發(fā)布時(shí)間：2018-08-11 18:21

【摘要】： 目的：探討米諾環(huán)素在L-谷氨酸誘導(dǎo)的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞損傷中的保護(hù)作用和分子機(jī)制。 方法：體外實(shí)驗(yàn),原代小鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞體外穩(wěn)定培養(yǎng)24h后,分為三組：對(duì)照組、L-谷氨酸組及L-谷氨酸+米諾環(huán)素組,相應(yīng)干預(yù)48h后分別觀察各組間神經(jīng)節(jié)細(xì)胞的存活率及神經(jīng)軸突生長(zhǎng)的差異。體內(nèi)實(shí)驗(yàn),將C57BL／6小鼠分為三組：對(duì)照組、L-谷氨酸組及L-谷氨酸+米諾環(huán)素組。前兩組小鼠腹腔內(nèi)注射生理鹽水(對(duì)照組,L-谷氨酸組),第三組腹腔內(nèi)注射米諾環(huán)素(L-谷氨酸+米諾環(huán)素組,60mg/kg),每天一次,連續(xù)7天,第二天時(shí),第一組玻璃體腔內(nèi)注射生理鹽水,后兩組小鼠玻璃體腔內(nèi)注射2ulL-谷氨酸(2mM),誘導(dǎo)視網(wǎng)膜神經(jīng)節(jié)細(xì)胞損傷。第八天采用組織免疫熒光法技術(shù)和激光共聚焦顯微鏡觀察β3tubulin陽性細(xì)胞數(shù)及視網(wǎng)膜GFAP蛋白表達(dá)以評(píng)估視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的損傷情況。Real-time PCR和Western blot法分別檢測(cè)視網(wǎng)膜組織中IFN-γ, IL-1, TNF-α與GFAP與Vimentin的mRNA及蛋白表達(dá)水平以初步探討損傷的分子機(jī)制。 結(jié)果：體外實(shí)驗(yàn)結(jié)果顯示：與對(duì)照組比較,L-谷氨酸組視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的存活率明顯降低,呈劑量和時(shí)間依賴性；神經(jīng)軸突生長(zhǎng)受抑制。米諾環(huán)素干預(yù)后可明顯減輕L-谷氨酸導(dǎo)致的上述效應(yīng)(P0.01)。動(dòng)物實(shí)驗(yàn)結(jié)果顯示,L-谷氨酸組小鼠神網(wǎng)膜神經(jīng)節(jié)細(xì)胞數(shù)目較對(duì)照組小鼠顯著減少,視網(wǎng)膜組織中GFAP的表達(dá)量明顯增加(P0.01)。米諾環(huán)素治療后可明顯改善L-谷氨酸誘導(dǎo)的神經(jīng)節(jié)細(xì)胞損傷并明顯降低其GFAP的表達(dá)(p0.01)。視網(wǎng)膜組織中IFN-γ、IL-1、NF-α與GFAP與Vimentin的mRNA及蛋白水平在L-谷氨酸組較對(duì)照組表達(dá)明顯增高,而米諾環(huán)素可顯著降低這些因子的表達(dá)。 結(jié)論：L-谷氨酸可誘導(dǎo)視網(wǎng)膜神經(jīng)節(jié)細(xì)胞損傷、抑制神經(jīng)軸突生長(zhǎng)和上調(diào)炎癥因子基因與蛋白的表達(dá),而米諾環(huán)素具有明顯改善L-谷氨酸所導(dǎo)致的視網(wǎng)膜神經(jīng)細(xì)胞損傷的作用。
[Abstract]:Aim: to investigate the protective effect and molecular mechanism of minocycline on L-glutamate induced retinal ganglion cell injury. Methods: the primary mouse retinal ganglion cells were cultured in vitro for 24 hours, and were divided into three groups: control group, L-glutamic acid group and L-glutamate minocycline group. The survival rate of ganglion cells and the growth of axons were observed after 48 h intervention. In vivo, C57BL/6 mice were divided into three groups: control group, L-glutamic acid group and L-glutamate minocycline group. The first two groups were intraperitoneally injected with normal saline (control group, L- glutamic acid group), and the third group with minocycline (L-glutamate minocycline group, 60 mg / kg), once a day for 7 days, the second day, the first group was injected with normal saline in vitreous cavity. In the latter two groups, the retinal ganglion cells were injured by intravitreal injection of 2 ulL-glutamic acid (2mM). On the 8th day, the number of 尾 3tubulin positive cells and the expression of retinal GFAP protein were observed by tissue immunofluorescence technique and laser confocal microscope to evaluate the damage of retinal ganglion cells. Real-time PCR and Western blot were used to detect the retinal group. The expression levels of mRNA and protein of IFN- 緯, IL-1, TNF- 偽 and GFAP and Vimentin were studied to explore the molecular mechanism of injury. Results: compared with the control group, the survival rate of retinal ganglion cells in L-glutamic acid group was significantly decreased in a dose-and time-dependent manner, and the growth of axons was inhibited. Minocycline can attenuate the above effects induced by L-glutamic acid (P0.01). The results of animal experiments showed that the number of retinal ganglion cells in the L- glutamic acid group was significantly lower than that in the control group, and the expression of GFAP in the retina was significantly increased (P0.01). Minocycline could significantly improve the injury of ganglion cells induced by L-glutamate and decrease the expression of GFAP (p0.01). The levels of mRNA and protein of IFN- 緯 IL-1NF- 偽, GFAP and Vimentin were significantly higher in L- glutamate group than in control group, but minocycline could significantly decrease the expression of these factors. ConclusionWow-L-glutamic acid can induce retinal ganglion cell injury, inhibit axon growth and up-regulate the expression of inflammatory factor gene and protein. Minocycline can obviously improve the damage of retinal nerve cells induced by L-glutamate.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R774

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