激活的小胶质细胞在大鼠视网膜缺血再灌注损伤中作用机制的研究

发布时间：2018-04-16 10:21

本文选题：视网膜缺血-再灌注损伤 + 小胶质细胞　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]通过大鼠前房穿刺灌注,建立不同时间点的视网膜缺血-再灌注模型,观察大鼠视网膜损伤后视网膜小胶质细胞的激活状态及不同时间点IL-1β,TNF-a,iNOs等炎性因子的分泌变化,分析小胶质细胞激活与神经细胞死亡的相互关系,并探讨活化小胶质细胞在损伤后释放炎性细胞因子的作用机制,旨在根据研究结论对于视网膜缺血性疾病的治疗带来一定的指导意义。[方法]将双眼成年的SPF级别的雄性SD大鼠,共105只,共105只眼,将其随机分为5组,分别为:正常组,假手术组,损伤后24h组,3d组,7d组,在相同实验条件下,建立视网膜缺血-再灌注模型,利用30-gauge针头进行前房穿刺,针头连接0.9%生理盐水,将瓶高升高至150cm,维持灌注60min后,拔除前房穿刺针头。在模型建立后24h, 3d及7d后取材,进行石蜡包埋切片,视网膜切片通过HE染色和凋亡实验,观察缺血损伤不同时间点,视网膜神经节细胞层的厚度改变及神经节细胞的凋亡情况。通过进行视网膜冰冻切片后,免疫荧光染色,特异性观察小胶质细胞的激活情况及缺血损伤后不同时间点小胶质细胞的激活比例。利用RT-PCR检测损伤后不同时间点的IL-1 β,TNF-a,iNOs等炎性因子的分泌情况与视网膜神经节细胞损伤的对应关系。通过以上步骤,分析视网膜缺血损伤不同时间节点,视网膜结构改变与小胶质细胞激活比例、激活后分泌的炎性因子的对应关系。[结果]对进行HE染色的视网膜石蜡切片观察,可发现:正常组与假手术组大鼠视网膜各层次结构清楚,各层细胞排列整齐,厚度均匀,神经节细胞层各细胞边缘清楚,缺血-再灌注损伤后24h,大鼠神经节细胞出现空泡变性,神经节细胞数量变少。缺血-再灌注损伤3d后,相较于对照组的大鼠视网膜神经节细胞层厚度,视网膜神经节细胞层变薄,神经节细胞数量减少,视网膜厚度变薄,缺血损伤后,神经节细胞层细胞排列紊乱,部分神经节细胞细胞可见明显空泡变性,胞浆内空泡形成,部分细胞核溶解,残存的神经节细胞细胞核染色深,视网膜内网层明显变薄。缺血-再灌注损伤7d后,视网膜神经节细胞层厚度较3d组的神经节细胞层厚度明显变薄,神经节细胞数量明显减少,大量细胞空泡变性,另外缺血-再灌注损伤大鼠视网膜内网层明显变薄,内核层细胞水肿明显,体积增大,染色变浅。视网膜凋亡实验结果显示正常组与假手术组仅表现背景荧光,无阳性细胞表达,而在缺血-再灌注损伤后24h,除红色背景荧光外,可见少量阳性细胞(P=0.2630.05,无明显统计学意义),阳性反应细胞逐渐增多,主要集中在视网膜内层,缺血-视网膜损伤3d后,可见阳性表达细胞最多(P=0. 0000. 05,差异存在统计学意义),缺血损伤7d后,可也见大量凋亡细胞,但凋亡细胞数量较3d组的减少(P=0.0020.05,差异存在统计学意义)视网膜Iba-1标记小胶质细胞,在正常组及假手术组中仅可在视网膜神经节细胞层看到静息状态的小胶质细胞,可见小胶质细胞胞体呈树枝状突起。缺血-再灌注损伤后24h,可见相比较于正常组及假手术组,小胶质细胞的数量明显增多,视网膜神经节层和外丛状层静息状态下的小胶质细胞大量激活,其中以视网膜神经节层为主,树枝状突起回缩,胞体变圆,内丛状层仍可见许多仍处于静息状态的小胶质细胞。在缺血-再灌注损伤3d后,可见视网膜神经节细胞层、内丛状层、外核层大量小胶质细胞激活。缺血损伤7d后,激活的小胶质细胞数量下降,但神经节细胞层及外核层仍可见大量激活的小胶质细胞。正常视网膜内IL-Iβ、TNF-α、iNOs的表达量较低,且与假手术组炎性因子的表达无明显统计学差异,缺血-再灌注损伤后24h,IL-1β、TNF-α、iNOs的mRNA表达量都大幅度增加(与正常对照组相比,P=0. 0000. 05,有统计学差异),并且随损伤时间的增加而增加,损伤后3d达到峰值后(与正常对照组相比,P=0. 0000. 05,有统计学差异),炎性因子的表达逐渐下降,损伤后7d,仍可见大量炎性因子表达。(与正常对照组相比,P=0. 0000. 05,有统计学差异)[结论]视网膜缺血-再灌注损伤后,随着损伤时间的延长,视网膜神经节细胞层厚度、内丛状层厚素及整体视网膜厚度越来越薄。同时,视网膜缺血可刺激视网膜小胶质细胞的增殖及迁移,视网膜中炎性因子IL-1β、TNF-α、iNOs的表达增加,且与视网膜神经节细胞凋亡程度、小胶质细胞活化程度相对应,因此,可认为,视网膜缺血-再灌注损伤后,小胶质细胞激活,且激活的小胶质细胞分泌炎性因子IL-1β、TNF-α、iNOs,炎性因子引起视网膜的炎症反应及相应结构的病理学改变。
[Abstract]:[Objective] by rat anterior chamber puncture perfusion, established at different time points of retinal ischemia-reperfusion model to observe retinal microglia retinal injury in rats after activation and different time points IL-1 beta, TNF-a, iNOs and other changes in the secretion of inflammatory factors, analysis of relationship between activation and neuronal cell death of microglia cells, and explore the mechanism of activated microglia release proinflammatory cytokines after injury, according to the conclusion of the study for the treatment of retinal ischemic disease has guiding significance.] some of the methods of male SD rat eyes adult SPF level, a total of 105, a total of 105 eyes were randomly divided into 5 group, respectively: normal group, sham operation group, injury group 24h, 3D group, 7d group, under the same experimental conditions, the establishment of retinal ischemia-reperfusion model of anterior chamber puncture with 30-gauge needle, needle is connected with the 0.9% students Instead, the bottle will rise up to 150cm, maintain perfusion after 60min removal of anterior chamber puncture needle. In the model of 24h, 3D and 7d respectively, were embedded in paraffin, HE staining and retinal slices through the experimental observation of apoptosis, ischemia at different time points, the apoptosis of retinal ganglion cell layer thickness change. And through the retinal ganglion cells after frozen section, immunofluorescence staining, observe the specificity of microglia activation and ischemic injury at different time points after activation of microglia was detected by RT-PCR. The proportion of injury at different time points after TNF-a, IL-1 beta, iNOs and other inflammatory factors secretion and retinal ganglion the relationship between cell damage. Through the above steps, the retinal ischemia injury in different time node analysis, the change of retinal structure and microglial activation ratio, inflammatory activation after secretion The results of the corresponding relationship. The factor of observation, HE staining of the retinal sections can be found: normal group and sham operation group rats retinal layers structure clearly, each layer of cells arranged in neat, uniform thickness, the edge of the cell ganglion cell