IL-1β、TNF-α、IFN-γ在大鼠弥漫性轴索损伤脑干中的表达

发布时间：2018-06-19 08:23

本文选题：弥漫性轴索损伤 + 促炎因子　；参考：《河北医科大学》2009年硕士论文

【摘要】： 目的:弥漫性轴索损伤(diffuse axonal injury,DAI)是指头部遭受特殊钝性外力产生加速运动时,在剪应力的作用下脑内发生的广泛分布的以神经轴索肿胀、断裂为特征的一系列病理生理变化,可单独出现也可同其他病理改变相伴发生。其典型表现为意识障碍,甚至可长期处于去大脑强直或植物生存状态,预后差。80%的交通工具所致的颅脑损伤和50%的严重颅脑损伤病人存在弥漫性轴索损伤,有学者提出1/3的颅脑损伤死亡案例与弥漫性轴索损伤有关[1,2]。弥漫性轴索损伤的致死率和致残率均高,发病机制复杂,临床却缺乏有效的检测手段。此外,弥漫性轴索损伤还可导致智能缺损、双侧感觉运动障碍、交感神经增强病症及癫痫等多种并发症[3～6],成为法医学损伤评定的难题。已有研究表明,弥漫性轴索损伤造成外伤后的脑功能障碍不仅是由于最初的机械外力对组织的剪切挤压等作用所致的原发性损伤,很大程度上与损伤后数小时甚至数天中发生的复杂的神经元“二次打击”(神经元继发性损伤)有关[7,8]。本实验室前期实验已经观察到弥漫性轴索损伤后大量的小胶质细胞被激活,离体实验也已经证实,促炎性细胞因子能激活小胶质细胞。但是,在弥漫性轴索损伤整体模型中,促炎因子的表达特点如何?其与小胶质细胞的活化关系如何,小胶质细胞的活化与轴索的继发性损伤的关系如何?其将值得深入研究。本实验旨在参照1994年[9]Marmarou A等的方法建立大鼠弥漫性轴索损伤模型,通过免疫组织化学方法,观察弥漫性轴索损伤后激活的小胶质细胞分布特点及小胶质细胞和神经元表达炎症介质IL-1β、TNF-α和IFN-γ在损伤后不同时间点表达的动态变化,探讨DAI中脑干区域促炎细胞因子IL-1β、TNF-α和IFN-γ的动态变化,及其与小胶质细胞激活的关系。方法:Sprague-Dawley大鼠(体重: 260±21g)随机分为11组:正常对照组、手术对照组、损伤组;包括打击后不同时相组:30min、1h、3h、6h、12h、24h、48h、72h、7d。参照Marmarous A等的方法制备大鼠脑弥漫性轴索损伤模型。正常对照组大鼠不作任何处理;手术对照组大鼠给予手术及打击前处理,但不予打击;损伤组按上述方法给予打击,根据打击后按不同时间给予10%水合氯醛麻醉后全身灌注固定取全脑,制作石蜡切片,采用HE染色、GLESS氏嗜银染色方法观察大鼠脑组织的病理改变。经以上方法验证符合标准的大鼠列入打击组,用免疫组织化学的方法测定脑组织中脑干损伤区IL-1β、IFN-γ和TNF-α的表达状况。统计数据采用均数士标准差(Mean士SD)表示,用SPSS统计分析软件进行统计学分析,各组均数的比较行单因素方差分析(ANOVA),用最小显著差法(LSD)作两两比较,以p0.05为有显著性差异。结果: 1成功建立弥漫性轴索损伤模型:常规HE染色观察,脑干的损伤最严重,因而采用连续切片对比观察脑干HE染色及GLESS氏嗜银染色的形态学改变,结果发现HE染色组织结构疏松水肿的部位,GLESS氏嗜银染色可见轴索的肿胀、扭曲等改变;而且水肿越严重,轴索肿胀、扭曲、断裂就越明显。 2β-APP的免疫组织化学染色:正常对照组和假手术对照组轴索无阳性表达,神经元胞体中可见弱表达。损伤组30min-6h组大鼠脑干神经元胞体、轴突可见多量β-APP的阳性表达;48h-72h时肿胀、断裂的轴索β-APP表达量达到高峰,周围多量小胶质细胞聚集。β-APP阳性表达是轴浆运输障碍的结果,也是神经元功能受损的表现。 3正常对照组与假手术对照组大鼠脑组织IL-1β弱表达,阳性神经元胞浆淡染,呈棕黄色;损伤后30min脑干腹侧及其深部阳性神经元和小胶质细胞数明显增高,与对照组比有显著性差异(P0.05)。阳性表达在6h达到高峰,其后神经元和小胶质细胞表达呈逐渐下降趋势,24h形成低谷,但较对照组仍然有显著性差异(P0.05),48h后神经元阳性细胞数表现呈上升趋势,与24h比有显著性差异(P0.05),在72h形成小高峰,7d阳性神经元和小胶质细胞数逐渐回落至低水平,较对照组仍有显著性差异(P0.05)。 4正常对照组与假手术对照组大鼠脑组织IFN-γ弱表达,阳性神经元淡染,呈棕黄色;损伤后30min脑干腹侧及其深部阳性神经元和小胶质细胞数明显增高,与对照组比有显著性差异(P0.05)。阳性表达在6h达到高峰,其后神经元和小胶质细胞表达呈逐渐下降趋势,在24h形成低谷,但较对照组仍然有显著性差异(P0.05),48h以后神经元阳性细胞数表现略呈上升趋势,与24h比有显著性差异(P0.05),在72h形成小高峰,7d阳性神经元和小胶质细胞数逐渐回落至低水平,较对照组仍有显著性差异(P0.05)。 5正常对照组与假手术对照组大鼠脑组织TNF-α弱表达,阳性神经元淡染,呈棕黄色;损伤后30min脑干腹侧及其深部阳性神经元和小胶质细胞数明显增高,与对照组比有统计学意义(P0.05)。阳性表达在6h达到高峰,其后神经元和小胶质细胞表达呈逐渐下降趋势,在24h形成低谷,但较对照组仍然有明显的差异(P0.05),48h以后神经元阳性细胞数表现略呈上升趋势,与24h比有显著性差异(P0.05),在72h形成小高峰,7d阳性神经元和小胶质细胞数逐渐回落至低水平,较对照组仍有显著性差异(P0.05)。结论:采用Marmarou A的自由落体撞击模型复制了大鼠弥漫性轴索损伤模型,观察到DAI后大鼠脑干轴索损伤区域IL-1β、TNF-α、IFN-γ的表达。结果显示,促炎性细胞因子IL-1β、TNF-α、IFN-γ在轴索损伤后表达增加,且呈一定的时间规律性,说明小胶质细胞活化和促炎性细胞因子的表达增加在DAI后继发性脑组织损伤中发挥一定得作用。
[Abstract]:Objective : diffuse axonal injury ( DAI ) refers to a series of pathological changes of axonal swelling and fracture in the brain under the action of shear stress .

