【作者】 杨瑞；

【导师】 陶陆阳；

【作者基本信息】 苏州大学 ， 法医学， 2010， 硕士

【摘要】 目的:研究E64d通过凋亡-自噬途径对TBI后神经细胞质膜完整性及神经功能的作用,并对其调节机制进行初步探讨。方法:采用改良的自由落体损伤装置建立小鼠定量脑外伤(traumatic brain injury, TBI)模型,实验组侧脑室注射E64d(cathepsinB和μ-calpain的特异性抑制剂,0.5μg/1μl)或等体积1%DMSO,并按伤后时间不同分为伤后1h,6h,12h,24h,和48h组,各组分别于处死前1h用腹腔注射法将碘化丙啶(propidium iodide, PI)注入各组小鼠体内,采用体视学显微镜计数各组损伤及其周边区(皮质与海马)阳性细胞数,研究组织蛋白酶对TBI引起的神经细胞质膜完整性破坏的影响;用组织缺损体积(lesion volume, LV)评估TBI后脑组织损害的情况,研究组织蛋白酶对TBI后脑组织损害的影响;采用行为学试验(Motor Test和Morris水迷宫试验)评估TBI后各组动物的运动和学习记忆的情况,研究组织蛋白酶对TBI后神经功能损害的影响。运用Western blot法和免疫组织荧光法观察TBI后各实验组脑组织中凋亡相关蛋白caspase-3,bcl-2,tBid,bax,自噬/溶酶体途径相关蛋白cathepsinB,LC3,P62的表达情况,进一步研究溶酶体酶对凋亡和自噬通路的影响以及两种通路的相互关系,期望为E64d对TBI后神经细胞质膜完整性及神经功能保护的调节机制提供理论依据。结果:PI计数,形态学、蛋白质学和行为学检测结果显示,与相对应各时段DMSO对照组比较,用E64d阻滞cathepsinB和μ-calpain后,损伤区PI阳性细胞数减少,缺损体积减小,运动和学习记忆功能有所改善;凋亡相关蛋白(caspase-3、tBid、bax)、自噬/溶酶体途径相关蛋白(cathepsinB、P62)的表达量降低,且以24h~48h最为显著(P<0.05);抗凋亡蛋白bcl-2和自噬相关蛋白LC3Ⅱ表达量显著增加(P<0.05)。结论1:TBI可以造成神经细胞质膜完整性破坏,TBI后24h~48h质膜完整性遭破坏的细胞数量达高峰,表现为PI阳性细胞显著增多;E64d可以减轻TBI造成的神经细胞质膜完整性破坏程度;2. E64d可以减小TBI后脑组织的缺损体积;3. TBI可引起运动和学习记忆功能损害,E64d可以明显改善TBI后运动和学习记忆功能;4.自噬/溶酶体途径、凋亡途径均参与脑外伤的病理过程;5.E64d抑制cathepsinB和μ-calpain后凋亡和自噬途径均受到了影响,凋亡受到明显抑制,而自噬却明显增多;6. E64d引起TBI后自噬过程增多而非自噬性细胞死亡增多,E64d通过抑制凋亡和促进自噬过程对神经细胞质膜完整及神经功能起保护作用。更多还原

【Abstract】 Objective: To investigate the effects of E64d (a cathepsin B andμ-calpain inhibitor) on TBI induced nerve cell plasmolemma permeability and neurological dysfunctionMethods: TBI model was established by weight drop device in adult mice based on procedures previously reported. Two hundreds mature male mice were randomly divided into DMSO (1μl) control group and E64d-(0.5μg/1μl) pretreated group for time course works of TBI. Fifty were used to assess the number of cell death by nuclear staining with Propidium Iodine (PI) for 1 h, 6 h, 12 h, 24 h, and 48 h. Mice post-TBI were pretreated with intraperitoneal injection of PI (0.4mg/ml) 1 h before sacrificed. Other animals were sacrificed at 1 h, 6 h, 12 h, 24 h, and 48 h time point after suffering TBI. The brain tissues were undergone immunofluorescence or Western blot analyses for caspase-3 activity, tbid, bcl-2, bax, lysosomal enzyme cathepsin B, and autophagy related protein LC3Ⅱ, P62. Brain tissue defect was assessed by Lesion Volume Test. Neurological function was detected by Motor Test and Morris Water Maze. E64d - and DMSO -pretreated mice subjected to TBI were also compared with each other respect to neurological function.Results: Lateral cerebral ventricle administration of E64d before TBI in mice resulted in improvement of neurological function when compared with DMSO control groups, as assessed by PI cell count, lesion volume and behavior test after TBI. caspase-3 activity, tbid, bcl-2, bax, lysosomal enzyme cathepsin B, and autophagy related protein LC3Ⅱ, P62 were quantified by immunoblotting. E64d pretreatment drastically reduced TBI induced caspase-3 activation, bax, tBid, P62 protein levels in the injured cortex and hippocampus (P < 0.05), and quantity analysis of LC3Ⅱ/LC3Ⅰshowed that TBI-induced activation of autophagy remarkably enhanced by E64d treatment(P < 0.05).Conclusion: 1. Lateral cerebral ventricle administration of cathepsinB andμ-calpain inhibitor E64d (0.5μg/1μl) could significantly attenuate nerve cell plasmolemma permeability and TBI-induced nerve cell death and dysfunction. 2. The effect of E64d on TBI induced nerve cell plasmolemma permeability and neuronal dysfunction may due to the crosstalk regulatory mechanism between autophagy and apoptosis.更多还原