本文選題：視網(wǎng)膜缺血-再灌注損傷 + 小膠質(zhì)細(xì)胞�。� 參考：《昆明醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[目的]通過大鼠前房穿刺灌注,建立不同時(shí)間點(diǎn)的視網(wǎng)膜缺血-再灌注模型,觀察大鼠視網(wǎng)膜損傷后視網(wǎng)膜小膠質(zhì)細(xì)胞的激活狀態(tài)及不同時(shí)間點(diǎn)IL-1β,TNF-a,iNOs等炎性因子的分泌變化,分析小膠質(zhì)細(xì)胞激活與神經(jīng)細(xì)胞死亡的相互關(guān)系,并探討活化小膠質(zhì)細(xì)胞在損傷后釋放炎性細(xì)胞因子的作用機(jī)制,旨在根據(jù)研究結(jié)論對于視網(wǎng)膜缺血性疾病的治療帶來一定的指導(dǎo)意義。[方法]將雙眼成年的SPF級別的雄性SD大鼠,共105只,共105只眼,將其隨機(jī)分為5組,分別為:正常組,假手術(shù)組,損傷后24h組,3d組,7d組,在相同實(shí)驗(yàn)條件下,建立視網(wǎng)膜缺血-再灌注模型,利用30-gauge針頭進(jìn)行前房穿刺,針頭連接0.9%生理鹽水,將瓶高升高至150cm,維持灌注60min后,拔除前房穿刺針頭。在模型建立后24h, 3d及7d后取材,進(jìn)行石蠟包埋切片,視網(wǎng)膜切片通過HE染色和凋亡實(shí)驗(yàn),觀察缺血損傷不同時(shí)間點(diǎn),視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層的厚度改變及神經(jīng)節(jié)細(xì)胞的凋亡情況。通過進(jìn)行視網(wǎng)膜冰凍切片后,免疫熒光染色,特異性觀察小膠質(zhì)細(xì)胞的激活情況及缺血損傷后不同時(shí)間點(diǎn)小膠質(zhì)細(xì)胞的激活比例。利用RT-PCR檢測損傷后不同時(shí)間點(diǎn)的IL-1 β,TNF-a,iNOs等炎性因子的分泌情況與視網(wǎng)膜神經(jīng)節(jié)細(xì)胞損傷的對應(yīng)關(guān)系。通過以上步驟,分析視網(wǎng)膜缺血損傷不同時(shí)間節(jié)點(diǎn),視網(wǎng)膜結(jié)構(gòu)改變與小膠質(zhì)細(xì)胞激活比例、激活后分泌的炎性因子的對應(yīng)關(guān)系。[結(jié)果]對進(jìn)行HE染色的視網(wǎng)膜石蠟切片觀察,可發(fā)現(xiàn):正常組與假手術(shù)組大鼠視網(wǎng)膜各層次結(jié)構(gòu)清楚,各層細(xì)胞排列整齊,厚度均勻,神經(jīng)節(jié)細(xì)胞層各細(xì)胞邊緣清楚,缺血-再灌注損傷后24h,大鼠神經(jīng)節(jié)細(xì)胞出現(xiàn)空泡變性,神經(jīng)節(jié)細(xì)胞數(shù)量變少。缺血-再灌注損傷3d后,相較于對照組的大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層厚度,視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層變薄,神經(jīng)節(jié)細(xì)胞數(shù)量減少,視網(wǎng)膜厚度變薄,缺血損傷后,神經(jīng)節(jié)細(xì)胞層細(xì)胞排列紊亂,部分神經(jīng)節(jié)細(xì)胞細(xì)胞可見明顯空泡變性,胞漿內(nèi)空泡形成,部分細(xì)胞核溶解,殘存的神經(jīng)節(jié)細(xì)胞細(xì)胞核染色深,視網(wǎng)膜內(nèi)網(wǎng)層明顯變薄。缺血-再灌注損傷7d后,視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層厚度較3d組的神經(jīng)節(jié)細(xì)胞層厚度明顯變薄,神經(jīng)節(jié)細(xì)胞數(shù)量明顯減少,大量細(xì)胞空泡變性,另外缺血-再灌注損傷大鼠視網(wǎng)膜內(nèi)網(wǎng)層明顯變薄,內(nèi)核層細(xì)胞水腫明顯,體積增大,染色變淺。視網(wǎng)膜凋亡實(shí)驗(yàn)結(jié)果顯示正常組與假手術(shù)組僅表現(xiàn)背景熒光,無陽性細(xì)胞表達(dá),而在缺血-再灌注損傷后24h,除紅色背景熒光外,可見少量陽性細(xì)胞(P=0.2630.05,無明顯統(tǒng)計(jì)學(xué)意義),陽性反應(yīng)細(xì)胞逐漸增多,主要集中在視網(wǎng)膜內(nèi)層,缺血-視網(wǎng)膜損傷3d后,可見陽性表達(dá)細(xì)胞最多(P=0. 0000. 05,差異存在統(tǒng)計(jì)學(xué)意義),缺血損傷7d后,可也見大量凋亡細(xì)胞,但凋亡細(xì)胞數(shù)量較3d組的減少(P=0.0020.05,差異存在統(tǒng)計(jì)學(xué)意義)視網(wǎng)膜Iba-1標(biāo)記小膠質(zhì)細(xì)胞,在正常組及假手術(shù)組中僅可在視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層看到靜息狀態(tài)的小膠質(zhì)細(xì)胞,可見小膠質(zhì)細(xì)胞胞體呈樹枝狀突起。缺血-再灌注損傷后24h,可見相比較于正常組及假手術(shù)組,小膠質(zhì)細(xì)胞的數(shù)量明顯增多,視網(wǎng)膜神經(jīng)節(jié)層和外叢狀層靜息狀態(tài)下的小膠質(zhì)細(xì)胞大量激活,其中以視網(wǎng)膜神經(jīng)節(jié)層為主,樹枝狀突起回縮,胞體變圓,內(nèi)叢狀層仍可見許多仍處于靜息狀態(tài)的小膠質(zhì)細(xì)胞。在缺血-再灌注損傷3d后,可見視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層、內(nèi)叢狀層、外核層大量小膠質(zhì)細(xì)胞激活。缺血損傷7d后,激活的小膠質(zhì)細(xì)胞數(shù)量下降,但神經(jīng)節(jié)細(xì)胞層及外核層仍可見大量激活的小膠質(zhì)細(xì)胞。正常視網(wǎng)膜內(nèi)IL-Iβ、TNF-α、iNOs的表達(dá)量較低,且與假手術(shù)組炎性因子的表達(dá)無明顯統(tǒng)計(jì)學(xué)差異,缺血-再灌注損傷后24h,IL-1β、TNF-α、iNOs的mRNA表達(dá)量都大幅度增加(與正常對照組相比,P=0. 0000. 05,有統(tǒng)計(jì)學(xué)差異),并且隨損傷時(shí)間的增加而增加,損傷后3d達(dá)到峰值后(與正常對照組相比,P=0. 0000. 05,有統(tǒng)計(jì)學(xué)差異),炎性因子的表達(dá)逐漸下降,損傷后7d,仍可見大量炎性因子表達(dá)。(與正常對照組相比,P=0. 0000. 05,有統(tǒng)計(jì)學(xué)差異)[結(jié)論]視網(wǎng)膜缺血-再灌注損傷后,隨著損傷時(shí)間的延長,視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層厚度、內(nèi)叢狀層厚素及整體視網(wǎng)膜厚度越來越薄。同時(shí),視網(wǎng)膜缺血可刺激視網(wǎng)膜小膠質(zhì)細(xì)胞的增殖及遷移,視網(wǎng)膜中炎性因子IL-1β、TNF-α、iNOs的表達(dá)增加,且與視網(wǎng)膜神經(jīng)節(jié)細(xì)胞凋亡程度、小膠質(zhì)細(xì)胞活化程度相對應(yīng),因此,可認(rèn)為,視網(wǎng)膜缺血-再灌注損傷后,小膠質(zhì)細(xì)胞激活,且激活的小膠質(zhì)細(xì)胞分泌炎性因子IL-1β、TNF-α、iNOs,炎性因子引起視網(wǎng)膜的炎癥反應(yīng)及相應(yīng)結(jié)構(gòu)的病理學(xué)改變。
