本文選題：甲醇中毒 + 視網(wǎng)膜��； 參考：《昆明醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的探索急性甲醇中毒大鼠視網(wǎng)膜神經(jīng)膠質(zhì)細(xì)胞的病理變化,為進(jìn)一步研究急性甲醇中毒導(dǎo)致視覺功能損害提供實(shí)驗(yàn)依據(jù)。 材料與方法將雌雄不限、體重(280+30)g的100只健康成年SD(Sprague-Dawley)大鼠隨機(jī)分為吸入等比混合氣體(N20/02)高劑量組(A組)、低劑量組(B組)、生理鹽水對照組(C組)及空氣生理鹽水對照組(D組),每組又隨機(jī)分為2h、12h、24h、72h、1w共五個(gè)時(shí)間點(diǎn),每個(gè)時(shí)間點(diǎn)5只,給予大鼠并分別灌胃高劑量甲醇溶液(2g/kg)、低劑量甲醇溶液(Jg/kg)和生理鹽水。采用氣相色譜法檢測大鼠血液中甲醇濃度。應(yīng)用免疫組織化學(xué)單標(biāo)和熒光雙標(biāo)染色觀察大鼠甲醇中毒后視網(wǎng)膜AQP4(水通道蛋白4)、GS(谷氨酰胺合成酶,視網(wǎng)膜Muller細(xì)胞標(biāo)記物)、GFAP(膠質(zhì)纖維酸性蛋白,星形膠質(zhì)細(xì)胞標(biāo)記物)、lectin(凝集素,小膠質(zhì)細(xì)胞標(biāo)記物)的病理學(xué)變化規(guī)律。 結(jié)果(1)大鼠血液中甲醇濃度檢測結(jié)果：大鼠血液甲醇濃度在A組2h、12h、24h,B組2h、12h分別顯著高于C、D組(p0.05),以2h濃度最高,A、B組72h、1w及C、D組血液中甲醇濃度未檢出。(2)大鼠視網(wǎng)膜病理學(xué)變化：①AQP4在A組2h、12h、24h表達(dá)增強(qiáng),A組視網(wǎng)膜AQP4陽性反應(yīng)平均光密度值分別高于B組及C、D組(p0.05,p0.01),特別是在2h,隨著時(shí)間延長逐漸降低。72h和1w與C、D組間差異無顯著性(p0.05)。AQP4在B組2h、12h表達(dá)增強(qiáng),B組視網(wǎng)膜AQP4陽性反應(yīng)平均光密度值顯著高于C、D組(p0.01,p0.05),24h后反應(yīng)逐漸下降,與C、D組之間是沒有顯著性差異(p0.05)。組織學(xué)上視網(wǎng)膜分為內(nèi)層(包括神經(jīng)纖維層、神經(jīng)節(jié)細(xì)胞層、內(nèi)叢狀層、內(nèi)核層)和外層(包括外叢狀層、外核層、外界膜、感光細(xì)胞層),在A組甲醇中毒后2h AQP4陽性反應(yīng)在視網(wǎng)膜內(nèi)層顯著增高,但在外層明顯降低。在A組內(nèi)層和外層間AQP4陽性反應(yīng)與其它組相比均有顯著性差異(p0.01,p0.05)。A組12h AQP4陽性反應(yīng)在視網(wǎng)膜內(nèi)層顯著增強(qiáng),與B、C、D組相比均有顯著性差異(p0.01,p0.05)；在視網(wǎng)膜外層A組12h AQP4陽性反應(yīng)明顯增強(qiáng),與C、D組相比有顯著性差異(p0.05),但是A、B組間無顯著性差異。24h、72h和1w AQP4陽性反應(yīng)在視網(wǎng)膜內(nèi)層和外層A、B、C組間蕪顯著性差異。②視網(wǎng)膜GS免疫陽性細(xì)胞在A組2h表達(dá)增強(qiáng)、在12h組達(dá)到高峰,與C組相比,有顯著統(tǒng)計(jì)學(xué)差異(F=6.592,p0.01),24h組GS表達(dá)逐漸減弱,但仍高于C組(p0.01)。大鼠急性甲醇中毒后GS免疫陽性細(xì)胞突起不連續(xù),細(xì)胞結(jié)構(gòu)紊亂。A組12h、24h,GS免疫陽性細(xì)胞表達(dá)高于B組(p0.05),72h、1w GS表達(dá)逐漸恢復(fù)至正常水平(p0.05)。在B組GS免疫陽性細(xì)胞表達(dá)2h開始增強(qiáng),12h達(dá)到高峰,高于C、D組(p0.05),24h以后GS表達(dá)逐漸恢復(fù)至正常。在視網(wǎng)膜內(nèi)層,GS免疫陽性細(xì)胞在A組12h表達(dá)最強(qiáng),高于C、D組(p0.05)；在視網(wǎng)膜外層,GS免疫陽性細(xì)胞與各組間相比,差異無統(tǒng)計(jì)學(xué)意義(p0.05)。③視網(wǎng)膜GFAP免疫表達(dá)在生理鹽水對照組主要表達(dá)于神經(jīng)纖維層和節(jié)細(xì)胞層,甲醇中毒A組2h可見GFAP免疫陽性細(xì)胞突起增粗增長,表達(dá)高于C、D組(p0.05),GFAP免疫陽性細(xì)胞在12h、24h沿內(nèi)叢狀層和內(nèi)核層一直延伸,以12h最為明顯,平均光密度值高于C、D組和B組(p0.01,p0.05),隨著時(shí)間的推移,GFAP免疫陽性細(xì)胞由內(nèi)核層伸出突起在內(nèi)、外界膜之間的表達(dá)逐漸減弱,A組72h和1w與C、D組間沒有顯著性差異(p0.05)。B組各時(shí)間點(diǎn)GFAP的表達(dá)變化與A組相對應(yīng)時(shí)間點(diǎn)相似,但陽性反應(yīng)較弱。