本文選題：羥基紅花黃色素A + 腦缺血再灌注��； 參考：《云南中醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:本研究通過線栓法阻塞右側(cè)大腦中動脈構(gòu)建大鼠缺血再灌注模型,在術(shù)前和術(shù)后分別尾靜脈給予羥基紅花黃色素A(HSYA)干預(yù),通過神經(jīng)功能缺損評分、腦梗死體積變化、免疫組化及蛋白質(zhì)印跡法等方法,檢測HSP70表達來探索HSYA對大腦中動脈缺血再灌注的預(yù)防和治療作用、對腦缺血再灌注對抗凋亡蛋白HSP70表達的影響,并進一步探討HSYA對缺血再灌注大鼠神經(jīng)保護的相關(guān)分子機制,為HSYA在腦梗死的治療上提供理論依據(jù)。方法:1.動物分組:將70只體重250-300g成年雄性SD大鼠隨機分為7組(即正常組、假手術(shù)組、模型組、術(shù)前5mg/kg組、術(shù)前10mg/kg組、術(shù)后5mg/kg組、術(shù)后10mg/kg組,n=10。2.缺血再灌注模型建立:局灶性腦缺血再灌注模型采用尼龍絲線阻塞右側(cè)大腦中動脈,通過將線栓有圓頭的一端由頸總動脈插入到頸內(nèi)動脈,進而到達大腦中動脈的起始端,阻塞大腦中動脈。阻塞2h后拔出栓線,再灌注24h。假手術(shù)組操作與之相同,但不阻塞大腦中動脈。3.給藥:術(shù)前5mg/kg組、術(shù)前10mg/kg組組于術(shù)前30min分別按5mg/kg、10mg/kg的劑量給予HSYA尾靜脈注射;術(shù)后5mg/kg組、術(shù)后10mg/kg組于術(shù)后分別按5mg/kg、10mg/kg的劑量給予HSYA尾靜脈注射;假手術(shù)組與模型組給予生理鹽水尾靜脈注射。4.神經(jīng)功能缺損評估:麻醉清醒后2h,采用五分法進行神經(jīng)功能缺損評分。5.組織學(xué)評估:所有大鼠再灌注24h后處死迅速取腦,采用TTC染色,用Image J軟件進行腦梗面積的測量,進而算出腦梗死比積;用免疫組化和蛋白質(zhì)印跡法檢測大鼠腦缺血再灌注后缺血半暗帶區(qū)HSP70的表達情況。結(jié)果:1.神經(jīng)功能評分示:HSYA(5mg/kg,10mg/kg)尾靜脈注射治療及預(yù)防用藥均可減少大鼠腦缺血再灌注后神經(jīng)功能缺損評分。與模型組相比,術(shù)前10mg/kg組、術(shù)后5mg/kg組、術(shù)后10mg/kg組神經(jīng)功能缺損評分明顯降低,差異具有統(tǒng)計學(xué)意義(P0.05)。術(shù)前5mg/kg組與模型組間的差異無統(tǒng)計學(xué)意義(P0.05)。2.腦梗死比積示:HSYA(5mg/kg,10mg/kg)的治療和預(yù)防用藥均可以明顯減少大鼠腦缺血再灌注后腦梗死比積,差異具有統(tǒng)計學(xué)意義(P0.05)。術(shù)后5mg/kg組、術(shù)后10mg/kg組腦梗死比積較術(shù)前5mg/kg組明顯減少,差異具有統(tǒng)計學(xué)意義(P0.05)。術(shù)后10mg/kg組治療給藥明顯優(yōu)于術(shù)后5mg/kg組預(yù)防給藥,差異具有統(tǒng)計學(xué)意義(P0.05)。3.免疫組化示:HSP70主要表達于細胞質(zhì)中,正常組陽性細胞表達量極少。假手術(shù)組較正常組表達稍多,但無明顯統(tǒng)計學(xué)差異(P0.05);腦缺血再灌注會導(dǎo)致大鼠腦組織半暗帶區(qū)陽性細胞大量增加,差異具有統(tǒng)計學(xué)意義(P0.01);給藥各組與模型組相比,陽性細胞表達明顯減少,且差異具有統(tǒng)計學(xué)意義(P0.01);與術(shù)前5mg/kg組相比,術(shù)后5mg/kg組及術(shù)后10mg/kg組陽性細胞表達明顯減少,差異具有統(tǒng)計學(xué)意義(P0.05);與術(shù)前10mg/kg組相比,術(shù)后10mg/kg組陽性細胞表達明顯減少,差異具有統(tǒng)計學(xué)意義(P0.01);與術(shù)后5mg/kg組相比,術(shù)后10mg/kg組陽性細胞表達明顯減少,差異具有統(tǒng)計學(xué)意義(P0.01)。4.蛋白質(zhì)印跡法示:與正常組相比,假手術(shù)組HSP70表達明顯增加,差異具有統(tǒng)計學(xué)意義(P0.01);與假手術(shù)相比,模型組HSP70表達明顯升高,差異具有統(tǒng)計學(xué)意義(P0.01);與術(shù)前5mg/kg組相比,術(shù)后5mg/kg組、術(shù)后10mg/kg組HSP70表達明顯減少,且差異具有統(tǒng)計學(xué)意義(P0.01);與術(shù)前10mg/kg組相比,術(shù)后5mg/kg組、術(shù)后10mg/kg組HSP70表達明顯增減少,且差異具有統(tǒng)計學(xué)意義(P0.01);與術(shù)后5mg/kg組相比,術(shù)后10mg/kg組HSP70表達明顯增高,且差異具有統(tǒng)計學(xué)意義(P0.01)。結(jié)論1.HSYA的治療及預(yù)防用藥均可減少大鼠腦缺血再灌注后神經(jīng)功能缺損評分,術(shù)前組10mg/kg、術(shù)后5mg/kg、10mg/kg劑量給藥可以明顯降低神經(jīng)功能缺損評分,改善大鼠肢體偏癱情況。2.HSYA的治療和預(yù)防用藥均可以明顯減少大鼠腦缺血再灌注后腦梗死體積,在同等劑量下(5mg/kg、10mg/kg),術(shù)后治療給藥優(yōu)于術(shù)前預(yù)防給藥。3.免疫組化和免疫蛋白印記法示:HSP70主要表達于細胞質(zhì)中,在正常組織中表達較少,手術(shù)損傷會導(dǎo)致HSP70表達稍增加。腦缺血再灌注后,大鼠腦組織半暗帶區(qū)HSP70蛋白大量增加。然而,HSYA預(yù)防及治療給藥均可減少SD大鼠腦缺血再灌注后缺血半暗帶區(qū)HSP70蛋白的表達。在同等劑量下(5mg/kg、10mg/kg),缺血再灌注后治療給藥降低HSP70表達的水平明顯優(yōu)于缺血前預(yù)防給藥,且術(shù)后10mg/kg治療組較術(shù)后5mg/kg組效果更佳。
[Abstract]:Objective: to block the right middle cerebral artery by blocking the right middle cerebral artery, the rat model of ischemia-reperfusion was constructed in this study. The tail vein was given hydroxysafflor A (HSYA) in the caudal vein before and after the operation, and the expression of HSP70 was detected by the methods of nerve function defect score, cerebral infarction volume, immunohistochemistry and Western blot, and HSYA was explored. The effect of the prevention and treatment of ischemia reperfusion in the middle