聚乙二醇对视神经损伤后视网膜神经节细胞的保护作用

发布时间：2018-06-20 19:46

本文选题：聚乙二醇 + 视神经损伤　；参考：《第四军医大学》2012年硕士论文

【摘要】：视神经损伤后,由于轴突逆行性退变，细胞膜和轴膜破裂，钙离子大量内流、氧化应激损伤、线粒体代谢功能紊乱、能量耗尽、活性酶丢失和炎症的发生，导致视网膜神经节细胞（节细胞）在轴突损伤后两周内大量死亡。抑制节细胞的死亡和减轻继发性损伤是视神经损伤后修复的重要的方面。视神经损伤后节细胞的存活是轴突再生的基础，促进节细胞在轴突损伤后的存活是神经修复研究的重要环节。抗氧化剂和离子通道阻断剂和炎症抑制剂等在视神经损伤后均能促进节细胞的存活。聚乙二醇（polyethyleneglycol,PEG）是无毒的亲水性高分子聚合物，可使细胞膜的脂类双分子层发生重组，促使细胞膜融合形成细胞块或多核细胞，从而修复损伤的细胞膜。PEG作为无毒性膜修复剂的优点，被广泛应用于神经修复的研究。George等通过全身或局部应用PEG于蚯蚓巨大神经纤维损伤处，发现PEG部分轴突穿越损伤区到达远侧段内并恢复一定的功能。有较多研究应用PEG修复脊髓损伤，发现PEG能修复破裂的生物膜，抑制细胞器线粒体膜的通透性转换和减少氧化应急损伤从而修复损伤的脊髓。Koob等还发现PEG能够减轻由大脑外伤性轴索损伤造成的β淀粉样前体蛋白(APP)的积聚。然而，PEG局部敷贴应用和静脉注射全身性给药的途径不同，是否会对中枢神经损伤后的保护作用产生显著影响，目前尚无确切的比较研究。尤其是未见PEG对视神经损伤后节细胞的保护作用的研究报道。本研究探讨局部与全身性不同给药途径的PEG，对视神经横断后节细胞保护作用的特异影响，以及PEG神经保护作用的机制，期望为PEG的临床应用提供新的基础研究的理论支持。本研究在完全切断成年大鼠单侧视神经后，在损伤视神经眶侧断端留置浸有PEG溶液的明胶海绵，或经尾静脉注射PEG溶液，比较术后不同存活时间两种不同给药途径PEG对损伤节细胞存活的影响。实验第一部分：比较局部和全身给药的PEG对成年大鼠视神经切断后节细胞存活的影响。72只成年SD大鼠左眼球后1.5mm处横断视神经，眶侧断端留置浸有荧光金的明胶海绵以逆行标记存活的节细胞。术后立即尾静脉注射1ml30%PEG（尾静脉注射PEG组）或等体积生理盐水（尾静脉注射盐水对照组），或在视神经眶侧断端留置浸有50%PEG（局部PEG组）或生理盐水（局部盐水对照组）的明胶海绵。四组动物（每组n=18）分别存活2d、7d或14d（每时间点n=6）后处死，取术侧视网膜，平铺计数存活节细胞并计算出节细胞密度。术后7d尾静脉注射PEG组存活节细胞平均密度(1121.4286±42.69/mm2)显著高于尾静脉注射盐水对照组(846.6667±58.19/mm2；P0.05)，而2d和14d时间点两组节细胞密度间无显著性差异（P0.05）；局部PEG组节细胞密度在各时间点与局部盐水对照组相比均无显著性差异(P0.05)；在7d点，尾静脉注射PEG组节细胞密度显著高于局部PEG组(774.43±50.49/mm2；P0.05)。提示PEG能在视神经切断后一定时间内延缓节细胞死亡，且这种神经保护作用有赖于PEG的给药途径。实验第二部分：探讨PEG对视神经损伤后节细胞保护作用的机制。24只成年大鼠左眼球后1.5mm处横断视神经，术后立即尾静脉注射1ml30%PEG或等体积生理盐水，分别存活2d和7d每个时间点(n=6)。断头处死动物后，摘除眼球行冰冻切片或组织匀浆，对视神经损伤后视网膜激活的小胶质细胞或巨噬细胞标志ED1以及视网膜氧化应激损伤指标之一硝基酪氨酸(nitrotyrosine，，NT)的活化水平行免疫组织化学荧光检测；对视神经损伤后视网膜炎症因子IL-1β和TNF-α的mRNA表达水平行qRT-PCR检测。实验结果显示，与对照组相比，尾静脉注射的PEG虽未能减少视神经损伤后各时间点TNF-α和IL-1β等炎症因子水平(P0.05)，但在视神经损伤后7天显著抑制了ED1阳性细胞的激活(P0.05)并减少硝基酪氨酸水平的升高(P0.05)。提示尾静脉注射的PEG通过减轻氧化应激损伤和减少小胶质细胞或巨噬细胞活化的方式，促进了视神经损伤后节细胞的存活。
[Abstract]:After the optic nerve injury, the retinal ganglion cells (ganglion cells) died and reduced in two weeks after axonal injury due to the retrograde degeneration of the axon, the rupture of the membrane and the axon membrane, the massive internal flow of calcium ions, the oxidative stress, the dysfunction of mitochondrial metabolism, the depletion of energy, the loss of active enzymes and the occurrence of inflammation. Light secondary injury is an important aspect of repair of optic nerve injury. The survival of the ganglion cells after optic nerve injury is the basis of axon regeneration. Promoting the survival of the ganglion cells after axonal injury is an important link in the study of nerve repair. Antioxidants and ion channel blockers and inflammatory agents can promote the joint after optic nerve injury. The survival of the cells.
Polyethyleneglycol (PEG) is a non-toxic and hydrophilic polymer, which can restructure the lipid bilayer of the cell membrane, promote the fusion of cell membrane to form cell blocks or multinuclear cells, and repair the damaged cell membrane.PEG as a non-toxic membrane repair agent, and is widely used in the study of neural repair,.George and so on. Through the systemic or local application of PEG to the damage of the massive earthworm nerve fibers, it was found that some of the PEG axons crossed the damaged area to the distal segment and recovered certain functions. There were more studies and applications of PEG to repair the spinal cord injury. It was found that PEG could repair the ruptured biological membrane, inhibit the permeability transformation of the cell membrane, and reduce the emergency oxidation damage. The repair of damaged spinal cord.Koob, and so on, also found that PEG can reduce the accumulation of beta amyloid precursor protein (APP) caused by traumatic axonal injury in the brain. However, the different approaches to PEG local application and intravenous injection of systemic administration will have a significant effect on the protective effect of central nerve injury. Comparative study, especially the study on the protective effect of PEG on ganglion cells after optic nerve injury.