[Abstract]:[Objective] by rat anterior chamber puncture perfusion, established at different time points of retinal ischemia-reperfusion model to observe retinal microglia retinal injury in rats after activation and different time points IL-1 beta, TNF-a, iNOs and other changes in the secretion of inflammatory factors, analysis of relationship between activation and neuronal cell death of microglia cells, and explore the mechanism of activated microglia release proinflammatory cytokines after injury, according to the conclusion of the study for the treatment of retinal ischemic disease has guiding significance.] some of the methods of male SD rat eyes adult SPF level, a total of 105, a total of 105 eyes were randomly divided into 5 group, respectively: normal group, sham operation group, injury group 24h, 3D group, 7d group, under the same experimental conditions, the establishment of retinal ischemia-reperfusion model of anterior chamber puncture with 30-gauge needle, needle is connected with the 0.9% students Instead, the bottle will rise up to 150cm, maintain perfusion after 60min removal of anterior chamber puncture needle. In the model of 24h, 3D and 7d respectively, were embedded in paraffin, HE staining and retinal slices through the experimental observation of apoptosis, ischemia at different time points, the apoptosis of retinal ganglion cell layer thickness change. And through the retinal ganglion cells after frozen section, immunofluorescence staining, observe the specificity of microglia activation and ischemic injury at different time points after activation of microglia was detected by RT-PCR. The proportion of injury at different time points after TNF-a, IL-1 beta, iNOs and other inflammatory factors secretion and retinal ganglion the relationship between cell damage. Through the above steps, the retinal ischemia injury in different time node analysis, the change of retinal structure and microglial activation ratio, inflammatory activation after secretion The results of the corresponding relationship. The factor of observation, HE staining of the retinal sections can be found: normal group and sham operation group rats retinal layers structure clearly, each layer of cells arranged in neat, uniform thickness, the edge of the cell ganglion cell layer clear, ischemia-reperfusion injury after 24h in rats, ganglion cells vacuolar degeneration of ganglion cells decreased. Ischemia reperfusion injury after 3D, thickness of retinal ganglion cells in rats compared with the control group, the retinal ganglion cell layer became thinner, reducing the number of ganglion cells, retinal thickness, ischemic injury, ganglion cell layer cells arranged in disorder, part of cells of ganglion cells was observed vacuolar degeneration and vacuolization in the cytoplasm, nucleus dissolved, the remnants of the ganglion cell nuclei staining deep, inner plexiform layer was thinner. Ischemia reperfusion injury after 7d, The thickness of the ganglion cell layer of retinal ganglion cell layer thickness was significantly thinner than that in group 3D, significantly reduced the number of ganglion cells, cell vacuolar degeneration, and ischemia reperfusion injury in rats retina network was significantly thinner, inl cell edema, increased volume, superficialdyeing. Experimental results show that the retinal apoptosis and normal group the sham operation group showed only background fluorescence, expression of positive cells in ischemia reperfusion injury after 24h, in addition to the red fluorescent background, positive cells (P=0.2630.05, not statistically significant), positive cells gradually increased, mainly concentrated in the inner retina ischemic retinal injury after 3D, the positive expression of most cells (P=0. 