康的主要疾病之一。因而找到一种可以改善缺血性损伤的治疗方法或者药物显得尤为重要。神经生长因子(NGF)和脑源性神经营养因子(BDNF)属于神经营养因子家族成员,对神经元生长发育、存活、分化均有重要作用;当脑缺血时,多种神经营养因子被激活,进一步催化下游信号分子,促进神经元再生及修复。抗凋亡蛋白Bcl-2和促凋亡蛋白Bax是两个重要的凋亡调控基因。在脑缺血后,凋亡相关基因被激活,Bax表达量增加,而Bcl-2表达量下降,同时激活大量细胞因子,引发神经元凋亡。甲状腺激素(T3)是在神经系统发育成熟过程中至关重要,同时其在成年及老年人的神经系统中亦至关重要,且可维持神经系统的兴奋性。研究发现,脑缺血后,甲状腺激素水平低下可能损害认知功能。但是其是否能够促进缺血性脑血管病的神经功能恢复及机制并无报道。因此,本研究通过建立大鼠大脑中动脉栓塞模型,观察甲状腺激素对缺血后NGF及BDNF,以及凋亡相关蛋白Bcl-2及Bax的影响,探讨甲状腺激素的对脑缺血再灌注损伤的作用及可能机制。目的观察甲状腺激素(T3)对大鼠大脑中动脉栓塞(MCAO)缺血再灌注损伤后NGF及BDNF的影响,并观察其对凋亡相关分子Bax、Bcl-2的影响,探讨甲状腺激素的神经保护作用及机制。方法1.SD大鼠随机分为4组,每组8只,分别为假手术组(Sham 1);假手术+T3组(Sham 2);缺血再灌注组(IR);缺血再灌注+T3组(T3)。应用线栓法制备大鼠大脑中动脉栓塞(MCAO)缺血再灌注模型,于缺血后1小时及再灌注后6小时给予腹腔注射甲状腺激素(T3)10μg/100g,生理盐水为安慰剂对照。术后24 h观察不同组别大鼠的神经功能评分情况,并进行TTC染色观察其梗死体积变化情况。2.收集不同组别大鼠缺血侧大脑皮质,通过RT-PCR检测脑源性神经营养因子NGF及BDNF的mRNA水平变化。3.收集不同组别大鼠缺血侧大脑皮质,通过Western Blot检测脑源性神经营养因子NGF及BDNF的蛋白表达变化。4.收集不同组别大鼠缺血侧大脑皮质,通过Western Blot检测抗凋亡蛋白Bcl-2及促凋亡蛋白Bax的蛋白表达变化。结果1.T3对脑缺血再灌注大鼠神经功能评分的影响缺血再灌注后24小时,Longa评分可见,假手术组及假手术+T3组均为0分,无神经功能缺损;而缺血再灌注组大鼠Longa评分(2.71±0.56),明显高于假手术及假手术+T3组(n=8,P0.05),且大鼠可见明显偏瘫样改变,躯体向偏瘫侧倾斜;给予T3处理的大鼠,其Longa评分(1.63±0.58),较缺血再灌注组减低(n=8,P0.05),同时偏瘫样改变亦有所缓解。2.T3对脑缺血再灌注大鼠脑组织梗死体积的影响缺血再灌注后24小时,计算梗死体积百分比可见,假手术组及假手术+T3组未见梗死灶,梗死体积百分比为0;而缺血再灌注组大鼠可见明显梗死灶,梗死体积百分比(45.12±2.32),明显高于假手术及假手术+T3组(n=8,P0.05);给予T3处理的大鼠,其梗死体积百分比(26.36±1.04),较缺血再灌注组减低(n=8,P0.05)。3.T3对脑缺血再灌注大鼠脑组织中NGF和BDNF mRNA水平的影响缺血再灌注后24小时,通过RT-PCR检测缺血侧大脑皮质NGF和BDNF mRNA的水平变化,可见,与假手术组及假手术+T3组相比,缺血再灌注组大鼠的NGF和BDNF mRNA水平均升高(n=8,P0.05);给予T3处理的大鼠,其NGF和BDNF mRNA水平较缺血再灌注大鼠进一步升高(n=8,P0.05)。4.T3对脑缺血再灌注大鼠脑组织中NGF和BDNF蛋白表达水平的影响缺血再灌注后24小时,通过Western Blot检测缺血侧大脑皮质NGF和BDNF蛋白水平变化,可见,与假手术组及假手术+T3组相比,缺血再灌注组大鼠的NGF和BDNF蛋白水平均升高(n=8,P0.05);给予T3处理的大鼠,其NGF和BDNF mRNA水平较缺血再灌注大鼠进一步升高(n=8,P0.05)。5.T3对脑缺血再灌注大鼠脑组织中Bcl-2和Bax表达水平的影响缺血再灌注后24小时,通过Western Blot检测缺血侧大脑皮质抗凋亡蛋白Bcl-2和促凋亡蛋白Bax水平变化,可见,与假手术组及假手术+T3组相比,缺血再灌注组大鼠的Bcl-2蛋白水平明显降低(n=8,P0.05);给予T3处理的大鼠,其Bcl-2水平较缺血再灌注大鼠升高(n=8,P0.05)。同时,与假手术组及假手术+T3组相比,缺血再灌注组大鼠的Bax蛋白水平明显升高(n=8,P0.05);给予T3处理的大鼠,其Bax水平较缺血再灌注大鼠降低(n=8,P0.05)。结论1.甲状腺激素T3能明显改善缺血再灌注损伤大鼠的神经功能障碍,且减少其梗死体积百分比,说明T3对脑缺血再灌注损伤大鼠具有保护及修复作用。2.甲状腺激素T3可使缺血再灌注损伤大鼠的NGF和BDNF的mRNA和蛋白水平均升高,促进神经元再生和修复。3.甲状腺激素T3可增加缺血再灌注损伤大鼠的抗凋亡蛋白Bcl-2,并降低促凋亡蛋白Bax的表达水平,从而抑制神经元的凋亡,促进神经功能的恢复。
[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.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743

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