本文選題：腦缺血 + 細(xì)胞骨架蛋白�。� 參考：《鄭州大學(xué)》2014年碩士論文

【摘要】：背景和目的 目前，人類預(yù)期壽命延長，，老齡化進(jìn)展提速，再加上人們生活水平提高后膳食組成結(jié)構(gòu)的改變和體育鍛煉的缺乏，使得缺血性腦血管病的發(fā)病率和患病率逐年增高，嚴(yán)重危害人們身心健康，因此研究和防治腦缺血已成為目前緊要任務(wù)。腦缺血區(qū)分缺血中心區(qū)及其周圍的缺血半暗帶區(qū)域，而缺血半暗帶區(qū)內(nèi)神經(jīng)元的損傷具有可逆性，因此促進(jìn)該區(qū)神經(jīng)元的可塑性有利于損傷后神經(jīng)元的修復(fù)，由于腦內(nèi)神經(jīng)細(xì)胞的存活及功能狀態(tài)與細(xì)胞骨架蛋白的動態(tài)變化有著密切關(guān)系，且細(xì)胞骨架蛋白可促進(jìn)神經(jīng)元的生長發(fā)育及其可塑性，因此研究如何增加細(xì)胞骨架蛋白在神經(jīng)修復(fù)中的作用，改善患者預(yù)后，具有重要的意義。 Sonic Hedgehog（Shh)信號轉(zhuǎn)導(dǎo)通路是hedgehog信號轉(zhuǎn)導(dǎo)途徑的主要的信號通路之一，近年研究發(fā)現(xiàn)Shh信號通路與細(xì)胞骨架蛋白重塑有密切關(guān)系。Shh信號通路的激活可促進(jìn)細(xì)胞骨架蛋白表達(dá)含量的增加，并增加其穩(wěn)定性。微管是細(xì)胞骨架體系中主要成分之一，α-tubulin是組成微管的重要組成成分。神經(jīng)系統(tǒng)中，微管蛋白不光是神經(jīng)細(xì)胞發(fā)生的標(biāo)志蛋白，亦是受損神經(jīng)元結(jié)構(gòu)和功能恢復(fù)的關(guān)鍵蛋白。微管結(jié)合蛋白-2（MAP-2）也是構(gòu)成細(xì)胞骨架系統(tǒng)的主要成分之一，是神經(jīng)元中的標(biāo)志性微管相關(guān)蛋白，與神經(jīng)元的再生、凋亡及可塑性有關(guān)。然而目前有關(guān)Sonic Hedgehog信號通路對腦卒中的研究在血管再生、氧化應(yīng)激方面有所涉及，而對細(xì)胞骨架蛋白的研究鮮見。 Shh信號通路主要由Shh、Ptch、Smo和Gli蛋白組成，其中Gli-1蛋白是Shh信號通路主要的下游靶基因，Shh不足時，Gli-1表達(dá)降低，Shh激活時，Gli-1表達(dá)增加，因此Gli-1常常作為檢測Shh信號通路激活的標(biāo)志性分子。Purmorphamine可與Shh信號通路中的Smo蛋白結(jié)合，并使Smo蛋白空間構(gòu)象發(fā)生相應(yīng)改變繼而激活Shh信號通路的下游分子，是Shh信號通路的特異性激動劑。本實(shí)驗(yàn)采用大鼠急性大腦中動腦脈缺血模型，觀察應(yīng)用purmorphamine激活Shh信號通路后缺血大鼠腦組織神經(jīng)元內(nèi)α-tubulin、MAP-2的表達(dá)水平變化，以探討Shh信號通路的激活對永久性腦缺血大鼠細(xì)胞骨架蛋白的影響，為缺血性腦血管病的治療提供新的線索。 材料與方法 選取健康成年雄性清潔級SD大鼠96只，體重220±30g，采用線栓法制作SD大鼠急性大腦中動脈腦缺血（MCAO）模型。將96只SD大鼠隨機(jī)分為三組：即假手術(shù)組、缺血組、藥物干預(yù)組，各組再分為1d、3d、7d、14d四個亞組，每個亞組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。