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  本文選題：腦缺血再灌注　切入點：溶酶體　出處：《南華大學(xué)》2015年碩士論文

【摘要】：目的本實驗通過模擬人類急性腦卒中的模型—大鼠大腦中動脈腦缺血再灌注模型(MCAO,middle cerebral artery occlusion),并運用Cathepsin L抑制劑Z-FY-DMK及Caspase-3蛋白抑制劑Z-DEVD-FMK分別進(jìn)行干預(yù),檢測并分析腦梗死體積、腦缺血再灌注后不同時間點Cathepsin L與Caspase-3蛋白的表達(dá)變化,探討Cathepsin L與Caspase-3蛋白在神經(jīng)細(xì)胞凋亡中的相互關(guān)系。方法雄性健康清潔級SD(Sprague-Dawley)大鼠162只,生長周期約10-12周,體重約260-300g。按照隨機(jī)數(shù)表法,將其分為假手術(shù)組(27只)和腦缺血再灌注組(簡稱模型組45只),抑制劑Z-FY-DMK組(簡稱CL干預(yù)組45只),抑制劑Z-DEVD-FMK(簡稱C-3干預(yù)組45只),假手術(shù)組按再灌注6h、12h、24h、48h分成4個亞組,每組6只大鼠(其中48小時組9只)。其余各組均按再灌注6h、12h、24h、48h分成4個亞組,每組10只大鼠(其中48小時組15只)。使用Longa線栓法制備局灶性右側(cè)大腦中動脈梗死模型,缺血2h后拔除線栓即形成在灌注,假手術(shù)組大鼠線栓插入深度為8-10mm,其余操作同模型組;CL干預(yù)組與C-3干預(yù)組分別采用側(cè)腦室穿刺法注射Cathepsin L特異性抑制劑Z-FY-DMK及Caspase-3蛋白抑制劑Z-DEVD-FMK(Z-FY-DMK:于MCAO前30分鐘注射入右側(cè)側(cè)腦室,濃度為20ug/1ul,共5ul,留針10min;Z-DEVD-FMK:術(shù)前30min注射入右側(cè)側(cè)腦室10ul,濃度為0.5ug/0.5ul,留針10min,等量的DMSO溶液注射入假手術(shù)組和模型組于同一時間點及部位。)其中假手術(shù)組取3只大鼠,模型組48h和干預(yù)組48h均取5只大鼠行TTC染色。按通用的神經(jīng)功能缺損評分方法(Longa,s5級評分法)評估神經(jīng)功能缺損癥狀;采用TTC染色法、TUNEL法及Western blotting法,分別檢測腦梗死體積、缺血側(cè)大腦皮質(zhì)神經(jīng)細(xì)胞凋亡變化及Cathepsin L與Caspase-3的蛋白表達(dá)的變化。結(jié)果1.模型成功率88.24%;模型組48h相對梗死體積為38.25±1.06%,CL干預(yù)組48h相對梗死體積為26.00±1.414%,C-3干預(yù)組48h相對梗死體積為29±1.414%。2.TTC染色中,48小時組時間點的各組腦組織中,假手術(shù)組未發(fā)現(xiàn)病灶,模型組、CL組、C-3組的缺血側(cè)下可見白色病灶,但CL組、C-3組相對梗死體積相對于模型組而言,明顯減少(P0.001),并且在CL干預(yù)組、C-3干預(yù)組的大鼠神經(jīng)功能缺損癥狀與體征也發(fā)生了明顯改善(P0.05)。3.在腦缺血側(cè)皮質(zhì)區(qū)的凋亡細(xì)胞檢測中,假手術(shù)組腦組織中凋亡細(xì)胞幾乎很少見,模型組6h可見凋亡神經(jīng)細(xì)胞,12h、24h、48h逐漸增多,呈上升趨勢;CL干預(yù)組和C-3干預(yù)組在6h、12h、24h、48h觀察到凋亡細(xì)胞與模型組對應(yīng)時間點相比較,有明顯下降(p0.05)。4.在腦缺血側(cè)皮質(zhì)區(qū)的Western blotting檢測相關(guān)蛋白中,假手術(shù)組的腦組織中可以檢測到少量的Cathepsin L蛋白表達(dá),模型組Cathepsin L蛋白,均在再灌注6小時開始上升,12h、24h達(dá)到高峰,48h仍保存高水平。與模型組比較,CL干預(yù)組與C-3干預(yù)組的Cathepsin L蛋白表達(dá)在各時間點均明顯減弱(p0.05)。5.在大鼠缺血側(cè)皮質(zhì)區(qū),假手術(shù)組可以檢測到少量的Caspase-3蛋白表達(dá),模型組Caspase-3蛋白,均在再灌注6小時開始上升,24h達(dá)到高峰,48h仍保存高水平。與模型組比較,CL干預(yù)組與C-3干預(yù)組的Caspase-3蛋白表達(dá)在各時間點均明顯減弱(p0.05)。結(jié)論1.大鼠腦缺血再灌注損傷后,Cathepsin L介導(dǎo)的Caspases凋亡通路中,可能與Caspase-3蛋白存在相互制約的關(guān)系。2.Z-FY-DMK可通過特異性抑制Cathepsin L介導(dǎo)的凋亡級聯(lián)反應(yīng),減少細(xì)胞的凋亡,減少腦梗死的體積。
[Abstract]:Objective to simulate the human brain stroke model in rat cerebral artery ischemia reperfusion model in this experiment (MCAO, middle cerebral artery occlusion), and the use of Cathepsin L inhibitor Z-FY-DMK and Caspase-3 inhibitor Z-DEVD-FMK protein were detected and analyzed by intervention, infarct volume, expression changes at different time points Cathepsin L and Caspase-3 protein and reperfusion after cerebral ischemia, to explore the relationship between Cathepsin L and Caspase-3 protein in apoptosis of nerve cells. Methods male healthy SD (Sprague-Dawley) 162 rats, the growth period of about 10-12 weeks, weighing about 260-300g. according to the random number table method, which can be divided into sham operation group (27 rats) and cerebral ischemia reperfusion group (model group 45), Z-FY-DMK group (CL inhibitor intervention group was 45 (C-3), Z-DEVD-FMK inhibitor intervention group 45 rats), sham operation group at reperfusion 6h, 12h, 24h 48h, divided into 4 subgroups, 6 rats in each group (including 48 hour = 9). The other groups according to reperfusion 6h, 12h, 24h, 48h are divided into 4 groups, 10 rats in each group (including 48 hour = 15). Occuluded method of focal middle cerebral artery infarction model using the Longa line, lack of blood 2h after removal of the suture is formed in the perfusion, the rats in the sham operation group the inserting depth is 