本文選題：黃芪甲苷 + 視神經(jīng)損傷��； 參考：《蚌埠醫(yī)學(xué)院》2013年碩士論文

【摘要】：研究背景神經(jīng)再生一直以來都是神經(jīng)科學(xué)研究的重點和難點。神經(jīng)再生是指特定條件下神經(jīng)元軸突再生，包括軸突出芽，生長和延伸，與靶細胞重建突觸關(guān)系，實現(xiàn)神經(jīng)支配，恢復(fù)功能。低等脊髓動物如魚類及兩棲動物的中樞神經(jīng)和周圍神經(jīng)都能夠再生，而哺乳動物只有周圍神經(jīng)可以再生，中樞神經(jīng)系統(tǒng)損傷后則無有效再生（Cajal1928）。視網(wǎng)膜是中樞神經(jīng)系統(tǒng)的外延，且由于視網(wǎng)膜的視神經(jīng)節(jié)細胞（retinalganglioncells,RGCs）的軸突集中成視神經(jīng)（opticnerve,ON），易于操作，便于對RGCs的再生進行定量分析，所以視網(wǎng)膜及視神經(jīng)廣泛運用于中樞神經(jīng)系統(tǒng)再生的研究。影響視神經(jīng)再生的因素包括：視網(wǎng)膜節(jié)細胞自身的再生潛力、周圍的抑制因素和促進因素的影響。視網(wǎng)膜神經(jīng)節(jié)細胞的再生主要圍繞這三個方面來實現(xiàn)，上調(diào)促進基因來重新啟動自身潛力，去除抑制因子的影響，清除瘢痕，增加神經(jīng)營養(yǎng)因子來促進再生。目前研究表明，神經(jīng)營養(yǎng)因子和鈣通道阻滯劑等對視神經(jīng)損傷后視網(wǎng)膜節(jié)細胞的存活和再生有促進作用。中藥對促進視網(wǎng)膜節(jié)細胞存活以及對中樞神經(jīng)系統(tǒng)神經(jīng)元存活和再生的研究具有重要意義。黃芪甲苷(astragalosideIVAST)是中藥黃芪的主要有效成分之一，是一種Ca2+內(nèi)流拮抗劑，具有舒張血管、抑制自由基生成、清除自由基、抗炎、抗氧化等作用。本實驗擬研究腹腔注射AST對軸突損傷后RGCs存活和再生的影響。 第一部分黃芪甲苷對視網(wǎng)膜節(jié)細胞存活的影響 目的研究黃芪甲苷對視神經(jīng)損傷后視網(wǎng)膜節(jié)細胞存活的影響。 方法選用SD大鼠，體重200～250g，動物隨機分組：正常組（n=5）、單純視神經(jīng)切斷組、視神經(jīng)切斷后AST處理組和生理鹽水處理的對照組，后三組又分為5、7、14d組（n=5）。量效關(guān)系組分4個AST劑量組10mg/kg、15mg/kg、20mg/kg、25mg/kg組（n=5）。 熒光金(fluorogold,FG)逆行標(biāo)記：正常對照組：上丘和外側(cè)膝狀體注射1.5ulFG逆行標(biāo)記RGCs，并且于左眼眶內(nèi)暴露視神經(jīng)，分層縫合，5d后取材。 視神經(jīng)損傷模型操作：損傷各組：經(jīng)FG逆行標(biāo)記RGCs5d后的大鼠，在手術(shù)顯微鏡下眶內(nèi)暴露視神經(jīng)，在球后2mm處切斷視神經(jīng)，分層縫合，于各時間點取材。 視網(wǎng)膜鋪片和計數(shù)：各觀察時間點，處死大鼠取出眼球，固定后剝離視網(wǎng)膜分鼻上、鼻下、顳上、顳下四部分鋪片，在熒光顯微鏡下，距視盤0.5、1.5、2.5mm處各拍攝200×的熒光照片各3張。用image-pro6.0軟件對照片上標(biāo)記的RGCs進行計數(shù)，求平均值，4個部分RGCs數(shù)量累加轉(zhuǎn)化成單位面積RGCs密度。 結(jié)果經(jīng)黃芪甲苷處理的動物，切斷視神經(jīng)5、7、14d后的存活節(jié)細胞的平均密度為（1359±193）/mm2，（1046±175）/mm2和（514±67）/mm2，與正常視網(wǎng)膜節(jié)細胞密度相比，其存活率分別為60.94%、46.90%和23.04%。5、7d、14d組的節(jié)細胞存活率增加了9.59%、11.27%、11.32%。經(jīng)方差分析，黃芪甲苷處理組與視神經(jīng)切斷組和生理鹽水對照組相比在視神經(jīng)切斷后不同時間段其P值均小于0.05，表明它們存在顯著性差異。 結(jié)論黃芪甲苷對視神經(jīng)損傷后視網(wǎng)膜節(jié)細胞存活有促進作用。 第二部分黃芪甲苷對視網(wǎng)膜節(jié)細胞再生的影響 目的研究黃芪甲苷對視網(wǎng)膜節(jié)細胞再生的影響。 方法選用SD大鼠，體重200～250g，動物隨機分組：生理鹽水對照組、AST處理組（各組又分為21、28d組）以及量效關(guān)系組（21d）n=5。視神經(jīng)再生模型操作：動物麻醉后手術(shù)顯微鏡下眶內(nèi)暴露視神經(jīng)，距球后1mm切斷視神經(jīng)，移植一段2cm自體坐骨神經(jīng)，取材前3d修剪移植的自體坐骨神經(jīng)為1.5cm并放置FG逆行標(biāo)記再生的視網(wǎng)膜節(jié)細胞，于21、28d取材。視網(wǎng)膜鋪片的RGCs計數(shù)同前。 結(jié)果經(jīng)黃芪甲苷處理組，術(shù)后3、4周再生視網(wǎng)膜節(jié)細胞平均密度為（978±99）/mm2和（546±61）/mm2，，與生理鹽水對照組相比較，t檢驗，存在顯著性差異(p0.05)。 結(jié)論黃芪甲苷對視神經(jīng)損傷后視網(wǎng)膜節(jié)細胞再生有促進作用。
[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.

【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R774

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