藥物干預(yù)組大鼠在模型制作完畢后按0.69mg/Kg的劑量給予purmorphamine溶液腹腔注射，假手術(shù)組及缺血組大鼠則在術(shù)后給予相應(yīng)劑量的單純DMF溶液（0.69mg/Kg）應(yīng)用相同的方式注射。行使手術(shù)后的大鼠清醒后，對其認(rèn)真觀察神經(jīng)功能缺損狀況并進(jìn)行神經(jīng)行為學(xué)評分，模型成功者于既定時間點(diǎn)將其處死，根據(jù)實(shí)驗(yàn)要求用相應(yīng)方法取出腦組織。本實(shí)驗(yàn)用免疫組織化學(xué)方法測定腦缺血后不同時間點(diǎn)各組大鼠腦組織α-tubulin的表達(dá)，用RT-PCR法測定不同時間點(diǎn)各組大鼠MAP-2、Gli-1的表達(dá)。 數(shù)據(jù)分析 采用SPSS21.0對數(shù)據(jù)進(jìn)行統(tǒng)計學(xué)處理，實(shí)驗(yàn)結(jié)果用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示。組間比較用單因素方差分析，兩兩比較用LSD檢驗(yàn)，檢驗(yàn)水準(zhǔn)取α=0.05。 結(jié)果 1.免疫組化法測定α-tubulin表達(dá)情況α-tubulin陽性表達(dá)反應(yīng)主要位于神經(jīng)元的胞質(zhì)及胞核中，也可表達(dá)于膠質(zhì)細(xì)胞及軸突。假手術(shù)組各時間點(diǎn)α-tubulin陽性反應(yīng)較強(qiáng)，缺血組第1d、3d下降較為明顯，隨著時間延長，陽性表達(dá)逐漸增加，7d、14d表達(dá)趨于穩(wěn)定。藥物干預(yù)組與缺血組相比，各時間點(diǎn)α-tubulin的表達(dá)均明顯增多，差異具有統(tǒng)計學(xué)意義。 2.用RT-PCR法測定MAP-2表達(dá)情況MAP-2的表達(dá)主要位于神經(jīng)元胞體和樹突。與假手術(shù)組相比，缺血組與藥物干預(yù)組MAP-2的表達(dá)都較低（p0.05）。缺血組MAP-2在缺血后表達(dá)降低，第1d、3d表達(dá)較低，隨缺血時間的延長，表達(dá)量增加，7d、14d表達(dá)增加趨于穩(wěn)定。給藥后，在缺血各時間點(diǎn)的表達(dá)與缺血組相比有明顯差異（p0.05））。 3.用RT-PCR法測定Gli-1表達(dá)情況假手術(shù)組腦組織內(nèi)因Shh信號通路未激活，Gli-1的表達(dá)水平很低，與假手術(shù)組比較，缺血組和藥物干預(yù)組Gli-1的表達(dá)增高，差異具有統(tǒng)計學(xué)意義（p0.05）。缺血組和藥物干預(yù)組Gli-1的表達(dá)在1d最高，之后有所下降，但表達(dá)量仍然較高。藥物干預(yù)組與缺血組各時間點(diǎn)相比，藥物干預(yù)組Gli-1的表達(dá)明顯增高，差異有統(tǒng)計學(xué)意義（p0.05）。 結(jié)論 1.應(yīng)用purmorphamine可有效促進(jìn)Shh信號通路中下游分子Gli-1的表達(dá)； 2.激活Shh信號通路可促進(jìn)缺血后腦組織內(nèi)α-tubulin、MAP-2表達(dá)增加，這可能是其減輕腦缺血后神經(jīng)損傷的機(jī)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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.3

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本文編號：1819693