【摘要】：背景及目的：近年來許多的動物實驗發(fā)現(xiàn)重復(fù)經(jīng)顱磁刺激（Repetitive Transcranial Magnetic Stimulation，rTMS）治療缺血性腦損傷，能促進(jìn)受損神經(jīng)細(xì)胞軸突再生、改善神經(jīng)軸突重塑的作用、促進(jìn)內(nèi)源性神經(jīng)細(xì)胞遷移并最終改善神經(jīng)損傷后的功能恢復(fù)，但具體機(jī)制尚不明確。本實驗?zāi)康?：體外培養(yǎng)原代神經(jīng)元細(xì)胞，建立神經(jīng)細(xì)胞缺氧缺糖（Oxygen/Glucose Deprivation）模型，體外模擬腦缺血缺氧，應(yīng)用MTT方法檢測磁刺激對缺血缺氧神經(jīng)細(xì)胞存活率的影響，應(yīng)用LDH（乳酸脫氫酶）的釋放量檢測磁刺激對缺血神經(jīng)細(xì)胞有無損傷，免疫細(xì)胞化學(xué)方法及RT-PCR方法檢測磁刺激缺血神經(jīng)元后Robo2和RhoA的表達(dá)變化，離體研究磁刺激對大鼠缺血缺氧神經(jīng)元的軸突再生、細(xì)胞活性和RhoA信號通路活動的影響，探討其可能機(jī)制。2：體外培養(yǎng)原代神經(jīng)元細(xì)胞，transwell小室體外模擬內(nèi)源性神經(jīng)元細(xì)胞的遷移，觀察磁刺激對神經(jīng)元細(xì)胞遷移的影響。 方法(第一部分)： 1.選取無菌分離15-18天胎齡SD胎鼠，，原代培養(yǎng)大鼠皮質(zhì)神經(jīng)元細(xì)胞。 2.取體外培養(yǎng)第6天SD胎鼠皮質(zhì)神經(jīng)元細(xì)胞免疫細(xì)胞化學(xué)方法鑒定。 3.原代皮質(zhì)神經(jīng)元接種培養(yǎng)板中，分組：(1)正常組(control，C)；(2)缺氧缺糖(Oxygen/Glucose Deprivation,OGD)組；(3)假刺激(shame，S)+缺氧缺糖組；(4)40%最大刺激強(qiáng)度(40% of maximum intensity of stimulation，M1)+缺氧缺糖組；(5)60%最大刺激強(qiáng)度(60% of maximum intensity ofstimulation，M2)+缺氧缺糖組。 4.于第6d MTT檢測細(xì)胞存活率，LDH釋放量檢測細(xì)胞損傷程度，免疫細(xì)胞化方法檢測各組細(xì)胞Robo2、RhoA的陽性表達(dá)，應(yīng)用RT-PCR檢測各組Robo2、RhoA表達(dá)量的變化。 5.細(xì)胞接種后第2d開始磁刺激，每天于固定時間刺激，3串/d，100次/串，每串間隔時間1min，連續(xù)刺激5d，頻率1HZ。 方法(第二部分)： 1.選取無菌分離15-18天胎齡SD胎鼠，原代培養(yǎng)大鼠皮質(zhì)神經(jīng)元細(xì)胞。 2.取體外培養(yǎng)第6天SD胎鼠皮質(zhì)神經(jīng)元細(xì)胞免疫細(xì)胞化學(xué)方法鑒定。 3.原代皮質(zhì)神經(jīng)元接種于transwell小室，分組：正常組、假刺激組、40%最大刺激強(qiáng)度組、60%最大刺激強(qiáng)度組；于第6d結(jié)晶紫染色遷移出的細(xì)胞。 4.細(xì)胞接種后第2d開始磁刺激，每天于固定時間刺激，3串/d，100次/串，每串間隔時間1min，連續(xù)刺激5d，頻率1HZ。 結(jié)果(第一部分)： 1.皮質(zhì)神經(jīng)元細(xì)胞離體培養(yǎng)6天，神經(jīng)細(xì)胞清晰可見，細(xì)胞貼壁良好，胞體較大，呈圓形、多極形，胞體折光性強(qiáng)，神經(jīng)突起相互聯(lián)絡(luò)成網(wǎng)。用NeuN（神經(jīng)元特異核蛋白）免疫細(xì)胞化學(xué)染色作分類計數(shù)，陽性神經(jīng)細(xì)胞約占(93.7土5.5)%。 2.鏡下觀察缺氧缺糖組細(xì)胞：OGD 30 min內(nèi)，神經(jīng)細(xì)胞形態(tài)較正常組無明顯變化；OGD 60min時，部分細(xì)胞皺縮、胞膜崩解、折光性差，LDH的釋放量明顯增加(P0.05)。 3.缺氧缺糖再灌注損傷后，OGD模型組的神經(jīng)細(xì)胞存活率較對照組顯著降低(P0.05)，而Robo2、RhoA的表達(dá)及細(xì)胞LDH釋放量較對照組顯著升高(P0.05)。假刺激組的細(xì)胞存活率、LDH釋放量、Robo2及RhoA的表達(dá)較OGD/R模型組無明顯變化(P0.05)，40%最大強(qiáng)度刺激組、60%最大強(qiáng)度刺激組的細(xì)胞存活率、Robo2的表達(dá)較OGD/R模型組明顯增加(P0.05)，40%最大強(qiáng)度刺激組、60%最大強(qiáng)度刺激組的LDH釋放量、RhoA的表達(dá)較OGD組明顯下降(P0.05)。 結(jié)果(第二部分): 結(jié)晶紫染色transwell小室遷移出的細(xì)胞，結(jié)果顯示：胞體、胞核均呈紫色，正常組、假刺激組遷移出的細(xì)胞數(shù)量無明顯差別(P0.05)，40%最大強(qiáng)度刺激組、60%最大強(qiáng)度刺激組較正常組及假刺激組遷移出的細(xì)胞數(shù)量明顯增加(P0.05)。 