It has been shown that the post - traumatic brain dysfunction caused by diffuse axonal injury is not only the primary injury due to the initial mechanical external force on the shear extrusion of the tissue , but also the complex neuron " secondary attack " ( secondary injury in neurons ) which occurred in several hours or even days after the injury . The experimental study of the present lab has shown that the pro - inflammatory cytokines can activate the microglial cells . However , the relationship between the activation of the microglial cells and the secondary injury of the axonal injury in the diffuse axonal injury is worth further study .

In this experiment , the dynamic changes of IL - 1尾 , TNF - 伪 and IFN - 纬 at different time points after injury were observed by immunohistochemical method , and the dynamic changes of IL - 1尾 , TNF - 伪 and IFN - 纬 were observed in DAI , and their relationship with microglial activation was discussed .

Methods : Sprague - Dawley rats ( body weight : 260 卤 21g ) were randomly divided into 11 groups : normal control group , operation control group and injury group .

In normal control group , no treatment was done in the normal control group ; the operation control group was given operation and pre - attack treatment , but it was not blow - down ; the injured group was treated by the above - mentioned method . According to the method , the pathological changes of brain tissue were observed with HE staining and GLESS ' s silver staining method . The expression of IL - 1尾 , IFN - 纬 and TNF - 伪 in brain stem cells was measured by immunohistochemical method .

Statistical analysis was carried out by SPSS statistical analysis software . The statistical analysis was carried out by SPSS statistical analysis software , and the mean difference of the mean number of each group was compared with that of the single factor analysis ( ANOVA ) , and the difference was significant between the two comparisons with the least significant difference method ( LSD ) .

Results : 1 A diffuse axonal injury model was established successfully : HE staining was observed and the damage of brain stem was the most serious .

There was no positive expression of 尾 - APP in normal control group and sham - operated control group .

The expression of IL - 1尾 in brain tissue of normal control group and sham - operated control group was significantly higher than that in control group ( P0.05 ) .

Compared with the control group , there was a significant difference in the number of neuron - positive neurons and microglial cells ( P0.05 ) .

The expression of TNF - 伪 in brain tissue of normal control group and sham - operated control group was significantly higher than that in control group ( P0.05 ) .

Conclusion : The expression of IL - 1尾 , TNF - 伪 and IFN - 纬 in the injured brain stem of rats after DAI was observed by the free fall impact model of Marmarou A . The results showed that the expression of IL - 1尾 , TNF - 伪 , IFN - 纬 increased after axonal injury , and the expression of pro - inflammatory cytokines IL - 1尾 , TNF - 伪 and IFN - 纬 increased after axonal injury .
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919

【参考文献】