本文選題：血管性癡呆 + 祛風通竅方��； 參考：《西南醫(yī)科大學》2017年碩士論文

【摘要】：目的:通過觀察祛風通竅方對血管性癡呆(VD)大鼠定位航行、空間探索能力,海馬CA1區(qū)神經(jīng)元細胞形態(tài),ChAT、Tau、Aβ、VEGF蛋白表達,IL-1β、IL-6、TNF-α濃度的影響,探討祛風通竅方對血管性癡呆模型大鼠學習記憶能力的保護作用機制。方法:1.分組:水迷宮篩選符合實驗要求大鼠,隨機分為假手術組、尼莫地平組、模型組、祛風通竅方高、中、低劑量組,每組大鼠各10只。2.給藥方法:高、中、低劑量組分別給予祛風通竅方26.8g/kg/d、13.4g/kg/d、6.7g/kg/d灌胃(按照等體積3ml的標準計算高、中、低劑量組大鼠灌胃藥物的濃度)。模型組、假手術組予以3ml生理鹽水灌胃。尼莫地平組予以尼莫地平混懸液6.25mg/kg/d灌胃。每天上午灌胃一次,連續(xù)30天。3.取材方法:配置戊巴比妥鈉濃度1%,對實驗大鼠進行腹腔麻醉。大鼠仰臥位固定于消毒實驗臺,開胸取血,離心,取上清液,-20℃冰箱保存,用于ELISA檢測IL-1β、IL-6、TNF-α的濃度。采集血液后,一部分大鼠迅速斷頭取出新鮮腦組織,將腦組織置于EP管中保存,用于半定量PCR及Western Blot檢測VEGF蛋白。另一部分大鼠進行灌注取腦,用于HE染色和免疫組化檢測ChAT、Tau、Aβ蛋白。4.觀察指標:Morris水迷宮檢測大鼠定位航行、空間探索能力;HE染色光鏡觀察大鼠海馬CA1區(qū)神經(jīng)元細胞形態(tài);免疫組化檢測ChAT、Tau、Aβ蛋白表達;ELISA法檢測IL-6、IL-1β、TNF-α濃度;半定量PCR法測定VEGFmRNA表達;Western Blot法檢測VEGF蛋白表達。結果:1.行為學:與假手術組比較,高、中、低劑量組、模型組、尼莫地平組大鼠EL延長,第i象限游泳的時間縮短,經(jīng)過原平臺范圍的次數(shù)減少,差異具有統(tǒng)計學意義(p0.05),高劑量組大鼠el隨著訓練時間延長而縮短,從第四天開始接近于假手術組。與模型組比較,高、中、低劑量組、尼莫地平組大鼠的el隨著訓練時間延長而縮短,第i象限游泳的時間延長,經(jīng)過原平臺范圍的次數(shù)增加,差異具有統(tǒng)計學意義(p0.05)。與尼莫地平組比較,低劑量組el隨著訓練時間延長而縮短,第i象限游泳的時間延長,經(jīng)過原平臺范圍的次數(shù)增加,但差異無統(tǒng)計學意義(p0.05);高、中劑量組大鼠el隨著訓練時間延長而縮短,第i象限游泳的時間延長,經(jīng)過原平臺范圍的次數(shù)增加,差異具有統(tǒng)計學意義(p0.05)。其中,以高劑量組大鼠行為學改善更為明顯,與中劑量組比較,差異具有統(tǒng)計學意義(p0.05)。2.he染色:同倍率光鏡下觀察,假手術組海馬ca1區(qū)神經(jīng)元細胞排列整齊,胞質(zhì)均質(zhì)紅染,胞核大而圓,核仁清晰明顯。模型組海馬ca1區(qū)神經(jīng)細胞排列紊亂,數(shù)量減少,細胞輪廓、胞核模糊,部分甚至看不到細胞。高、中劑量組神經(jīng)細胞排列比較規(guī)則,細胞輪廓、核膜與核仁比較清晰,且高劑量組神經(jīng)細胞形態(tài)優(yōu)于中劑量組,更為接近正常形態(tài)。低劑量組和尼莫地平組的神經(jīng)元細胞形態(tài)介于模型組與中劑量組之間。3.chat、tau、aβ蛋白檢測:chat、tau、aβ陽性細胞均表現(xiàn)為胞漿內(nèi)呈現(xiàn)棕黃色的沉淀物。(1)chat蛋白:與假手術組比較,模型組、各給藥組chat蛋白mod值有所降低(p0.05)。與模型組比較,各給藥組chat蛋白mod值有所升高(p0.05)。與尼莫地平組比較,低劑量組chat蛋白mod值升高,但差異無統(tǒng)計學意義(p0.05);高、中劑量組chat蛋白mod值有所升高,差異有統(tǒng)計學意義(p0.05)。其中,以高劑量組升高最為明顯,與中劑量比較,差異有統(tǒng)計學意義(p0.05)(2)tau蛋白:與假手術組比較,模型組、各給藥組tau蛋白mod值有所升高(p0.05)。與模型組比較,各給藥組tau蛋白mod值降低(p0.05)。與尼莫地平組比較,低劑量組tau蛋白mod值降低,但差異無統(tǒng)計學意義(p0.05);高、中劑量組tau蛋白mod值有所降低,差異有統(tǒng)計學意義(p0.05)。其中,以高劑量組降低最為明顯,與中劑量組比較,差異有統(tǒng)計學意義(p0.05)。(3)aβ蛋白:與假手術組比較,模型組、各給藥組aβ蛋白mod值有所升高(p0.05)。與模型組比較,各給藥組aβ蛋白mod值有所降低(p0.05)。與尼莫地平組比較,低劑量組aβ蛋白mod值降低,但差異無統(tǒng)計學意義(p0.05);高、中劑量組aβ蛋白mod值有所降低,差異有統(tǒng)計學意義(p0.05)。其中,以高劑量組降低最為明顯,與中劑量組比較,差異有統(tǒng)計學意義(p0.05)。4.il-6、il-1β、tnf-α檢測:與假手術組比較,模型組、各給藥組il-6、il-1β、tnf-α濃度有所升高(p0.05)。與模型組比較,各給藥組il-6、il-1β、tnf-α濃度降低(p0.05)。與尼莫地平組比較,高、中、低劑量組il-6、il-1β、tnf-α濃度有所降低(p0.05)。其中,以高劑量組降低最為明顯,與中劑量組比較,差異具有統(tǒng)計學意義。5.vegfmrna表達檢測:與假手術組相比,模型組、各給藥組vegfmrna表達有所增加(p0.05)。與模型組比較,各給藥組vegfmrna表達有所增加(p0.05)。與尼莫地平組比較,低劑量組vegfmrna表達增加,但差異無統(tǒng)計學意義(p0.05);高、中劑量組vegfmrna表達有所增加,差異有統(tǒng)計學意義(p0.05)。其中,以高劑量組增加最為明顯,與中劑量組比較,差異有統(tǒng)計學意義(p0.05)。6.vegf蛋白表達檢測:與假手術組相比,模型組、各給藥組vegf蛋白表達有所增加(p0.05)。與模型組比較,各給藥組vegf蛋白表達有所增加(P0.05)。與尼莫地平組比較,低劑量組VEGF蛋白表達增加,但差異無統(tǒng)計學意義(P0.05);高、中劑量組VEGF蛋白表達有所增加,差異有統(tǒng)計學意義(P0.05)。其中,以高劑量組增加最為明顯,與中劑量組比較,差異有統(tǒng)計學意義(P0.05)。結論:1.血管性癡呆大鼠存在學習記憶能力的下降;海馬CA1區(qū)神經(jīng)元細胞形態(tài)紊亂,ChAT蛋白表達降低,Tau、Aβ蛋白表達升高,IL-6、IL-1β、TNF-α的濃度升高,VEGF表達增加。2.祛風通竅方能有效改善VD大鼠的行為學,保護VD大鼠海馬CA1區(qū)神經(jīng)元細胞形態(tài)。3.祛風通竅方對血管性癡呆模型大鼠的作用機制可能與以下有關:(1)上調(diào)VD大鼠ChAT的表達,改善膽堿能系統(tǒng)的功能;(2)抑制VD大鼠腦內(nèi)Tau蛋白過度磷酸化,降低Aβ蛋白表達,減少神經(jīng)元的損害;(3)降低VD大鼠血清中炎性細胞因子IL-6、IL-1β、TNF-α的濃度,從而抑制VD大鼠缺血區(qū)的炎性反應;(4)能刺激VEGF的表達,從而促進缺血時血管的新生,修復缺血損傷的神經(jīng)。4.祛風通竅方改善血管性癡呆大鼠學習記憶能力存在一定量效關系。
