NogoA-PirB通路对脑出血大鼠轴突再生和神经功能的影响

发布时间：2018-01-13 23:31

本文关键词：NogoA-PirB通路对脑出血大鼠轴突再生和神经功能的影响　出处：《西南医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:通过构建大鼠脑出血模型,观察NogoA和Pir B两种蛋白在脑出血大鼠脑组织中不同时间点的定量表达、PirB表达的时空特点、轴突再生情况、大鼠神经功能缺损情况,探讨PirB的表达对脑出血损伤大鼠轴突再生和神经功能的影响、NogoA和PirB蛋白表达的相关性,推测Nogo A-PirB通路与神经功能缺损的相关性,探讨脑出血损伤后脑组织的修复机制,进一步探索脑出血后治疗靶点,为临床治疗研究提供理论依据。方法:雄性SD大鼠75只,随机分为假手术(Sham)组(n=25)和脑出血(ICH)组(n=50),两组分别按断头取脑时间点不同分为1d、3d、7d、14d、28d共5个亚组,各时间点脑出血大鼠随机分为Western Blot组和组织免疫荧光组。Sham组大鼠颅骨钻孔后不注射血液,脑出血组以大鼠自体鼠尾动脉不凝血50ul,经改良二次注入法建立大鼠脑出血模型,术后3小时通过神经功能评分判定造模成功与否。造模成功后,各时间点大鼠采用Garcia神经功能障碍评分方法进行评分后断头取脑。Western Blot组脑组织取出后立即液氮冷冻,随后转入-80℃冰箱冻存备用,研磨组织提取蛋白后,采用蛋白免疫印迹法(Western-blot)测定PirB和NogoA蛋白的表达;组织免疫荧光组大鼠深度麻醉后予以PBS和4%多聚甲醛灌注后取脑,取出脑组织经后续处理后行石蜡切片和冰冻切片,分别行HE染色和免疫荧光。结果:(1)、脑出血组神经功能缺失评分明显低于对应时间点Sham组,以3d组神经功能缺失评分最低,以上结果统计学差异具有显著性(P0.05)。(2)脑组织形态结构改变:Sham组脑组织中可见神经细胞完整。脑出血组1d亚组病灶内充满红细胞,病灶周围水肿明显伴少量炎细胞浸润;3d亚组血肿红细胞减少,病灶周围神经元肿胀坏死明显,大量炎细胞浸润;7d亚组血肿进一步吸收,周围水肿有所减轻,炎细胞减少,胶质细胞大量增生;14d、28d亚组血肿吸收,见明显胶原纤维增生及修复瘢痕。(3)NogoA和PirB定量分析:脑出血组各时间点NogoA、PirB表达均高于对应时间点的Sham组;二者在脑出血后表达明显增加,并在脑出血后7天达到表达高峰,持续高水平表达至28d,差异具有统计学意义(P0.05)。(4)、双标免疫荧光:PirB在脑出血组各时间点均有表达,主要表达在神经元细胞、轴突及星形胶质细胞;脑出血组出现明显轴突断裂、紊乱等损伤表现,轴突长度较正常对照组明显缩短,持续至脑出血后28天无明显改善,与Sham组差异具有统计学意义(P0.05)。(5)、脑出血组各时间点NogoA、PirB表达与神经功能评分和轴突长度呈负相关。结论:(1)正常成年大鼠脑组织中可见少量NogoA、PirB表达,主要表达于神经元及星形胶质细胞,维持神经功能的稳定。(2)脑出血可诱导NogoA、PirB表达增加,NogoA-PirB通路可抑制轴突再生,阻碍脑出血后的神经功能恢复。
[Abstract]:Objective: to construct a rat model of intracerebral hemorrhage, the expression of NogoA and Pir were two B in the quantitative protein at different time points in rats with intracerebral hemorrhage, spatial and temporal characteristics of PirB expression, axonal regeneration, the neurological deficit in rats, to explore the influence of the injured axon regeneration and neural function of rats with cerebral hemorrhage. The expression of PirB, NogoA and PirB protein expression, speculated that the correlation between Nogo A-PirB pathway and neural function defect, explore the repair mechanism of the injured brain tissue in cerebral hemorrhage, cerebral hemorrhage and further explore the therapeutic targets, provide a theoretical basis for clinical treatment. Methods: 75 male SD rats were randomly divided into sham operation (Sham) group (n=25) and cerebral hemorrhage (ICH) group (n=50), two groups were decapitated at different time points of 1D, 3D, 7d, 14d, 28d a total of 5 sub groups, each time point in cerebral hemorrhage rats were randomly divided into Western group and Blot group of immune The fluorescent group.Sham rats after injection of blood drilling skull, cerebral hemorrhage group with rat tail artery blood autologous 50ul modified two injection method to establish the rat model of intracerebral hemorrhage, 3 hours after the operation to determine the model is successful or not by the neurological score. After the success of the model, each time point rats with Garcia nerve dysfunction score method were decapitated after.Western Blot in brain tissue were removed immediately after freezing in liquid nitrogen, then transferred to the -80 C refrigerator and cryopreserved, grinding tissue protein extraction, by Western blotting (Western-blot) to detect the expression of PirB and NogoA protein; immunohistochemical rats after deep anesthesia to be PBS and 4% paraformaldehyde perfusion after brain, brain tissue removed by subsequent treatment after paraffin and frozen sections were stained by HE staining and immunofluorescence. Results: (1), cerebral hemorrhage group nerve function lack of clear evaluation Significantly lower than that of the corresponding time points in Sham group, 3D group with neurological deficit score was the lowest, the above results have significant difference (P0.05). (2) morphological changes of brain tissue, brain tissue Sham was found in nerve cells. The complete group of 1D subgroup of lesions filled with red blood cells of cerebral hemorrhage, edema around the lesion with obvious a small amount of inflammatory cell infiltration; 3D subgroup hematoma decreased red blood cells around the lesion, swelling of neuron necrosis, inflammatory cell infiltration; 7d subgroup hematoma further absorption, peripheral edema reduced, inflammatory cells decreased, glial cell proliferation; 14d, absorption of 28d subgroup of hematoma, obvious hyperplasia of collagen fiber and scar repair (. 3) PirB: quantitative analysis of NogoA and NogoA group at different time points of cerebral hemorrhage, the expression of PirB was higher than that of Sham group at corresponding time points; the two was significantly increased in cerebral hemorrhage after expression, and the expression peak in up to 7 days after cerebral hemorrhage, the sustained high level table Up to 28d, the difference was statistically significant (P0.05). (4), double immunofluorescence: the expression of PirB in the group at each time point were cerebral hemorrhage, mainly expressed in neurons, axons and astrocytes; cerebral hemorrhage group showed axonal fracture, disorder of injury, axonal length was significantly shortened and until 28 days after cerebral hemorrhage had no obvious improvement, with statistically significant difference with Sham group (P0.05). (5), NogoA group at different time points of cerebral hemorrhage, the expression of PirB was negatively correlated with the score of nerve function and axon length. Conclusion: (1) in brain tissue of rats with a small amount of NogoA, normal adult the expression of PirB was mainly expressed in neurons and astrocytes, nerve function to maintain stability. (2) NogoA can induce cerebral hemorrhage, increase the expression of PirB, NogoA-PirB pathway can inhibit axon regeneration, hindrance to God after intracerebral hemorrhage by function recovery.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.34

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