【摘要】：目的： 腦血管疾病(cerebrovascular disease)是一類嚴重影響人類身體健康的疾病,在全球,腦血管疾病是致死和致殘的主要疾病之一,尤其是臨床上較常見的缺血性腦血管病(ischemic cerebrovascular disease, ICVD)。ICVD的主要發(fā)病機制主要包括能量耗竭、酸中毒,氧自由基產生、損害,一氧化氮和一氧化氮合成酶,鈣超載,炎癥因子,細胞凋亡調控因子,興奮性氨基酸(excitatory amino acid, EAA)。其中EAA是眾多發(fā)病機制中最為關鍵的一個,它能引起其他機制的產生。谷氨酸(glutamate, Glu)是中樞神經系統(tǒng)中含量最高的興奮性氨基酸,而胞外過多的Glu是會加重腦缺血損傷。據報告,補陽還五湯(Buyang Huanwu Decoction, BYHWD)能減少神經細胞外的谷氨酸濃度,而Glu的清除主要依賴于星形膠質細胞上谷氨酸轉運體-1(glutamate transporter-1, GLT-1或GLT1)和谷氨酰胺合成酶(glutamine synthetase, GS)。所以,本實驗主要運用免疫組織熒光化學和蛋白印跡western-blot等技術,探索補陽還五湯能否增加腦缺血后星形膠質細胞GLT1和GS蛋白的表達,從而減少細胞外谷氨酸的濃度；除此,本實驗還對其作用機制進行初步探索,希望能為補陽還五湯治療腦缺血提供更多的實驗依據。 方法： 1.補陽還五湯對局灶性腦缺血星形膠質細胞GLT1和GS表達的影響 健康成年的SD大鼠,采用大腦中動脈栓塞(middle cerebral artery occlusion, MCAO)法制備模型,手術結束大鼠清醒后,進行神經功能評分,評分合格的大鼠隨機分為模型組(1d、3d、5d、7d)和藥物組(1d、3d、5d、7d),各組大鼠再缺血2h再灌24h后給藥,其中模型組每次給予2ml的生理鹽水灌胃,2次/d,藥物組給予每次補陽還五湯(16g/kg)灌胃,2次/d。各組實驗結束后,將動物進行全身灌注,取出大腦制備冰凍切片,采用免疫組織化學染色法和免疫組織熒光化學染色法分別檢測各組GLT1和GS的表達,并用免疫熒光雙標法對GLT1和膠質細胞原纖維酸性蛋白(glial fibrillary acidic protein, GFAP)、GS和GFAP的表達進行檢測,顯微鏡下觀察并獲取圖片,用ImageJ進行圖片分析。 2.補陽還五湯影響局灶性腦缺血星形膠質細胞GLT1和GS表達的機制研究 健康成年的SD大鼠,隨機分為假手術組、模型組、補陽還五湯組(BYHWD)、補陽還五湯聯(lián)合垂體腺苷酸環(huán)化酶激活多肽拮抗劑(pituitary adenylate cyclase activating polypeptide (6-38), PACAP(6-38))組(BYHWD+PACAP(6-38)),每組18只。各組大鼠在缺血2h后尾靜脈注射2次藥物,分別為再灌前的15min和再灌后48h,其中假手術組、模型組和BYHWD組給予1ml生理鹽水,BYHWD+PACAP(6-38)給予PACAP(6-38)(1.49mmol/ml).此外,各組大鼠在缺血2h再灌24h后灌胃給藥,假手術組和模型組每次給予2ml的生理鹽水灌胃,2次/d, BYHWD組和BYHWD+PACAP(6-38)組給予補陽還五湯(16g/kg/次,2次/d),各組連續(xù)給藥3d。動物在最后一次灌胃給藥后12h內取材,其中每組抽取3只制備冰凍切片,采用免疫組織熒光化學法檢鋇PACAP38. GLT1、GS和GFAP的表達；剩下的動物麻醉脫臼處死,取出大腦,冰上分離缺血側大腦的海馬組織,采用western-blot蛋白印跡法檢鋇GLT1、GS和GFAP的表達,紫外分光光度法檢測谷氨酸濃度。 結果： 腦缺血后海馬CA1區(qū)GLT1和GS的表達都是先升高后下降,藥物組的兩種蛋白表達均比同時間點的模型組高,且具有差異(P0.01)；而給藥3d后的GLT1和GS表達明顯比其他給藥組高(P0.01)。 腦缺血后,BYHWD組大鼠海馬CA1區(qū)的PACAP表達明顯比模型組高(P0.01),而BYHWD+PACAP(6-38)組大鼠缺血側大腦海馬GLT1和GS蛋白表達比BYHWD組的弱；并且BYHWD+PACAP(6-38)組的谷氨酸濃度與BYHWD組的相比有所升高；此外,GFAP在BYHWD+PACAP(6-38)組的表達比BYHWD組強。 結論： 補陽還五湯能通過PACAP介導星形膠質細胞GLTl和GS表達,減少缺血后谷氨酸濃度,從而保護神經元。
[Abstract]:Objective:
Cerebrovascular disease (cvd) is a kind of disease that seriously affects human health. globally, CVD is one of the main diseases causing death and disability, especially ischemic cerebrovascular disease (ICVD). the main pathogenesis of icVD mainly includes energy depletion, acid depletion. Poisoning, oxygen free radical production, damage, nitric oxide and nitric oxide synthase, calcium overload, inflammatory factors, apoptosis regulators, excitatory amino acids (EAA). EAA is one of the most important pathogenesis, which can cause other mechanisms. Glutamate (Glu) is the central nervous system. Buyang Huanwu Decoction (BYHWD) has been reported to reduce the concentration of extracellular glutamate, and glutamate transporter-1 (GLT-1) or GLT-1 (glutamate transporter-1, GLT-1) is the most abundant excitatory amino acid in astrocytes. In this study, immunohistochemistry and Western-blot were used to explore whether Buyang Huanwu decoction could increase the expression of GLT1 and GS protein in astrocytes after cerebral ischemia, thereby reducing the concentration of extracellular glutamate. We hope to provide more experimental evidence for the treatment of cerebral ischemia by buyanghuan Five Decoction.
