视网膜胶质细胞在大鼠视神经夹持损伤后的作用研究
发布时间：2018-11-08 21:17
【摘要】：[目的]建立不同时间点大鼠视神经夹持损伤模型,观察大鼠视神经损伤后视网膜炎症因子和胶质细胞的反应,探讨视神经损伤后视网膜炎症因子和胶质细胞在视神经夹持损伤中的作用,为临床治疗外伤性视神经损伤的相关疾病提供新的思路和方法。[方法]采用50 g力度的视神经夹持镊建立大鼠视神经夹持损伤模型;将99只成年SPF级雌性SD大鼠随机分成正常组(n=3)、手术组(n=48)和假手术组(n=48)。手术组做视神经夹持损伤模型,假手术组仅分离暴露视神经,但不行夹持。手术组与假手术组按损伤后时间点分别在1d、3d、5 d、7 d进行取材,每个时间点共12只大鼠。Western blot实验(n=4)分别观察视网膜IL-1β、TNF-α在相应时间点的蛋白表达水平;采用免疫荧光组织化学染色法(n=4)观察大鼠视神经损伤后不同时间点视神经及视网膜上胶质细胞的变化情况;并采用免疫荧光共定位(n=4)观察视网膜TNF-α IL-1β与胶质细胞的共表达情况。[结果]Western blot实验结果显示:视神经夹持损伤后IL-1β、TNF-α的表达均发生变化。IL-1β在3d的表达量达峰值,与假手术组相比差异具有统计学意义(p0.05),其它时间点的差异较假手术组相比无统计学意义(p0.05);TNF-α在损伤后1d表达量明显上升,3d达峰值,5-7d稍有下降但差异较假手术组相比均有统计学意义(均为p0.05)。视神经上胶质细胞免疫荧光单染结果显示:视神经夹持损伤后视神经上的细胞结构排列不整齐,分布不均匀,且夹持部位细胞缺失。视神经夹持损伤后小胶质细胞的绿色荧光信号表达较假手术组相比明显增强,且分布的小胶质细胞数量增加;星形胶质细胞与假手术组相比,GFAP荧光信号表达也有所增强,轴突变长。视网膜上小胶质细胞和星形胶质细胞免疫荧光单染结果显示:视神经夹持损伤后胶质细胞的形态、数量和分布发生变化,小胶质细胞主要分布在视网膜神经节细胞层和内核层,损伤后小胶质细胞1d突触变短变粗,3d胞体变圆、突触变短,二级分支减少或消失,5-7 d出现活化的阿米巴状,且数量上,活化的小胶质细胞数量明显增多,差异与假手术组相比具有统计学意义(p0.05);而星形胶质细胞主要分布在视网膜神经节细胞层之外,在夹持损伤后被激活,表现为胞体变得肥大,轴突增粗,GFAP荧光表达增强。免疫荧光共定位显示:视神经损伤后小胶质细胞出现活化现象,视网膜中TNF-α、IL-1β的表达明显,且TNF-α、IL-1β主要分布于视网膜中的小胶质细胞中。而由星形胶质细胞分泌的IL-1β在3d表达明显,但在视网膜星形胶质细胞中未检测到TNF-α的表达。[结论]1)视神经夹持损伤后视网膜炎症因子IL-1β和TNF-α表达上升;2)视神经损伤后视神经、视网膜小胶质细胞和星形胶质细胞均出现激活现象;3)小胶质细胞和星形胶质细胞活化后均可表达IL-1β,视网膜中的TNF-α可能主要来源于小胶质细胞。
[Abstract]:[objective] to establish a rat model of optic nerve clamp injury at different time points and to observe the response of inflammatory factors and glial cells to retinal inflammation after optic nerve injury in rats. To explore the role of retinal inflammatory factors and glial cells in optic nerve clamping injury after optic nerve injury, and to provide new ideas and methods for clinical treatment of traumatic optic nerve injury related diseases. [methods] the optic nerve clamp injury model was established by 50 g forceps, 99 adult SPF female SD rats were randomly divided into normal group (n = 3), operation group (n = 48) and sham operation group (n = 48). The optic nerve clamp injury model was made in the operation group, but only the optic nerve was isolated in the sham operation group, but it could not be clamped. In the operation group and the sham operation group, the retinal IL-1 尾 was observed in 12 rats by. Western blot test (N4) at 1 day, 3 days, 5 days and 7 days after injury, respectively. The protein expression level of TNF- 伪 at the corresponding time points; The changes of optic nerve and retinal glial cells at different time points after optic nerve injury in rats were observed by immunofluorescence histochemical staining (nb4). The co-expression of TNF- 伪 IL-1 尾 and glial cells in retina was observed by immunofluorescence co-localization (nb4). [results] the results of Western blot experiment showed that the expression of IL-1 尾 and TNF- 伪 were all changed after optic nerve clamping injury. The expression of IL-1 尾 reached its peak value on the 3rd day, and the difference was statistically significant compared with the sham operation group (p0.05). There was no significant difference in other time points compared with sham operation group (p0.05). The expression of TNF- 伪 increased significantly at 1 day after injury, reached its peak at 3 days, and decreased slightly at 5 to 7 days, but the difference was statistically significant compared with that in sham operation group (all p0. 05). The results of immunofluorescence single staining of glial cells on the optic nerve showed that the structure of the optic nerve was irregular, the distribution was not even, and the cells were missing in the gripping position after the injury of optic nerve. The expression of green fluorescence signal in microglia after optic nerve clamp injury was significantly higher than that in sham operation group, and the number of distributed microglia was increased. The expression of GFAP fluorescence signal was also increased in astrocytes compared with sham operation group, and the axon became longer. The results of immunofluorescence single staining of microglia and astrocytes in the retina showed that the morphology, number and distribution of glial cells were changed after optic nerve clamping injury, and microglia were mainly distributed in the retinal ganglion cell layer and the nuclear layer. After injury, the synapses of the microglia became shorter and thicker at day 1, the cell body became round at day 3, the synapse became shorter, the secondary branches decreased or disappeared, and the activated amoeba appeared at 5-7 days, and the number of activated microglia increased obviously. The difference was statistically significant compared with sham operation group (p0.05). The astrocytes mainly distributed outside the retinal ganglion cell layer and were activated after clamping injury, which showed that the cell body became hypertrophic, axon thickened, and the GFAP fluorescence expression increased. Immunofluorescence co-localization showed that the microglia were activated after optic nerve injury, the expression of TNF- 伪 and IL-1 尾 in retina was obvious, and TNF- 伪 and IL-1 尾 were mainly distributed in microglia of retina. However, IL-1 尾 secreted by astrocytes was significantly expressed on day 3, but no expression of TNF- 伪 was detected in retinal astrocytes. [conclusion] 1) the expression of IL-1 尾 and TNF- 伪 increased after optic nerve clamp injury, 2) the activation of optic nerve, retinal microglia and astrocytes was observed after optic nerve injury. 3) IL-1 尾 was expressed in both microglia and astrocytes, and TNF- 伪 in retina probably originated from microglia.
