调节性T细胞在小鼠脑创伤模型中功能作用的实验研究

发布时间：2018-03-10 15:32

本文选题：创伤性脑损伤　切入点：调节性T细胞　出处：《天津医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究目的:通过观察分析调节性T细胞在颅脑创伤后的变化情况及其对颅脑创伤(Traumatic Brain Injury,TBI)后神经功能恢复情况的影响,明确其在脑创伤中的功能作用,并初步探讨其潜在的作用机制。研究方法:本实验选用清洁级健康成年雄性C57B1/6小鼠共计184只,按照随机数字表法将其随机分为实验组1(空白对照组,n=46只)、实验组2(假手术组,n=46只)、实验组3(单纯TBI组,n=46只)和实验组4(TBI+CD25抗体组,n=46只)。分组后,我们建立小鼠液压打击颅脑创伤模型,并应用改良神经功能评分法和HE染色,评价其行为学功能和脑组织病理变化。我们应用流式细胞术和免疫组织化学染色技术首先分别观察了各组小鼠(n=30只)的调节性T细胞于伤后1天、3天、5天、7天和14天在外周脾脏和脑组织中的变化情况;其次,通过应用Morris水迷宫技术,在造模后7天时,观察了各组小鼠(n=10只)认知功能的差异;再次,应用伊文思蓝测定了各组小鼠(n=6只)伤后3天的血脑屏障渗透率;最后,我们应用免疫组织化学和TUNEL染色技术检测了各组小鼠脑组织中MPO+中性粒细胞和凋亡细胞的变化情况。研究结果:液压打击后,各创伤组小鼠脑组织HE染色均有典型TBI后病理变化,伤后12小时,各创伤组小鼠间神经功能评分无差异,但随着伤情的渐渐恢复,差异逐渐显现。与实验组2比较,实验组3小鼠脾脏中调节性T细胞的数量于3天时降低(P0.5),5天时达谷底(P0.05),之后有所回升,但仍不能恢复正常;同时巧合的是实验组3小鼠脑组织中的调节性T细胞于3天时出现(P0.05),并于5天时达高峰(P0.05),之后表达减少,但仍有表达;而在实验组4小鼠中,调节性T细胞则无论在脾脏中还是在脑组织中均表达很少。脑创伤后,实验组3和4小鼠的逃避潜伏期明显长于实验组2(P0.05),实验组4小鼠的逃避潜伏期长于实验组3(P0.05),而各组小鼠之间游泳速度无差异(P0.05)。较之于实验组3小鼠,实验组4小鼠血脑屏障损伤更为严重,伊文思蓝渗透率更高(P0.05)。在脑创伤后3天时,MPO+中性粒细胞在脑组织损伤灶周围及坏死组织中大量聚集,凋亡细胞则主要分布在半影区。与实验组3小鼠相比,实验组4小鼠脑组织中MPO+中性粒细胞和凋亡细胞数量均显著增加(P0.05)。实验组1和2小鼠在各项指标检测中均无差异,未见明显依文思蓝渗漏,MPO+中性粒细胞和凋亡细胞数也都表达在正常水平。研究结论:小鼠脑创伤后,调节性T细胞可能会透过损伤的血脑屏障由外周组织进入脑实质,并参与继发性脑损伤的发生发展。它可能通过抑制中性粒细胞的浸润,来缓解血脑屏障的损伤和减少神经细胞的凋亡,从而改善小鼠脑创伤后神经功能的预后。
[Abstract]:Objective: to observe and analyze the changes of regulatory T cells after traumatic brain injury (TBI) and its effect on the recovery of nerve function after traumatic Brain injury (TBI). Methods: 184 healthy male C57B1 / 6 mice of clean grade were selected in this experiment. They were randomly divided into experimental group (n = 46), control group (n = 46), sham operation group (n = 46), experimental group (n = 46) and test group (n = 46), and group 4 (n = 46) were divided into two groups: the control group (n = 46), the control group (n = 46), the control group (n = 46), the control group (n = 46) and the control group (n = 46). We established a mouse model of traumatic brain injury by hydraulic beating, and applied modified neurological function scoring method and HE staining. To evaluate the behavioral function and pathological changes of brain tissue, we first observed the regulatory T cells of 30 mice in each group by flow cytometry and immunohistochemical staining. The changes of peripheral spleen and brain tissue on the 14th day; Secondly, by using Morris water maze technique, we observed the difference of cognitive function of 10 mice in each group on the 7th day after model making; third, we used Evans blue to measure the blood-brain barrier permeability 3 days after injury in 6 mice of each group; finally, we measured the permeability of blood-brain barrier 3 days after injury. The changes of MPO neutrophils and apoptotic cells in brain tissues of mice in each group were detected by immunohistochemistry and TUNEL staining. There were typical pathological changes in brain tissue of each trauma group after TBI, but there was no difference in neurological function score between each trauma group 12 hours after injury, but the difference gradually appeared with the recovery of injury, and the difference was compared with experimental group 2. The number of regulatory T cells in the spleen of experimental group 3 mice decreased at the 3rd day, and reached the bottom of the valley at 5 days, and then increased, but still could not return to normal. Coincidentally, the regulatory T cells in the brain of the experimental group 3 mice appeared P0.05A on the 3rd day, reached the peak at the 5th day, then decreased, but still expressed, while in the experimental group 4 mice, Regulatory T cells were rarely expressed in the spleen or in the brain. The escape latency of experimental group 3 and 4 mice was longer than that of experimental group 2 (P0.05), and that of experimental group 4 mice was longer than that of experimental group 3 (P0.05), but there was no difference in swimming speed between each group of mice. In experimental group 4, the damage of blood-brain barrier was more serious, and Evans blue permeability was higher than P0.050.The MPO neutrophils accumulated in the brain tissue around the injured area and in the necrotic tissue 3 days after brain trauma. Compared with experimental group 3, the number of MPO neutrophils and apoptotic cells in brain tissue of experimental group 4 mice increased significantly (P 0.05). The number of MPO neutrophils and apoptotic cells were also expressed at normal level. Conclusion: after brain trauma, regulatory T cells may pass through the injured blood-brain barrier into the brain parenchyma. It may alleviate the damage of blood-brain barrier and decrease the apoptosis of nerve cells by inhibiting the infiltration of neutrophils, and thus improve the prognosis of neural function after brain trauma in mice.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R651.15;R-332

【相似文献】