cerebral artery, the effect of cerebral ischemia reperfusion against the expression of apoptotic protein HSP70, and further explore the molecular mechanism of HSYA on the neuroprotection of ischemia reperfusion rats, and provide a theoretical basis for the treatment of HSYA in cerebral infarction. Methods: 1. animals were divided into 70 weight male adult male S. D rats were randomly divided into 7 groups (normal group, sham operation group, model group, pre operation 5mg/kg group, group 10mg/kg, group 5mg/kg, 10mg/kg group after operation, n=10.2. ischemia reperfusion model. The focal cerebral ischemia reperfusion model was blocked by nylon thread to the right middle cerebral artery, by inserting one end of the thread with a round head of the neck to the common carotid artery. " The internal carotid artery, which then reached the beginning of the middle cerebral artery, blocked the middle cerebral artery and blocked the middle cerebral artery. After blocking 2h, the thrombus line was pulled out and the 24h. sham operation group operated the same, but did not block the.3. administration of the middle cerebral artery: before operation, group 5mg/kg, before operation, the 10mg/kg group was given 5mg/ kg, 10mg/kg dose to the tail vein of HSYA; 5mg/kg after the operation. Group 10mg/kg was given HSYA tail vein injection at the dose of 5mg/kg and 10mg/kg after operation respectively. The sham operation group and the model group were given the.4. nerve function defect of the tail vein of physiological saline: 2h after anesthesia sober, five points method was used to evaluate the neurological deficit score.5. histology evaluation: all rats were executed after 24h and executed quickly to get the brain. TTC staining was used to measure the area of cerebral infarction with Image J software, and then the cerebral infarction specific product was calculated. The expression of HSP70 in the ischemic penumbra region after cerebral ischemia and reperfusion was detected by immunohistochemistry and Western blotting. Results: the 1. nerve function score showed that the HSYA (5mg/kg, 10mg/kg) tail vein injection treatment and the preventive medication can reduce the large amount. Compared with the model group, the scores of nerve function defect in group 10mg/kg, group 5mg/kg and group 10mg/kg were significantly lower than those in the model group (P0.05). There was no statistically significant difference between the 5mg/kg group and the model group before operation (P0.05).2. infarction ratio: HSYA (5mg/kg, 10mg/kg). Treatment and prevention of drugs can significantly reduce the cerebral infarction ratio after cerebral ischemia and reperfusion in rats. The difference has statistical significance (P0.05). After operation, the cerebral infarction ratio in group 5mg/kg after operation is significantly lower than that of group 5mg/kg (P0.05). The treatment of 10mg/kg in group 10mg/kg is obviously better than that of group 5mg/kg after operation. The difference was statistically significant (P0.05).3. immunohistochemical staining: HSP70 was mainly expressed in the cytoplasm, and the expression of positive cells in the normal group was very few. The sham operation group was a little more expressed than the normal group, but there was no significant