黄芪甲苷对视网膜节细胞存活和再生的影响

发布时间：2018-04-29 07:48

本文选题：黄芪甲苷 + 视神经损伤　；参考：《蚌埠医学院》2013年硕士论文

【摘要】：研究背景神经再生一直以来都是神经科学研究的重点和难点。神经再生是指特定条件下神经元轴突再生，包括轴突出芽，生长和延伸，与靶细胞重建突触关系，实现神经支配，恢复功能。低等脊髓动物如鱼类及两栖动物的中枢神经和周围神经都能够再生，而哺乳动物只有周围神经可以再生，中枢神经系统损伤后则无有效再生（Cajal1928）。视网膜是中枢神经系统的外延，且由于视网膜的视神经节细胞（retinalganglioncells,RGCs）的轴突集中成视神经（opticnerve,ON），易于操作，便于对RGCs的再生进行定量分析，所以视网膜及视神经广泛运用于中枢神经系统再生的研究。影响视神经再生的因素包括：视网膜节细胞自身的再生潜力、周围的抑制因素和促进因素的影响。视网膜神经节细胞的再生主要围绕这三个方面来实现，上调促进基因来重新启动自身潜力，去除抑制因子的影响，清除瘢痕，增加神经营养因子来促进再生。目前研究表明，神经营养因子和钙通道阻滞剂等对视神经损伤后视网膜节细胞的存活和再生有促进作用。中药对促进视网膜节细胞存活以及对中枢神经系统神经元存活和再生的研究具有重要意义。黄芪甲苷(astragalosideIVAST)是中药黄芪的主要有效成分之一，是一种Ca2+内流拮抗剂，具有舒张血管、抑制自由基生成、清除自由基、抗炎、抗氧化等作用。本实验拟研究腹腔注射AST对轴突损伤后RGCs存活和再生的影响。第一部分黄芪甲苷对视网膜节细胞存活的影响目的研究黄芪甲苷对视神经损伤后视网膜节细胞存活的影响。方法选用SD大鼠，体重200～250g，动物随机分组：正常组（n=5）、单纯视神经切断组、视神经切断后AST处理组和生理盐水处理的对照组，后三组又分为5、7、14d组（n=5）。量效关系组分4个AST剂量组10mg/kg、15mg/kg、20mg/kg、25mg/kg组（n=5）。荧光金(fluorogold,FG)逆行标记：正常对照组：上丘和外侧膝状体注射1.5ulFG逆行标记RGCs，并且于左眼眶内暴露视神经，分层缝合，5d后取材。视神经损伤模型操作：损伤各组：经FG逆行标记RGCs5d后的大鼠，在手术显微镜下眶内暴露视神经，在球后2mm处切断视神经，分层缝合，于各时间点取材。视网膜铺片和计数：各观察时间点，处死大鼠取出眼球，固定后剥离视网膜分鼻上、鼻下、颞上、颞下四部分铺片，在荧光显微镜下，距视盘0.5、1.5、2.5mm处各拍摄200×的荧光照片各3张。用image-pro6.0软件对照片上标记的RGCs进行计数，求平均值，4个部分RGCs数量累加转化成单位面积RGCs密度。结果经黄芪甲苷处理的动物，切断视神经5、7、14d后的存活节细胞的平均密度为（1359±193）/mm2，（1046±175）/mm2和（514±67）/mm2，与正常视网膜节细胞密度相比，其存活率分别为60.94%、46.90%和23.04%。5、7d、14d组的节细胞存活率增加了9.59%、11.27%、11.32%。经方差分析，黄芪甲苷处理组与视神经切断组和生理盐水对照组相比在视神经切断后不同时间段其P值均小于0.05，表明它们存在显著性差异。结论黄芪甲苷对视神经损伤后视网膜节细胞存活有促进作用。第二部分黄芪甲苷对视网膜节细胞再生的影响目的研究黄芪甲苷对视网膜节细胞再生的影响。方法选用SD大鼠，体重200～250g，动物随机分组：生理盐水对照组、AST处理组（各组又分为21、28d组）以及量效关系组（21d）n=5。视神经再生模型操作：动物麻醉后手术显微镜下眶内暴露视神经，距球后1mm切断视神经，移植一段2cm自体坐骨神经，取材前3d修剪移植的自体坐骨神经为1.5cm并放置FG逆行标记再生的视网膜节细胞，于21、28d取材。视网膜铺片的RGCs计数同前。结果经黄芪甲苷处理组，术后3、4周再生视网膜节细胞平均密度为（978±99）/mm2和（546±61）/mm2，，与生理盐水对照组相比较，t检验，存在显著性差异(p0.05)。结论黄芪甲苷对视神经损伤后视网膜节细胞再生有促进作用。
[Abstract]:Neural regeneration is the key and difficult point in neuroscience research. Nerve regeneration refers to the regeneration of axonal axons under specific conditions, including axon buds, growth and extension, reconstructing synapses with the target cells, realizing innervation and restoring function. The central nerves and peripheral nerves of lower spinal animals such as fish and amphibian. The nerve can regenerate, but only the peripheral nerve can be regenerated in mammals, and the central nervous system has no effective regeneration (Cajal1928). The retina is the epitaxy of the central nervous system, and the axons of the retinal ganglion cells (retinalganglioncells, RGCs) are concentrated into the optic nerve (opticnerve, ON), easy to operate and facilitate the RGC. The regeneration of S is quantitatively analyzed, so the retina and optic nerve are widely used in the study of the regeneration of the central nervous system. The factors affecting the regeneration of the optic nerve include: the regeneration potential of the retinal ganglion cells, the surrounding factors and the influence of promoting factors. The regeneration of the retinal ganglion cells is mainly implemented around these three aspects. The current study shows that neurotrophic factors and calcium channel blockers have a promoting effect on the survival and rebirth of retinal ganglion cells after optic nerve injury. It is of great significance to live and study the survival and regeneration of neurons in the central nervous system. Astragaloside (astragalosideIVAST) is one of the main effective components of Astragalus membranaceus. It is an Ca2+ antagonist with diastolic blood vessels, inhibition of free radical generation, free radical scavenging, anti-inflammatory and antioxidant effects. This experiment is to study intraperitoneal injection. The effect of AST on the survival and regeneration of RGCs after axon injury.