[Abstract]:Objective: To observe the effect of dispel Tong Qiao Decoction on the location of vascular dementia (VD) rats, space exploration ability, neuron cell morphology in hippocampus CA1 region, ChAT, Tau, A beta, VEGF protein expression, IL-1 beta, IL-6, and TNF- alpha concentration, and explore the mechanism of protective effect of dispelling wind and Tong Fang on learning and memory ability of vascular idiot model rats. Methods: 1. groups: The water maze screening rats were randomly divided into sham operation group, nimodipine group, model group, high, low dose group, and 10.2. methods in each group: high, middle, and low dose groups were given 26.8g/kg/d, 13.4g/kg/d, and 6.7g/kg/d respectively (high, medium and low dosage according to the standard of equal volume 3ml. The model group, the model group, the sham operation group was given 3ml physiological saline for gastric perfusion. Nimodipine group was treated with nimodipine suspension 6.25mg/kg/d. One time every morning, 30 days of continuous.3. extraction method: the concentration of pentobarbital sodium was 1%, the experimental rats were anesthetized with abdominal cavity. The supine position of rats was fixed at the disinfectant test table. Blood, centrifuge, centrifuge, take the supernatant, save the supernatant, -20 C refrigerator and used for ELISA to detect the concentration of IL-1 beta, IL-6, TNF- alpha. After collecting the blood, some rats quickly cut out the fresh brain tissue and put the brain tissue in the EP tube and used for the semi quantitative PCR and Western Blot detection VEGF protein. Another part of the rats was perfused to take the brain for HE dyeing and in the HE staining. Immunohistochemistry test ChAT, Tau, A beta protein.4. observation index: Morris water maze test rats navigation, space exploration ability; HE staining light microscope observation of the hippocampus CA1 region neuron cell morphology; immunohistochemistry detection ChAT, Tau, A beta protein expression; ELISA method for IL-6, beta, alpha concentration; semi quantitative determination method The OT method was used to detect the expression of VEGF protein. Results: 1. behavior: compared with the sham group, the EL in the high, middle, low dose group, the model group, the nimodipine group was prolonged, the time of the swimming in the I quadrant shortened, the difference was statistically significant (P0.05) after the frequency of the original platform range (P0.05), and the El in the high dose group shortened with the training time, from fourth. Compared with the model group, the El in the high, middle and low dose group, the rats in Nimodipine group shortened with the time of training, the time of swimming in the I quadrant was prolonged, and the difference was statistically significant (P0.05). Compared with the nimodipine group, the low dose group El was prolonged with the training time. The duration of swimming in the I quadrant was lengthened, and the number of swimming in the original platform increased, but the difference was not statistically significant (P0.05). The El in the middle dose group shortened with the training time, the time of the I quadrant was prolonged, and the difference