SHH的激活对永久性脑缺血大鼠细胞骨架蛋白α-tubulin、MAP-2表达的影响

发布时间：2018-04-29 11:17

本文选题：脑缺血 + 细胞骨架蛋白　；参考：《郑州大学》2014年硕士论文

【摘要】：背景和目的目前，人类预期寿命延长，，老龄化进展提速，再加上人们生活水平提高后膳食组成结构的改变和体育锻炼的缺乏，使得缺血性脑血管病的发病率和患病率逐年增高，严重危害人们身心健康，因此研究和防治脑缺血已成为目前紧要任务。脑缺血区分缺血中心区及其周围的缺血半暗带区域，而缺血半暗带区内神经元的损伤具有可逆性，因此促进该区神经元的可塑性有利于损伤后神经元的修复，由于脑内神经细胞的存活及功能状态与细胞骨架蛋白的动态变化有着密切关系，且细胞骨架蛋白可促进神经元的生长发育及其可塑性，因此研究如何增加细胞骨架蛋白在神经修复中的作用，改善患者预后，具有重要的意义。 Sonic Hedgehog（Shh)信号转导通路是hedgehog信号转导途径的主要的信号通路之一，近年研究发现Shh信号通路与细胞骨架蛋白重塑有密切关系。Shh信号通路的激活可促进细胞骨架蛋白表达含量的增加，并增加其稳定性。微管是细胞骨架体系中主要成分之一，α-tubulin是组成微管的重要组成成分。神经系统中，微管蛋白不光是神经细胞发生的标志蛋白，亦是受损神经元结构和功能恢复的关键蛋白。微管结合蛋白-2（MAP-2）也是构成细胞骨架系统的主要成分之一，是神经元中的标志性微管相关蛋白，与神经元的再生、凋亡及可塑性有关。然而目前有关Sonic Hedgehog信号通路对脑卒中的研究在血管再生、氧化应激方面有所涉及，而对细胞骨架蛋白的研究鲜见。 Shh信号通路主要由Shh、Ptch、Smo和Gli蛋白组成，其中Gli-1蛋白是Shh信号通路主要的下游靶基因，Shh不足时，Gli-1表达降低，Shh激活时，Gli-1表达增加，因此Gli-1常常作为检测Shh信号通路激活的标志性分子。Purmorphamine可与Shh信号通路中的Smo蛋白结合，并使Smo蛋白空间构象发生相应改变继而激活Shh信号通路的下游分子，是Shh信号通路的特异性激动剂。本实验采用大鼠急性大脑中动脑脉缺血模型，观察应用purmorphamine激活Shh信号通路后缺血大鼠脑组织神经元内α-tubulin、MAP-2的表达水平变化，以探讨Shh信号通路的激活对永久性脑缺血大鼠细胞骨架蛋白的影响，为缺血性脑血管病的治疗提供新的线索。材料与方法选取健康成年雄性清洁级SD大鼠96只，体重220±30g，采用线栓法制作SD大鼠急性大脑中动脉脑缺血（MCAO）模型。将96只SD大鼠随机分为三组：即假手术组、缺血组、药物干预组，各组再分为1d、3d、7d、14d四个亚组，每个亚组8只大鼠。Purmorphamine在使用之前用二甲基甲酰胺（DMF）溶液使之完全溶解。药物干预组大鼠在模型制作完毕后按0.69mg/Kg的剂量给予purmorphamine溶液腹腔注射，假手术组及缺血组大鼠则在术后给予相应剂量的单纯DMF溶液（0.69mg/Kg）应用相同的方式注射。行使手术后的大鼠清醒后，对其认真观察神经功能缺损状况并进行神经行为学评分，模型成功者于既定时间点将其处死，根据实验要求用相应方法取出脑组织。本实验用免疫组织化学方法测定脑缺血后不同时间点各组大鼠脑组织α-tubulin的表达，用RT-PCR法测定不同时间点各组大鼠MAP-2、Gli-1的表达。数据分析采用SPSS21.0对数据进行统计学处理，实验结果用均数±标准差(x±s)表示。组间比较用单因素方差分析，两两比较用LSD检验，检验水准取α=0.05。结果 1.免疫组化法测定α-tubulin表达情况α-tubulin阳性表达反应主要位于神经元的胞质及胞核中，也可表达于胶质细胞及轴突。假手术组各时间点α-tubulin阳性反应较强，缺血组第1d、3d下降较为明显，随着时间延长，阳性表达逐渐增加，7d、14d表达趋于稳定。药物干预组与缺血组相比，各时间点α-tubulin的表达均明显增多，差异具有统计学意义。 2.用RT-PCR法测定MAP-2表达情况MAP-2的表达主要位于神经元胞体和树突。与假手术组相比，缺血组与药物干预组MAP-2的表达都较低（p0.05）。缺血组MAP-2在缺血后表达降低，第1d、3d表达较低，随缺血时间的延长，表达量增加，7d、14d表达增加趋于稳定。给药后，在缺血各时间点的表达与缺血组相比有明显差异（p0.05））。 3.用RT-PCR法测定Gli-1表达情况假手术组脑组织内因Shh信号通路未激活，Gli-1的表达水平很低，与假手术组比较，缺血组和药物干预组Gli-1的表达增高，差异具有统计学意义（p0.05）。缺血组和药物干预组Gli-1的表达在1d最高，之后有所下降，但表达量仍然较高。药物干预组与缺血组各时间点相比，药物干预组Gli-1的表达明显增高，差异有统计学意义（p0.05）。结论 1.应用purmorphamine可有效促进Shh信号通路中下游分子Gli-1的表达； 2.激活Shh信号通路可促进缺血后脑组织内α-tubulin、MAP-2表达增加，这可能是其减轻脑缺血后神经损伤的机制之一。