0.05, there were statistically significant differences), ischemic damage after 7d, to see a large number of apoptotic cells can also, but the number of apoptotic cells was decreased in the 3D group (P=0.0020.05, difference There are different statistically significant) retinal Iba-1 labeled microglia, only to see the resting state of microglial cells in the retinal ganglion cell layer in normal group and sham operation group, visible microglia cells with dendritic processes. Ischemia reperfusion injury after 24h, visible compared with the normal group and sham operation group. The number of microglia increased significantly, a large number of activated retinal ganglion layer and outer plexiform layer of resting microglia, the retinal ganglion layer, dendritic retraction, rounded cell body, inner plexiform layer still visible many still in the resting state of microglial cells in ischemia-reperfusion. Reperfusion injury after 3D, retinal ganglion cell layer, inner plexiform layer, outer nuclear layer, a large number of activated microglia. 7d after ischemic injury, activated microglia cells decreased, but the ganglion cell layer and the outer nuclear layer still To see a large number of activated microglia in the normal retina. IL-I beta, TNF- alpha, iNOs expression was low, and there were no significant differences between sham operation group and expression of inflammatory factors, ischemia-reperfusion injury after 24h, IL-1 beta, TNF- alpha, iNOs mRNA expression were significantly increased (compared to with the normal control group, P=0. 0.05, there were significant differences), and increased with the increase of injury, injury after 3D after reaching the peak (compared with the normal control group P=0. 0.05, there were significant differences), expression of inflammatory factors decreased gradually, 7d after injury, there were a lot of inflammatory factor expression (. Compared with the normal control group P=0. 0.05, there were significant differences) [Conclusion] retinal injury after ischemia reperfusion, with the extension of injury, retinal ganglion cell layer thickness, inner plexiform layer thickness and the overall retinal thickness is becoming thinner and thinner. At the same time, retinal ischemia can sting The proliferation and migration induced retinal microglia in the retina, inflammatory factor IL-1 alpha beta, TNF-, increase the expression of iNOs and apoptosis of retinal ganglion cells, corresponding to the degrees, activation of microglia so that damage to the retina after ischemia reperfusion, microglia activation, and the activation of the small glial cells secrete inflammatory factor IL-1 beta, TNF- alpha, iNOs, inflammatory factors induced by inflammatory reaction and corresponding pathological retinal morphology change.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R774.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Wen-Qin Xu;Yu-Sheng Wang;;The role of Toll-like receptors in retinal ischemic diseases[J];International Journal of Ophthalmology;2016年09期

2 李娟娟;李燕;湯志偉;;激活的小膠質(zhì)細(xì)胞在大鼠視網(wǎng)膜缺血再灌損傷模型中的作用[J];眼科新進(jìn)展;2015年01期

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本文編號：1758474