was statistically significant (P0.05) through the number of the original platform range (P0.05). The behavior improvement of rats was more obvious. Compared with the middle dose group, the difference was statistically significant (P0.05).2.he staining: under the same rate light microscope, the neuron cells in the hippocampal CA1 area of the sham operation group were arranged neatly, the cytoplasm homogeneous red dye, the nucleus large and round, the nucleolus clear and obvious. The neurons in the hippocampus CA1 area were arranged in disorder, and the number of cells decreased. The outline, the nucleus of the cell was blurred, and the cells were not even visible. The nerve cells in the middle dose group were arranged more regularly, the cell outline, the nuclear membrane and the nucleolus were clearer, and the morphology of the nerve cells in the high dose group was better than the middle dose group, and it was closer to the normal form. The neurons in the low dose group and the nimodipine group were between the model group and the middle dose group. .3.chat, tau, a beta protein detection: chat, tau, a beta positive cells showed brown yellow sediment in the cytoplasm. (1) chat protein: compared with the sham group, the model group, the mod value of chat protein in each group decreased (P0.05). Compared with the model group, chat protein Mod value increased. The mod value of chat protein in the group increased, but the difference was not statistically significant (P0.05). The mod value of chat protein in the middle dose group was higher, the difference was statistically significant (P0.05). Among them, the high dose group was the most obvious, and the difference was statistically significant (P0.05) (2) tau protein: the comparison with the sham operation group, the model group, and the tau protein m in each administration group. The OD value increased (P0.05). Compared with the model group, the tau protein mod value of each group was reduced (P0.05). Compared with the nimodipine group, the tau protein mod value of the low dose group decreased, but the difference was not statistically significant (P0.05); the value of tau protein mod value in the middle dose group decreased, and the difference was statistically significant (P0.05). Compared with the middle dose group, the difference was statistically significant (P0.05). (3) a beta protein: compared with the sham group, the mod value of a beta protein in the model group was higher (P0.05). Compared with the model group, the A beta protein mod value decreased (P0.05). Compared with the nimodipine group, the A beta protein mod value decreased in the low dose group, but the difference was not statistically significant (p0.). 05); high, middle dose group A beta protein mod value decreased, the difference was statistically significant (P0.05), among which, the most obvious reduction in high dose group, compared with the middle dose group, the difference was statistically significant (P0.05).4.il-6, IL-1 beta, tnf- alpha detection: compared with the sham operation group, the model group, the concentration of IL-6, IL-1 beta, tnf- alpha in each administration group increased (P0.05). And model Compared with the nimodipine