本文選題：聚乙二醇 + 視神經(jīng)損傷　； 參考：《第四軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：視神經(jīng)損傷后,由于軸突逆行性退變，細胞膜和軸膜破裂，鈣離子大量內(nèi)流、氧化應(yīng)激損傷、線粒體代謝功能紊亂、能量耗盡、活性酶丟失和炎癥的發(fā)生，導(dǎo)致視網(wǎng)膜神經(jīng)節(jié)細胞（節(jié)細胞）在軸突損傷后兩周內(nèi)大量死亡。抑制節(jié)細胞的死亡和減輕繼發(fā)性損傷是視神經(jīng)損傷后修復(fù)的重要的方面。視神經(jīng)損傷后節(jié)細胞的存活是軸突再生的基礎(chǔ)，促進節(jié)細胞在軸突損傷后的存活是神經(jīng)修復(fù)研究的重要環(huán)節(jié)�？寡趸瘎┖碗x子通道阻斷劑和炎癥抑制劑等在視神經(jīng)損傷后均能促進節(jié)細胞的存活。 聚乙二醇（polyethyleneglycol,PEG）是無毒的親水性高分子聚合物，可使細胞膜的脂類雙分子層發(fā)生重組，促使細胞膜融合形成細胞塊或多核細胞，從而修復(fù)損傷的細胞膜。PEG作為無毒性膜修復(fù)劑的優(yōu)點，被廣泛應(yīng)用于神經(jīng)修復(fù)的研究。George等通過全身或局部應(yīng)用PEG于蚯蚓巨大神經(jīng)纖維損傷處，發(fā)現(xiàn)PEG部分軸突穿越損傷區(qū)到達遠側(cè)段內(nèi)并恢復(fù)一定的功能。有較多研究應(yīng)用PEG修復(fù)脊髓損傷，發(fā)現(xiàn)PEG能修復(fù)破裂的生物膜，抑制細胞器線粒體膜的通透性轉(zhuǎn)換和減少氧化應(yīng)急損傷從而修復(fù)損傷的脊髓。Koob等還發(fā)現(xiàn)PEG能夠減輕由大腦外傷性軸索損傷造成的β淀粉樣前體蛋白(APP)的積聚。然而，PEG局部敷貼應(yīng)用和靜脈注射全身性給藥的途徑不同，是否會對中樞神經(jīng)損傷后的保護作用產(chǎn)生顯著影響，目前尚無確切的比較研究。尤其是未見PEG對視神經(jīng)損傷后節(jié)細胞的保護作用的研究報道。 本研究探討局部與全身性不同給藥途徑的PEG，對視神經(jīng)橫斷后節(jié)細胞保護作用的特異影響，以及PEG神經(jīng)保護作用的機制，期望為PEG的臨床應(yīng)用提供新的基礎(chǔ)研究的理論支持。本研究在完全切斷成年大鼠單側(cè)視神經(jīng)后，在損傷視神經(jīng)眶側(cè)斷端留置浸有PEG溶液的明膠海綿，或經(jīng)尾靜脈注射PEG溶液，比較術(shù)后不同存活時間兩種不同給藥途徑PEG對損傷節(jié)細胞存活的影響。 實驗第一部分：比較局部和全身給藥的PEG對成年大鼠視神經(jīng)切斷后節(jié)細胞存活的影響。72只成年SD大鼠左眼球后1.5mm處橫斷視神經(jīng)，眶側(cè)斷端留置浸有熒光金的明膠海綿以逆行標記存活的節(jié)細胞。術(shù)后立即尾靜脈注射1ml30%PEG（尾靜脈注射PEG組）或等體積生理鹽水（尾靜脈注射鹽水對照組），或在視神經(jīng)眶側(cè)斷端留置浸有50%PEG（局部PEG組）或生理鹽水（局部鹽水對照組）的明膠海綿。四組動物（每組n=18）分別存活2d、7d或14d（每時間點n=6）后處死，取術(shù)側(cè)視網(wǎng)膜，平鋪計數(shù)存活節(jié)細胞并計算出節(jié)細胞密度。術(shù)后7d尾靜脈注射PEG組存活節(jié)細胞平均密度(1121.4286±42.69/mm2)顯著高于尾靜脈注射鹽水對照組(846.6667±58.19/mm2；P0.05)，而2d和14d時間點兩組節(jié)細胞密度間無顯著性差異（P0.05）；局部PEG組節(jié)細胞密度在各時間點與局部鹽水對照組相比均無顯著性差異(P0.05)；在7d點，尾靜脈注射PEG組節(jié)細胞密度顯著高于局部PEG組(774.43±50.49/mm2；P0.05)。提示PEG能在視神經(jīng)切斷后一定時間內(nèi)延緩節(jié)細胞死亡，且這種神經(jīng)保護作用有賴于PEG的給藥途徑。 實驗第二部分：探討PEG對視神經(jīng)損傷后節(jié)細胞保護作用的機制。24只成年大鼠左眼球后1.5mm處橫斷視神經(jīng)，術(shù)后立即尾靜脈注射1ml30%PEG或等體積生理鹽水，分別存活2d和7d每個時間點(n=6)。斷頭處死動物后，摘除眼球行冰凍切片或組織勻漿，對視神經(jīng)損傷后視網(wǎng)膜激活的小膠質(zhì)細胞或巨噬細胞標志ED1以及視網(wǎng)膜氧化應(yīng)激損傷指標之一硝基酪氨酸(nitrotyrosine，，NT)的活化水平行免疫組織化學(xué)熒光檢測；對視神經(jīng)損傷后視網(wǎng)膜炎癥因子IL-1β和TNF-α的mRNA表達水平行qRT-PCR檢測。實驗結(jié)果顯示，與對照組相比，尾靜脈注射的PEG雖未能減少視神經(jīng)損傷后各時間點TNF-α和IL-1β等炎癥因子水平(P0.05)，但在視神經(jīng)損傷后7天顯著抑制了ED1陽性細胞的激活(P0.05)并減少硝基酪氨酸水平的升高(P0.05)。提示尾靜脈注射的PEG通過減輕氧化應(yīng)激損傷和減少小膠質(zhì)細胞或巨噬細胞活化的方式，促進了視神經(jīng)損傷后節(jié)細胞的存活。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R774.6

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