本文關鍵詞： 腦保護 腦缺血 腦心通 凋亡 Akt　出處：《河北醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的：腦梗死對人類的健康已然造成嚴重威脅，通過探討其病理生理學機制，來尋求有效治療是當前慢病防治研究的重點之一。近幾年研究發(fā)現(xiàn)，細胞凋亡是缺血后腦損傷的重要病理生理機制之一。調(diào)控腦缺血后凋亡相關基因已成為臨床治療新的靶點。大量的基礎體內(nèi)外實驗和臨床對照研究發(fā)現(xiàn)，腦心通（NXT）具有抗炎、抗氧化、抗凋亡等多種作用，因而，本實驗設計在小鼠大腦中動脈閉塞（middle cerebral arteryocclusion，MCAO）所致局灶腦缺血模型上，觀察腦心通對缺血急性期腦組織是否具有保護作用及其對局部腦缺血后大腦皮層Akt、CREB以及Bcl-2表達是否有影響。 方法：采用雄性成年CD-1小鼠，應用改良Longa線栓法建立小鼠右側(cè)MCAO模型。實驗動物隨機分為假手術組（Sham）、模型組（MCAO）、溶劑對照組（Vehicle）、腦心通小劑量組（MCAO+NXT360mg/kg, NXT-L）、腦心通大劑量組（MCAO+NXT540mg/kg, NXT-H）、抑制劑組（MCAO+NXT540mg/kg+LY294002，NXT-LY）。術后24h對小鼠進行神經(jīng)功能評分、測定腦組織的含水量，測定腦梗死體積，應用免疫組化、蛋白印記、實時定量逆轉(zhuǎn)錄多聚酶鏈反應和免疫熒光共定位技術測定Akt、CREB和Bcl-2的表達。應用PI3-K抑制劑LY294002觀察其對上述指標表達的影響。 結(jié)果： 模型組、溶劑對照組、腦心通組小鼠均出現(xiàn)左側(cè)肢體偏癱。腦心通大劑量組的神經(jīng)功能評分明顯低于溶劑對照組（P 0.05），腦心通小劑量組神經(jīng)功能評分減低，但與溶劑對照組相比無明顯差異。在腦缺血后24h，腦心通大劑量組與溶劑對照組相比差異有統(tǒng)計學意義（35.28%±5.92%v.s.46.54%±4.24%, P 0.05）。腦心通小劑量組與溶劑對照組差異無統(tǒng)計學意義。正常組無梗死灶。腦心通大劑量組梗死體積比溶劑對照組明顯減小，差異有統(tǒng)計學意義（81.35%±1.75%vs.85.55%±0.93%,P 0.05）。與溶劑對照組相比，腦心通小劑量組梗死體積無明顯減小。 腦缺血24h后，模型組Akt、CREB及Bcl-2的活性形式表達較正常組減低（P 0.05）。腦心通大劑量組p-Akt、p-CREB、Bcl-2蛋白及Akt、CREB、Bcl-2信使RNA表達升高，與溶劑對照組相比有統(tǒng)計學意義（P 0.05）。腦心通小劑量組Akt、CREB及Bcl-2表達也升高，但與溶劑對照組相比，結(jié)果無統(tǒng)計學意義。與腦心通大劑量組相比，抑制劑組p-Akt、p-CREB蛋白表達明顯下降，結(jié)果有統(tǒng)計學意義（P 0.05）。抑制劑組Bcl-2蛋白表達下降，與腦心通大劑量組相比，結(jié)果無統(tǒng)計學意義。 結(jié)論：腦缺血后腦組織中Akt、CREB及Bcl-2的活性形式表達降低。大劑量腦心通可以改善神經(jīng)癥狀，，減輕腦水腫，減輕腦梗死體積，保護局灶性缺血腦組織。應用PI3-k抑制劑LY294002后，缺血區(qū)腦組織p-Akt、p-CREB蛋白表達下調(diào)，可部分逆轉(zhuǎn)腦心通的保護作用。腦心通對缺血性腦血管病的保護作用可能與上調(diào)Akt、CREB、Bcl-2的活性形式表達有關。
[Abstract]:Objective: cerebral infarction has posed a serious threat to human health. It is one of the key points in the study of prevention and treatment of chronic diseases to seek effective treatment by exploring its pathophysiological mechanism. Apoptosis is one of the important pathophysiological mechanisms of brain injury after ischemia. Regulation of apoptosis-related genes after cerebral ischemia has become a new target of clinical therapy. Therefore, this experiment was designed on the model of focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in mice. To observe the protective effect of Naoxintong on cerebral tissue in acute phase of ischemia and its effect on the expression of CREB and Bcl-2 in cerebral cortex after local cerebral ischemia. Methods: male adult CD-1 mice were used. The right side MCAO model of mice was established by modified Longa thread embolization method. The experimental animals were randomly divided into sham-operated group, model group, control group, Naoxintong small dose group, MCAO NXT 360 mg / kg, NXT-LJ, Naoxintong large dose group MCAO NXT 540 mg / kg, NXT-HU, inhibitor group, MCAO NXT540mg/kg LY294002NXT-LY. after operation, MCAO NXT 360mg / kg, NXT-LT 540mg / kg, NXT-HU, MCAO NXT540mg/kg LY294002NXT-LY. The neurological function of mice was evaluated at 24 hours. To measure the water