Method:
1. the effect of buyanghuanfive Decoction on the expression of GLT1 and GS in astrocytes after focal cerebral ischemia
Healthy adult SD rats were divided into model group (1d, 3d, 5d, 7d) and drug group (1d, 3d, 5d, 7d) by middle cerebral artery occlusion (MCAO). The rats in each group were given Buyang Huanwu Decoction (16g/kg) twice a day, and the rats in each group were given Buyang Huanwu Decoction (16g/kg) twice a day. The expression of GLT1 and glial fibrillary acidic protein (GFAP), GS and GFAP were detected by fluorescence double labeling. The pictures were observed under microscope and analyzed by ImageJ.
2. the mechanism of Buyang huanfive Decoction on the expression of GLT1 and GS in astrocytes after focal cerebral ischemia
Healthy adult SD rats were randomly divided into sham operation group, model group, BYHWD group, pituitary adenylate cyclase activating polypeptide (6-38), PACAP (6-38) group (BYHWD + PACAP (6-38), 18 rats in each group were injected into tail vein 2 hours after ischemia. The rats in sham operation group, model group and BYHWD group were given 1 ml normal saline, BYHWD + PACAP (6-38) were given PACAP (6-38) (1.49 mmol/ml). In addition, the rats in each group were given 2 ml normal saline after 2 h of ischemia and 24 h of reperfusion respectively. HWD+PACAP(6-38) group was given Buyang Huanwu Decoction(16g/kg/time,twice/day) for 3 days. Animals were taken within 12 hours after the last intragastric administration. Three of them were taken from each group to prepare frozen sections and the expression of barium PACAP38.GLT1,GS and GFAP were detected by immunohistochemistry. The expression of barium GLT1, GS and GFAP in hippocampus of ischemic brain was detected by Western blot and the concentration of glutamate was detected by ultraviolet spectrophotometry.
Result:
After cerebral ischemia, the expression of GLT1 and GS in CA1 area of hippocampus increased first and then decreased, and the expression of GLT1 and GS in drug group was higher than that in model group at the same time point (P 0.01).
After cerebral ischemia, the expression of PACAP in hippocampal CA1 region of BYHWD group was significantly higher than that of model group (P 0.01), while the expression of GLT1 and GS in hippocampus of BYHWD + PACAP (6-38) group was weaker than that of BYHWD group, and the concentration of glutamate in BYHWD + PACAP (6-38) group was higher than that in BYHWD + PACAP (6-38) group. Group YHWD is strong.
Conclusion:
Buyang Huanwu Decoction can protect neurons by reducing glutamate concentration after ischemia through PACAP-mediated GLTl and GS expression in astrocytes.
【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R277.7

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