Cathepsin L介导的细胞凋亡在大鼠脑缺血再灌注后与Caspase-3的关系

发布时间：2018-03-27 18:19

  本文选题：脑缺血再灌注　切入点：溶酶体　出处：《南华大学》2015年硕士论文

【摘要】：目的本实验通过模拟人类急性脑卒中的模型—大鼠大脑中动脉脑缺血再灌注模型(MCAO,middle cerebral artery occlusion),并运用Cathepsin L抑制剂Z-FY-DMK及Caspase-3蛋白抑制剂Z-DEVD-FMK分别进行干预,检测并分析脑梗死体积、脑缺血再灌注后不同时间点Cathepsin L与Caspase-3蛋白的表达变化,探讨Cathepsin L与Caspase-3蛋白在神经细胞凋亡中的相互关系。方法雄性健康清洁级SD(Sprague-Dawley)大鼠162只,生长周期约10-12周,体重约260-300g。按照随机数表法,将其分为假手术组(27只)和脑缺血再灌注组(简称模型组45只),抑制剂Z-FY-DMK组(简称CL干预组45只),抑制剂Z-DEVD-FMK(简称C-3干预组45只),假手术组按再灌注6h、12h、24h、48h分成4个亚组,每组6只大鼠(其中48小时组9只)。其余各组均按再灌注6h、12h、24h、48h分成4个亚组,每组10只大鼠(其中48小时组15只)。使用Longa线栓法制备局灶性右侧大脑中动脉梗死模型,缺血2h后拔除线栓即形成在灌注,假手术组大鼠线栓插入深度为8-10mm,其余操作同模型组;CL干预组与C-3干预组分别采用侧脑室穿刺法注射Cathepsin L特异性抑制剂Z-FY-DMK及Caspase-3蛋白抑制剂Z-DEVD-FMK(Z-FY-DMK:于MCAO前30分钟注射入右侧侧脑室,浓度为20ug/1ul,共5ul,留针10min;Z-DEVD-FMK:术前30min注射入右侧侧脑室10ul,浓度为0.5ug/0.5ul,留针10min,等量的DMSO溶液注射入假手术组和模型组于同一时间点及部位。)其中假手术组取3只大鼠,模型组48h和干预组48h均取5只大鼠行TTC染色。按通用的神经功能缺损评分方法(Longa,s5级评分法)评估神经功能缺损症状;采用TTC染色法、TUNEL法及Western blotting法,分别检测脑梗死体积、缺血侧大脑皮质神经细胞凋亡变化及Cathepsin L与Caspase-3的蛋白表达的变化。结果1.模型成功率88.24%;模型组48h相对梗死体积为38.25±1.06%,CL干预组48h相对梗死体积为26.00±1.414%,C-3干预组48h相对梗死体积为29±1.414%。2.TTC染色中,48小时组时间点的各组脑组织中,假手术组未发现病灶,模型组、CL组、C-3组的缺血侧下可见白色病灶,但CL组、C-3组相对梗死体积相对于模型组而言,明显减少(P0.001),并且在CL干预组、C-3干预组的大鼠神经功能缺损症状与体征也发生了明显改善(P0.05)。3.在脑缺血侧皮质区的凋亡细胞检测中,假手术组脑组织中凋亡细胞几乎很少见,模型组6h可见凋亡神经细胞,12h、24h、48h逐渐增多,呈上升趋势;CL干预组和C-3干预组在6h、12h、24h、48h观察到凋亡细胞与模型组对应时间点相比较,有明显下降(p0.05)。4.在脑缺血侧皮质区的Western blotting检测相关蛋白中,假手术组的脑组织中可以检测到少量的Cathepsin L蛋白表达,模型组Cathepsin L蛋白,均在再灌注6小时开始上升,12h、24h达到高峰,48h仍保存高水平。与模型组比较,CL干预组与C-3干预组的Cathepsin L蛋白表达在各时间点均明显减弱(p0.05)。5.在大鼠缺血侧皮质区,假手术组可以检测到少量的Caspase-3蛋白表达,模型组Caspase-3蛋白,均在再灌注6小时开始上升,24h达到高峰,48h仍保存高水平。与模型组比较,CL干预组与C-3干预组的Caspase-3蛋白表达在各时间点均明显减弱(p0.05)。结论1.大鼠脑缺血再灌注损伤后,Cathepsin L介导的Caspases凋亡通路中,可能与Caspase-3蛋白存在相互制约的关系。2.Z-FY-DMK可通过特异性抑制Cathepsin L介导的凋亡级联反应,减少细胞的凋亡,减少脑梗死的体积。
[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.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R743.3

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中国硕士学位论文全文数据库 前1条

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