layer clear, ischemia-reperfusion injury after 24h in rats, ganglion cells vacuolar degeneration of ganglion cells decreased. Ischemia reperfusion injury after 3D, thickness of retinal ganglion cells in rats compared with the control group, the retinal ganglion cell layer became thinner, reducing the number of ganglion cells, retinal thickness, ischemic injury, ganglion cell layer cells arranged in disorder, part of cells of ganglion cells was observed vacuolar degeneration and vacuolization in the cytoplasm, nucleus dissolved, the remnants of the ganglion cell nuclei staining deep, inner plexiform layer was thinner. Ischemia reperfusion injury after 7d, The thickness of the ganglion cell layer of retinal ganglion cell layer thickness was significantly thinner than that in group 3D, significantly reduced the number of ganglion cells, cell vacuolar degeneration, and ischemia reperfusion injury in rats retina network was significantly thinner, inl cell edema, increased volume, superficialdyeing. Experimental results show that the retinal apoptosis and normal group the sham operation group showed only background fluorescence, expression of positive cells in ischemia reperfusion injury after 24h, in addition to the red fluorescent background, positive cells (P=0.2630.05, not statistically significant), positive cells gradually increased, mainly concentrated in the inner retina ischemic retinal injury after 3D, the positive expression of most cells (P=0. 0.05, there were statistically significant differences), ischemic damage after 7d, to see a large number of apoptotic cells can also, but the number of apoptotic cells was decreased in the 3D group (P=0.0020.05, difference There are different statistically significant) retinal Iba-1 labeled microglia, only to see the resting state of microglial cells in the retinal ganglion cell layer in normal group and sham operation group, visible microglia cells with dendritic processes. Ischemia reperfusion injury after 24h, visible compared with the normal group and sham operation group. The number of microglia increased significantly, a large number of activated retinal ganglion layer and outer plexiform layer of resting microglia, the retinal ganglion layer, dendritic retraction, rounded cell body, inner plexiform layer still visible many still in the resting state of microglial cells in ischemia-reperfusion. Reperfusion injury after 3D, retinal ganglion cell layer, inner plexiform layer, outer nuclear layer, a large number of activated microglia. 7d after ischemic injury, activated microglia cells decreased, but the ganglion cell layer and the outer nuclear layer still To see a large number of activated microglia in the normal retina. IL-I beta, TNF- alpha, iNOs expression was low, and there were no significant differences between sham operation group and expression of inflammatory factors, ischemia-reperfusion injury after 24h, IL-1 beta, TNF- alpha, iNOs mRNA expression were significantly increased (compared to with the normal control group, P=0. 0.05, there were significant differences), and increased with the increase of injury, injury after 3D after reaching the peak (compared with the normal control group P=0. 0.05, there were significant differences), expression of inflammatory factors decreased gradually, 7d after injury, there were a lot of inflammatory factor expression (. Compared with the normal control group P=0. 0.05, there were significant differences) [Conclusion] retinal injury after ischemia reperfusion, with the extension of injury, retinal ganglion cell layer thickness, inner plexiform layer thickness and the overall retinal thickness is becoming thinner and thinner. At the same time, retinal ischemia can sting The proliferation and migration induced retinal microglia in the retina, inflammatory factor IL-1 alpha beta, TNF-, increase the expression of iNOs and apoptosis of retinal ganglion cells, corresponding to the degrees, activation of microglia so that damage to the retina after ischemia reperfusion, microglia activation, and the activation of the small glial cells secrete inflammatory factor IL-1 beta, TNF- alpha, iNOs, inflammatory factors induced by inflammatory reaction and corresponding pathological retinal morphology change.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R774.1

【参考文献】