content of brain tissue and the volume of cerebral infarction, immunohistochemistry and protein imprinting were used. The expression of Bcl-2 and CREB was detected by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence co-localization technique. The effect of LY294002 on the expression of these indexes was observed by PI3-K inhibitor LY294002. Results:. The model group, solvent control group and Naoxintong group all showed left limb hemiplegia, the scores of nerve function in the high dose group of Naoxintong group were significantly lower than those in the solvent control group (P 0.05), and the scores of nerve function in the small dose group of Naoxintong group were lower than those in the control group. But there was no significant difference compared with the solvent control group at 24 hours after cerebral ischemia, the difference between the high dose group and the solvent control group was 35.28% 鹵5.92v.s.46.54% 鹵4.240.There was no significant difference between the small dose group of Naoxintong and the solvent control group. The infarct volume in the high dose group of Naoxintong group was significantly smaller than that in the solvent control group. The difference was statistically significant (81.35% 鹵1.75 vs 85.55% 鹵0.93%, P 0.05). Compared with the solvent control group, the infarct volume in the small dose group of Naoxintong did not decrease significantly. After 24 hours of cerebral ischemia, the active forms of Bcl-2 and Bcl-2 in the model group were lower than those in the normal group (P 0.05), and the expression of p-Akttou p-CREBN Bcl-2 protein and the RNA expression of the RNA in the large dose of Naoxintong group were higher than those in the normal group. Compared with the solvent control group, the expression of Bcl-2 and Bcl-2 in the low dose group of Naoxintong was significantly higher than that in the control group, but there was no significant difference between the two groups. Compared with the high dose group of Naoxintong, the protein expression of p-Aktna-p-CREB was significantly decreased in the inhibitor group. Results the expression of Bcl-2 protein in the inhibitor group was significantly lower than that in the high dose group of Naoxintong. Conclusion: after cerebral ischemia, the expression of Bcl-2 and CREB in brain tissue is decreased. High dose Naoxintong can improve the neurological symptoms, reduce the cerebral edema, reduce the volume of cerebral infarction, protect the focal ischemic brain tissue. After the application of LY294002, the PI3-k inhibitor can be used. The down-regulation of p-Aktna-p-CREB protein expression in ischemic brain tissue may partially reverse the protective effect of Naoxintong on ischemic cerebrovascular disease, which may be related to the up-regulation of the active form of Akton-CREB-Bcl-2.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R743.31

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