在視網(wǎng)膜內(nèi)層,A組GFAP免疫陽性細(xì)胞表達(dá)2h增多、12h達(dá)到高峰、24h下降,但均高于C、D組(p0.05)；B組視網(wǎng)膜內(nèi)層GFAP免疫陽性細(xì)胞12h、24h表達(dá)高于C、D組(p0.05)。GFAP表達(dá)在A、B組72h和1w逐漸降低,GFAP免疫陽性細(xì)胞伸出的突起較長,直達(dá)外界膜。④GS與GFAP,AQP4與GS免疫熒光雙標(biāo)染色顯示：在視網(wǎng)膜內(nèi)層內(nèi)界膜到內(nèi)核層可見GS與GFAP有絲狀的共表達(dá)；Muller細(xì)胞胞體位于的內(nèi)核層,突起貫穿內(nèi)、外界膜,可見胞體及突起均與AQP4有共表達(dá),表明Muller細(xì)胞表達(dá)AQP4。⑤視網(wǎng)膜lectin熒光染色顯示：A組12h、24h、72h及B組24h、72h,lectin日性細(xì)胞數(shù)量逐漸增多,在72h達(dá)高峰。一個(gè)顯著的特點(diǎn)是,它們從視網(wǎng)膜內(nèi)層向視網(wǎng)膜外層遷移突起增粗變短,與C、D組有顯著性差異(F=17.664,p0.01),A、B組間差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。在視網(wǎng)膜內(nèi)層,A組12h、24h、72h,B組72h可見lectin陽性細(xì)胞表達(dá)在神經(jīng)節(jié)細(xì)胞層,內(nèi)叢狀層,內(nèi)核層,數(shù)量以72h組最多,與生理鹽水對照組相比有顯著性差異(p0.01)。在視網(wǎng)膜外層,A組24h、72h,B組72h lectin陽性細(xì)胞數(shù)量較生理鹽水對照組增多(p0.01),其余各組間差異無顯著性。 結(jié)論(1)大鼠吸入等比混合氣體(N2O/O2)并分別灌胃高、低劑量甲醇溶液能成功復(fù)制急性甲醇中毒高、低劑量大鼠模型。(2)大鼠視網(wǎng)膜內(nèi)層Muller細(xì)胞早期谷氨酰胺合成酶增多,水通道蛋白4表達(dá)異常增高及異常表達(dá)膠原纖維酸性蛋白,可能與視網(wǎng)膜水腫有關(guān),程度與甲醇中毒劑量有關(guān)；小膠質(zhì)細(xì)胞被激活,從視網(wǎng)膜內(nèi)層遷移到外層,一方面可能對視網(wǎng)膜Muller細(xì)胞和其它細(xì)胞具有保護(hù)作用,另一方面也可能加重視網(wǎng)膜炎癥反應(yīng),造成神經(jīng)損害,有待進(jìn)一步研究。
[Abstract]:Objective to explore the pathological changes of retinal glial cells in rats with acute methanol poisoning, and to provide an experimental basis for further study of visual impairment caused by acute methanol poisoning.
Materials and methods 100 healthy adult SD (Sprague-Dawley) rats of body weight (280+30) g were randomly divided into high dose group of inhaled isometric mixed gas (N20/02), low dose group (A group), low dose group (B group), normal saline control group (C group) and air physiological saline group (D group), each group was randomly divided into 2h, 12h, 24h, and every five time points, each At a time point, 5 rats were given a high dose of methanol solution (2g/kg), a low dose of methanol solution (Jg/kg) and normal saline. The concentration of methanol in the blood of rats was detected by gas chromatography. The retinal AQP4 (water channel protein 4) and GS (glutamine) were observed by immunohistochemistry and fluorescence double staining. Synthetase, retina Muller cell marker), GFAP (glial fibrillary acidic protein, astrocyte marker), and pathological changes of lectin (lectin, microglia markers).