group, the concentration of IL-6, IL-1 beta, and tnf- a decreased (P0.05) in the high, middle and low dose groups compared with the nimodipine group (P0.05). Compared with the high dose group, the concentration of IL-6, IL-1 beta and tnf- alpha was lower (P0.05). Compared with the middle dose group, the difference was statistically significant.5.vegfmrna expression detection: compared with the sham operation group, the model group was compared with the sham group, and the model group was compared with the sham group. The expression of VEGFmRNA in the drug group increased (P0.05). Compared with the model group, the expression of VEGFmRNA in each group increased (P0.05). Compared with the nimodipine group, the expression of VEGFmRNA in the low dose group increased, but the difference was not statistically significant (P0.05); the expression of VEGFmRNA in the middle dose group increased, and the difference was statistically significant (P0.05). Among them, the high dose group was in the high dose group. The increase was most obvious, compared with the middle dose group, the difference was statistically significant (P0.05).6.vegf protein expression detection: compared with the sham group, the expression of VEGF protein in the model group was increased (P0.05). Compared with the model group, the expression of VEGF protein in each group was increased (P0.05). The expression of VEGF protein in the low dose group was compared with the nimodipine group and the expression of VEGF protein in the low dose group. The difference was not statistically significant (P0.05), and the expression of VEGF protein in the middle dose group increased, and the difference was statistically significant (P0.05). Among them, the increase in the high dose group was the most obvious, and the difference was statistically significant (P0.05) compared with the middle dose group (P0.05). Conclusion: 1. the decrease of learning and memory ability in the rats with vascular dementia and the neurons in the hippocampus CA1 area neurons. The cell morphologic disorder, the expression of ChAT protein, the increase of Tau, A beta protein expression, the increase of IL-6, IL-1 beta, TNF- alpha, the increase of VEGF expression to improve the behavior of VD rats, and protect the mechanism of the neuronal cell morphology of the CA1 region of the hippocampus of VD rats to the mechanism of vascular dementia model rats may be the following. (1) up regulating the expression of ChAT in VD rats and improving the function of the cholinergic system; (2) inhibiting the excessive phosphorylation of Tau protein in the brain of VD rats, reducing the expression of A beta protein and reducing the damage of neurons; (3) reducing the concentration of inflammatory cytokines IL-6, IL-1 beta and TNF- alpha in serum of VD rats, thus inhibiting the inflammatory response in the ischemic region of VD rats; (4) stimulates VEGF. The expression of.4. can promote the regeneration of blood vessels during ischemia, and repair the ischemic injury of the nerve to improve the learning and memory ability of vascular dementia rats.
【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285.5;R-332

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