8-10mm, the same with the model group; CL intervention group and C-3 intervention group respectively by lateral ventricle puncture injection of Cathepsin L specific inhibitor Z-FY-DMK and Caspase-3 inhibitor Z-DEVD-FMK (Z-FY-DMK: in 30 minutes before MCAO injection into the right lateral ventricle, the concentration of 20ug/1ul, 5ul, Z-DEVD-FMK: for 10min; preoperative 30min injected into the lateral ventricle of 10ul, concentration of 0.5ug/0.5ul, for 10min, the same amount of DMSO solution was injected into the sham operation group and model group at the same time and location) in sham operation. Group 3 rats, model group and 48h intervention group 48h 5 rats were stained with TTC. According to the general method of nerve function defect score (Longa, S5 grade score) to assess neurological symptoms; using TTC staining method, TUNEL method and Western blotting method were used to detect the cerebral infarction volume, change. The expression of Cathepsin in ischemic changes and neuronal apoptosis in cerebral cortex of L and Caspase-3 protein. Results the success rate of the model 1. 88.24%; model group 48h relative infarct volume was 38.25 + 1.06%, CL group 48h the relative infarct volume was 26 + 1.414%, C-3 group 48h on the infarct volume was 29 + 1.414%.2.TTC staining in the brain. Each group 48 hour time points in the sham operation group showed no lesions, the model group, CL group, C-3 group showed ischemic white lesions, but the CL group, C-3 group, the relative infarct volume compared with the model group, was significantly reduced (P0.001), and in CL Pre group, C-3 intervention group rats neurological symptoms and signs was significantly improved (P0.05) to detect the apoptosis of.3. in the cerebral ischemic cortex in the brain tissue of sham group of apoptotic cells is very rare. The model group 6h showed the nerve cell apoptosis, 12h, 24h, 48h gradually increased. Increased; CL intervention group and C-3 intervention group in 6h, 12h, 24h, 48h of apoptotic cells compared with the model group at the same time, decreased significantly (P0.05) associated protein Western blotting.4. was detected in ischemic cortex in the sham operation group, the brain tissue was detected in expression a small amount of Cathepsin L protein, L protein Cathepsin in the model group, both in the 6 hours of reperfusion began to rise, 12h, reached the peak at 24h, 48h still kept high level. Compared with the model group, the expression of CL intervention group and C-3 intervention group Cathepsin L protein at each time point were significantly decreased (P0.05 .5.) in the rat cortex in ischemic area, the sham operation group can detect the expression of a small amount of Caspase-3 protein, Caspase-3 protein in the model group, both in the 6 hours of reperfusion began to rise, and reached the peak at 24h, 48h still kept high level. Compared with the model group, CL intervention group and C-3 expression of Caspase-3 protein in the intervention group time points were significantly decreased (P0.05). The injury of cerebral ischemia reperfusion in rats after the conclusion of the 1., Caspases Cathepsin L mediated apoptosis pathway in the apoptosis cascade reaction of.2.Z-FY-DMK may exist mutual restriction between the specific inhibition of Cathepsin mediated L and Caspase-3 protein, reduce apoptosis, decrease the infarct volume.

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R743.3

【參考文獻(xiàn)】

中國碩士學(xué)位論文全文數(shù)據(jù)庫 前1條

1 項蓓;Cathepsin L在6-OHDA損傷SH-SY5Y細(xì)胞中的作用[D];蘇州大學(xué);2009年

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本文編號：1672682