difference (P0.05). The cerebral ischemia reperfusion could lead to a large increase in the positive cells in the semi dark zone of the brain tissue of the rats, and the difference was statistically significant. Significance (P0.01). Compared with the model group, the expression of positive cells decreased significantly, and the difference was statistically significant (P0.01). Compared with the pre operation 5mg/kg group, the expression of positive cells in group 5mg/kg and 10mg/kg group decreased significantly (P0.05). The expression of positive cells in 10mg/kg group after operation was compared with that of group 10mg/kg before operation, and the expression of positive cells in 10mg/kg group after operation. The difference was statistically significant (P0.01). Compared with group 5mg/kg, the expression of positive cells in group 10mg/kg decreased significantly after operation, and the difference was statistically significant (P0.01).4. Western blot showed that the expression of HSP70 in the sham operation group increased significantly compared with the normal group, and the difference was statistically significant (P0.01); compared with the sham operation, the model group HS was compared. The expression of P70 was significantly higher, and the difference was statistically significant (P0.01). Compared with group 5mg/kg, HSP70 expression in group 10mg/kg after operation was significantly decreased and the difference was statistically significant (P0.01). Compared with group 10mg/kg, the expression of HSP70 expression in group 5mg/kg and 10mg/kg group after operation was significantly increased, and the difference was statistically significant (P0.01). The expression of HSP70 in group 10mg/kg after operation was significantly higher than that of group 5mg/kg, and the difference was statistically significant (P0.01). Conclusion the treatment and prevention of 1.HSYA can reduce the score of nerve function defect after cerebral ischemia-reperfusion in rats, 10mg/kg before operation, 5mg/kg and 10mg/kg dosage after operation can obviously reduce the score of nerve function defect. The treatment and prevention of hemiplegia in the good rat's limbs.2.HSYA can significantly reduce the volume of cerebral infarction after cerebral ischemia and reperfusion in rats, at the same dose (5mg/kg, 10mg/kg). The postoperative treatment is better than the pre operation prophylaxis.3. immunohistochemistry and immunoglobulin imprinting method: HSP70 is mainly expressed in the cytoplasm and expressed in normal tissues. Less, surgical injury may lead to a slight increase in the expression of HSP70. After cerebral ischemia and reperfusion, the HSP70 protein in the semi dark zone of the rat brain increases greatly. However, HSYA prevention and treatment can reduce the expression of HSP70 protein in the ischemic penumbra region of SD rats after cerebral ischemia reperfusion. At the same dose (5mg/kg, 10mg/kg), after ischemia-reperfusion treatment The level of HSP70 expression was significantly better than that before ischemia, and the 10mg/kg treatment group had better effect than the 5mg/kg group after operation.

【學(xué)位授予單位】：云南中醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R743.33

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