This study explored the specific effects of PEG on the local and systemic drug delivery, and the specific effects of the protection of the ganglion cells after the optic nerve transection, as well as the mechanism of the protective effect of PEG nerve. We hope to provide a new theoretical support for the clinical application of PEG. This study is in the damage of the orbital side of the optic nerve after the unilateral optic nerve is completely cut off from the adult rats. The broken end of indwelling gelform soaked in PEG solution, or by intravenous injection of PEG solution, compare the different postoperative survival time of two different routes of administration of PEG on the survival of injury cells.
The first part of the experiment: the effect of PEG on the survival of the ganglion cells after the optic nerve transection in adult rats compared with the local and systemic administration in adult rat.72, the left eyeball in the adult SD rats was transected in the left eyeball at 1.5mm, and the fluorescent gold gelatin sponge was placed in the orbital part of the orbital side to mark the surviving ganglion cells. Immediately after the operation, the tail vein was injected with the tail vein injection. The four groups of animals (each group of n=18) survived 2D, 7d or 14d (each time n=6) were killed, and the four groups of animals (each group n=18) survived 2D, 7d or 14d (n=6) of 14d (each time point n=6). The average density of the surviving ganglion cells in the PEG group after the 7d tail vein injection (1121.4286 + 42.69/mm2) was significantly higher than that in the saline control group (846.6667 + 58.19/mm2; P0.05), but there was no significant difference between the two groups of 2D and 14d time points (P0.05), and the local PEG group cell density at each time point was higher than that in the 14d time point. There was no significant difference in the local saline control group (P0.05), and at the 7d point, the ganglion cell density in the tail vein PEG group was significantly higher than that of the local PEG group (774.43 + 50.49/mm2; P0.05). It suggested that PEG could delay the ganglion cell death in a certain time after the optic nerve was cut off, and this neuroprotective effect depended on the way of administration of PEG.
The second part of the experiment: To explore the mechanism of the protective effect of PEG on the ganglion cells after the optic nerve injury..24 transected the optic nerve at the 1.5mm of the left eyeball of the adult rats. After the operation, the tail vein was injected with 1ml30%PEG or equal volume of physiological saline immediately after the operation. The survival of 2D and 7d at each time point (n=6). After the dead animals at the end of the broken head, the eyeball was removed and the frozen section or tissue homogenization was removed. The activation level of ED1 and nitrotyrosine (NT), one of the indicators of retinal oxidative stress injury after optic nerve injury, was detected by immunohistochemistry. The level of mRNA expression of retinal inflammatory factor IL-1 beta and TNF- alpha after optic nerve injury was determined by qRT-PCR The results showed that, compared with the control group, PEG failed to reduce the level of TNF- A and IL-1 beta (P0.05) at all time points after optic nerve injury, but it significantly inhibited the activation of ED1 positive cells (P0.05) and decreased the level of nitro tyrosine (P0.05) at 7 days after optic nerve injury. PEG promotes the survival of ganglion cells after optic nerve injury by alleviating oxidative stress and reducing the activation of microglia or macrophages.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.6

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