[Abstract]:Background and purpose
At present, the prolongation of life expectancy, the increase of the progress of aging, the change of the structure of dietary composition and the lack of physical exercise after the improvement of people's living standards make the incidence and morbidity of ischemic cerebrovascular disease increase year by year, which seriously endangers the physical and mental health of the people. Therefore, the research and prevention of cerebral ischemia has become the urgent task at present. Ischemia distinguishes the ischemic center from the ischemic penumbra region and its surrounding area, and the damage of neurons in the ischemic half dark zone is reversible, so promoting the plasticity of neurons in this area is beneficial to the repair of neurons after injury. The survival and function of neurons in the brain are closely related to the dynamic changes of the cytoskeleton protein. It is of great significance to study how to increase the role of cytoskeleton protein in the repair of nerve and to improve the prognosis of the patients.
Sonic Hedgehog (Shh) signal transduction pathway is one of the main signaling pathways of hedgehog signal transduction pathway. In recent years, it is found that the Shh signaling pathway is closely related to the remodeling of cytoskeleton protein, and the activation of.Shh signaling pathway can promote the increase of cytoskeleton protein expression and increase its stability. Microtubule is a cytoskeleton system. One of the main components, alpha -tubulin is an important component of the microtubule. In the nervous system, microtubulin is not only a marker for the occurrence of nerve cells, but also a key protein for the structure and function recovery of damaged neurons. Microtubule binding protein -2 (MAP-2) is also one of the main components of the bone frame system of the cells. It is a marker in the neuron. Sexual microtubule related proteins are related to the regeneration, apoptosis and plasticity of neurons. However, the study of Sonic Hedgehog signaling pathway is involved in vascular regeneration and oxidative stress, and the study of cytoskeleton protein is rare.