Part I effects of astragaloside IV on survival of retinal ganglion cells
Objective to study the effects of astragaloside IV on retinal ganglion cell survival after optic nerve injury.
Methods SD rats were selected and weighed 200 to 250g. The animals were randomly divided into normal group (n=5), simple optic nerve cutting group, AST treatment group and saline treatment group after optic nerve cut off, and the latter three groups were divided into 5,7,14d group (n=5). The dose effect group was divided into 4 AST doses group 10mg/ kg, 15mg/kg, 20mg/kg, 25mg/kg group.
Fluorogold (FG) retrograde labeling: the normal control group: the upper colliculus and the lateral geniculate body were injected with 1.5ulFG retrograde RGCs, and the optic nerve was exposed in the left orbit, sutured stratified, and after 5D.
The operation of optic nerve injury model: the rats were injured after the FG retrograde labeling of RGCs5d, the optic nerve was exposed in the orbit under the operation microscope, the optic nerve was cut off at 2mm after the ball, and the material was collected at every time point.
Retina sheet and count: each time point was observed, the rat was taken out of the eye, and then the retina was stripped to the nose, the nose, the superior temporal and the infratemporal, four slices of 200 x of each photograph were taken from the 0.5,1.5,2.5mm of the disc, and the image-pro6.0 software was used to count the RGCs. The average value was 4. The amount of RGCs is converted to RGCs density per unit area.
Results the average density of the surviving ganglion cells after the treatment of Astragalus glucoside was (1359 + 193) /mm2, (1046 + 175) /mm2 and (514 + 67) /mm2, and the survival rate was 60.94%, 46.90% and 23.04%.5,7d compared with normal retinal ganglion cell density, and the survival rate of ganglion cells in group 14d increased 9.59%, 11.27%, 11.32%.. Difference analysis, the P value of the astragaloside treatment group compared with the optic neurotomy group and the normal saline control group was less than 0.05 in different time periods after the optic nerve cut off, indicating that there were significant differences.
Conclusion astragaloside can promote the survival of retinal ganglion cells after optic nerve injury.
The second part is astragaloside IV on the regeneration of retinal ganglion cells.
Objective to study the effects of astragaloside IV on retinal ganglion cell regeneration.
Methods SD rats, weighing 200 to 250g, were randomly divided into two groups: normal saline control group, AST treatment group (divided into 21,28d group) and quantitative effect group (21d) n=5. optic nerve regeneration model operation: after the anaesthetized operation microscope, the optic nerve was exposed in the orbit, the optic nerve was cut off from the ball after the ball and a 2cm autologous sciatic nerve was transplanted. The autologous sciatic nerve of the anterior 3D pruning was 1.5cm and the FG retrograde regenerated retinal ganglion cells were placed in 21,28d. The RGCs count of the retina sheet was counted as the same before.
Results the average density of regenerated retinal ganglion cells in 3,4 weeks after the operation was (978 + 99) /mm2 and (546 + 61) /mm2, compared with the normal saline control group and t test, there were significant differences (P0.05).
Conclusion astragaloside can promote the regeneration of retinal ganglion cells after optic nerve injury.

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R774

【参考文献】