結(jié)論： (l)磁刺激能提高缺氧缺糖神經(jīng)元的存活率，降低缺氧缺糖神經(jīng)元LDH的釋放，對缺氧缺糖神經(jīng)元有直接的神經(jīng)保護(hù)作用。(2)高強(qiáng)低頻磁刺激通過促進(jìn)Robo2表達(dá)的上調(diào),而促進(jìn)受損神經(jīng)元軸突再生。(3)高強(qiáng)低頻磁刺激能抑制受損神經(jīng)元RhoA的表達(dá)，可能是磁刺激促進(jìn)軸突再生的機(jī)制之一。(4)高強(qiáng)低頻磁刺激能促進(jìn)體外神經(jīng)細(xì)胞遷移。
[Abstract]:BACKGROUND & OBJECTIVE: In recent years, many animal experiments have found that repeated transcranial magnetic stimulation (rTMS) is used to treat ischemic brain injury, and can promote the axonal regeneration of damaged nerve cells and improve the role of nerve axon remodeling. The mechanism of promoting the migration of endogenous nerve cells and ultimately improving the function of nerve injury is not clear. Objective: To study the effects of magnetic stimulation on the survival rate of hypoxic-ischemic neurons in cultured primary neuronal cells in vitro, in order to establish an oxygen-deficient (Oxygen/ Gluctose Department) model of nerve cells, to simulate the cerebral ischemia and hypoxia in vitro, and to use the MTT method to detect the effect of magnetic stimulation on the survival rate of the ischemic and hypoxic cells. The effects of magnetic stimulation on the expression of Robo2 and RhoA following the magnetic stimulation were detected by the release of LDH (lactate dehydrogenase), and the changes of the expression of Robo2 and RhoA were detected by the method of immunocytochemistry and RT-PCR. The effect of magnetic stimulation on the cell migration of the neurons was observed in vitro, and the effect of magnetic stimulation on the migration of the neurons was observed. Method (Part 1): 1. Select sterile separation of 15-18 days of fetal-age SD rat, and primary culture of the cortical neurons of the rat Cell. 2. Immunocytochemical method for the in vitro culture of the 6-day SD rat cortical neuron cell Identification. 3. Primary cortical neurons were inoculated into a culture plate with a group of (1) a normal group (control, C); (2) a hypoxia-deficient (OGD) group; (3) a sham, S, and a hypoxia-deficient group; (4) a maximum intensity of stimulation of 40% (40% of maximum intensity of stimulat ion,M1)+緙烘哀緙虹硸緇勶紱(5)60%鏈

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