【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】：R774.6
[Abstract]:[objective] to establish a rat model of optic nerve clamp injury at different time points and to observe the response of inflammatory factors and glial cells to retinal inflammation after optic nerve injury in rats. To explore the role of retinal inflammatory factors and glial cells in optic nerve clamping injury after optic nerve injury, and to provide new ideas and methods for clinical treatment of traumatic optic nerve injury related diseases. [methods] the optic nerve clamp injury model was established by 50 g forceps, 99 adult SPF female SD rats were randomly divided into normal group (n = 3), operation group (n = 48) and sham operation group (n = 48). The optic nerve clamp injury model was made in the operation group, but only the optic nerve was isolated in the sham operation group, but it could not be clamped. In the operation group and the sham operation group, the retinal IL-1 尾 was observed in 12 rats by. Western blot test (N4) at 1 day, 3 days, 5 days and 7 days after injury, respectively. The protein expression level of TNF- 伪 at the corresponding time points; The changes of optic nerve and retinal glial cells at different time points after optic nerve injury in rats were observed by immunofluorescence histochemical staining (nb4). The co-expression of TNF- 伪 IL-1 尾 and glial cells in retina was observed by immunofluorescence co-localization (nb4). [results] the results of Western blot experiment showed that the expression of IL-1 尾 and TNF- 伪 were all changed after optic nerve clamping injury. The expression of IL-1 尾 reached its peak value on the 3rd day, and the difference was statistically significant compared with the sham operation group (p0.05). There was no significant difference in other time points compared with sham operation group (p0.05). The expression of TNF- 伪 increased significantly at 1 day after injury, reached its peak at 3 days, and decreased slightly at 5 to 7 days, but the difference was statistically significant compared with that in sham operation group (all p0. 05). The results of immunofluorescence single staining of glial cells on the optic nerve showed that the structure of the optic nerve was irregular, the distribution was not even, and the cells were missing in the gripping position after the injury of optic nerve. The expression of green fluorescence signal in microglia after optic nerve clamp injury was significantly higher than that in sham operation group, and the number of distributed microglia was increased. The expression of GFAP fluorescence signal was also increased in astrocytes compared with sham operation group, and the axon became longer. The results of immunofluorescence single staining of microglia and astrocytes in the retina showed that the morphology, number and distribution of glial cells were changed after optic nerve clamping injury, and microglia were mainly distributed in the retinal ganglion cell layer and the nuclear layer. After injury, the synapses of the microglia became shorter and thicker at day 1, the cell body became round at day 3, the synapse became shorter, the secondary branches decreased or disappeared, and the activated amoeba appeared at 5-7 days, and the number of activated microglia increased obviously. The difference was statistically significant compared with sham operation group (p0.05). The astrocytes mainly distributed outside the retinal ganglion cell layer and were activated after clamping injury, which showed that the cell body became hypertrophic, axon thickened, and the GFAP fluorescence expression increased. Immunofluorescence co-localization showed that the microglia were activated after optic nerve injury, the expression of TNF- 伪 and IL-1 尾 in retina was obvious, and TNF- 伪 and IL-1 尾 were mainly distributed in microglia of retina. However, IL-1 尾 secreted by astrocytes was significantly expressed on day 3, but no expression of TNF- 伪 was detected in retinal astrocytes. [conclusion] 1) the expression of IL-1 尾 and TNF- 伪 increased after optic nerve clamp injury, 2) the activation of optic nerve, retinal microglia and astrocytes was observed after optic nerve injury. 3) IL-1 尾 was expressed in both microglia and astrocytes, and TNF- 伪 in retina probably originated from microglia.
【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】：R774.6
【参考文献】
相关期刊论文 前7条
1 江姿潞;王芬;谢滔;李伟彦;;分泌脂质蛋白-2激活小胶质细胞对大鼠抑郁症发病机制的影响[J];医学研究生学报;2015年10期
2 贾s，
本文编号：2319709
本文链接：https://www.wllwen.com/yixuelunwen/wuguanyixuelunwen/2319709.html
最近更新
教材专著