The Shh signaling pathway is mainly composed of Shh, Ptch, Smo and Gli protein, and Gli-1 protein is the main downstream target gene of Shh signaling pathway. When Shh is insufficient, Gli-1 expression is reduced and Gli-1 expression increases when Shh activates. Therefore Gli-1 often acts as a marker to detect the activation of the signaling pathway. It is a specific agonist for the Shh signaling pathway, which changes the spatial conformation of the Smo protein and then activates the downstream molecules of the Shh signaling pathway and is a specific agonist in the Shh signaling pathway. In this experiment, the rat cerebral ischemia model in the acute brain was used to observe the expression of alpha -tubulin and MAP-2 in the neurons of the cerebral tissue of the ischemic rat after the purmorphamine activation of the Shh signaling pathway. The effect of the activation of Shh signaling pathway on the cytoskeleton of permanent cerebral ischemia rats was studied to provide a new clue for the treatment of ischemic cerebrovascular disease.
Materials and methods
96 healthy adult male clean SD rats and weight 220 + 30g were selected to make SD rat model of acute middle cerebral artery cerebral ischemia (MCAO) by thread thrombus. 96 SD rats were randomly divided into three groups: sham operation group, ischemia group, and drug intervention group, each group was divided into 1D, 3D, 7d, 14d four subgroups, and 8 rats in each subgroup were used in.Purmorphamine. Two methylformamide (DMF) solution was used to completely dissolve it. The rats in the drug intervention group were given the purmorphamine solution intraperitoneally at the dosage of 0.69mg/Kg after the model was finished. The sham operation group and the ischemic group were given the same dosage of the same dose of DMF solution (0.69mg/ Kg) in the same way after the operation. After the rat was awake, the nerve function defect was carefully observed and the neurobehavioral score was carried out. The model success was executed at a given time. The brain tissue was removed with the corresponding method according to the experimental requirements. The expression of alpha -tubulin in the brain tissue of each group of rats after cerebral ischemia was measured by immunohistochemical method, and RT-PC was used to determine the expression of the brain tissue. R method was used to detect the expression of MAP-2 and Gli-1 in different groups of rats at different time points.
Data analysis
The data were statistically processed with SPSS21.0, and the experimental results were expressed with mean standard deviation (x + s). A single factor analysis of variance was used for comparison between groups. 22 was compared with LSD test, and a level of alpha =0.05. was tested.
Result
1. the positive expression of alpha -tubulin expression in the expression of alpha -tubulin was mainly located in the cytoplasm and nucleus of neurons, but also expressed in glial cells and axons. The positive reaction of alpha -tubulin at each time point in sham operation group was stronger. The decrease of 1D and 3D in the ischemic group was more obvious. The positive expression gradually increased with the prolongation of time, and the expression of 7D, 14d expression gradually. The expression of -tubulin in the drug intervention group was significantly higher than that in the ischemic group at different time points, and the difference was statistically significant.
2. the expression of MAP-2 expression of MAP-2 was mainly located in the cell body and dendrite by RT-PCR method. Compared with the sham group, the expression of MAP-2 in the ischemic group and the drug intervention group was lower (P0.05). The expression of MAP-2 in the ischemic group decreased after ischemia, the expression of 1D, 3D was lower, and the expression increased with the prolongation of the ischemia time, and the expression of 7D and 14d tended to be stable. After administration, there was a significant difference in the expression at different time points in ischemia group compared with ischemia group (P0.05).
3. RT-PCR method was used to determine the expression of Gli-1 in the sham operation group. The Shh signal pathway was not activated and the expression level of Gli-1 was very low. Compared with the sham operation group, the expression of Gli-1 in the ischemic group and the drug intervention group was higher, the difference was statistically significant (P0.05). The expression of Gli-1 in the ischemic group and the drug dry group was the highest, but then decreased, but the table decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the table was decreased, but the Compared with the ischemic group, the expression of Gli-1 in the drug intervention group was significantly higher than that in the ischemic group at different time points (P0.05).
conclusion
1. the application of purmorphamine can effectively promote the expression of Gli-1 in the downstream of Shh signaling pathway.
2. activation of Shh signaling pathway can increase the expression of -tubulin and MAP-2 in brain tissue after ischemia, which may be one of the mechanisms to alleviate nerve injury after cerebral ischemia.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

【参考文献】