Results (1) the test results of the concentration of methanol in the blood of rats: the concentration of methanol in the blood of rats in group A 2h, 12h, 24h, B group 2h, 12h was significantly higher than that of C, D group (P0.05), 2h concentration was the highest, and the concentration of methanol in the blood was not detected. (1) the pathological changes in retina of rats: (1) The average optical density of sexual response was higher than that of B group and C, group D (P0.05, P0.01), especially in 2H, and gradually decreased.72h and 1W and C with time, and there was no significant difference between D groups (P0.05). There is no significant difference between the groups (P0.05). Histologically, the retina is divided into the inner layer (including the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the core layer) and the outer layer (including the outer plexiform layer, outer nucleus, external membrane, photoreceptor layer). In the group A, the positive reaction of 2H AQP4 in the inner layer of the retina significantly increases after the methanol intoxication, but the outer layer is obviously reduced. The positive reaction of AQP4 between the inner layer and the outer layer of the A group was significantly different from the other groups (P0.01, P0.05), and the positive reaction of 12h AQP4 in the.A group was significantly enhanced in the retina, and there were significant differences (P0.01, P0.05) with B, C and D groups. 05), however, there was no significant difference between A and B groups.24h, 72h and 1W AQP4 positive reactions in the inner and outer layers of A, B, and C group. 2. The expression of GS immunoreactive cells in the retina was enhanced in A group, and reached a peak in the group of 2H. 0.01. After acute methanol poisoning, GS immunoreactive cells were discontinuous, and the expression of 12h, 24h, GS immunoreactive cells in group.A was higher than that in group B (P0.05), and the expression of 72h and 1W GS gradually recovered to the normal level (P0.05). In the inner layer of the retina, the expression of GS immunoreactive cells in the A group was the strongest, higher than that of C, D group (P0.05), and in the outer retina, the GS immunoreactive cells were not significantly different from each other (P0.05). (3) the GFAP immune expression in the retina was mainly expressed in the nerve fiber layer and the ganglion layer, and the methanol in the saline control group was mainly expressed in the nerve fiber layer and the ganglion cell layer. 2H positive cells of GFAP immunoreactive cells grew thicker than C, D group (P0.05), GFAP immunoreactive cells in 12h, 24h along the inner plexiform layer and core layer, and 12h most obvious, the average optical density value was higher than C, the D group and the group (GFAP) protruded out with the kernel layer over time. There was no significant difference between the outer membrane and the 72h and 1W in the A group (P0.05). The expression of GFAP in the.B group was similar to that in the A group, but the positive reaction was weak. In the inner layer of the retina, the GFAP immunoreactive cells in the A group expressed more 2H, reaching the peak and decreasing. The expression of GFAP immunoreactive cells in the inner retina of the retina was higher than that of C, and the expression of 24h in group D (P0.05).GFAP expressed in A, B group 72h and 1W gradually decreased, and the protuberance extended by the GFAP immunoreactive cells was longer and directly reached the outer membrane. The expression of Muller cell body is located in the core layer, penetration, external membrane, visible cell body and protuberance are co expressed with AQP4, indicating that Muller cells express AQP4. retinal lectin fluorescence staining: A group 12h, 24h, 72h and B 24h, 72h, the number of daily cells in the peak. The migration of the retina from the inner retina to the outer layer of the retina became thicker and shorter. There was a significant difference between the group of C and D (F=17.664, P0.01), A, and B group had statistical significance (P0.05). In the inner retina, A group 12h, 24h, 72h. There was a significant difference in the saline control group (P0.01). In the outer retina, the number of 72h lectin positive cells in group A, 24h, 72h and B increased (P0.01) in the saline control group (P0.01), and there was no significant difference between the other groups.
Conclusion (1) rats inhaled the isometric mixture of gas (N2O/O2) and gavage respectively. The low dose methanol solution could successfully replicate the high and low dose rat models of acute methanol poisoning. (2) the increase of glutamine synthetase in the early Muller cells in the inner retina of the rats, the abnormal increase of the aquaporin 4 and the abnormal expression of the collagen fibrillary acidic protein, may be associated with the abnormal expression of collagen fibrillary acidic protein. The degree of retinal edema is related to the dose of methanol poisoning. Microglia is activated and migrated from the inner layer of the retina to the outer layer. On the one hand, it may have protective effect on the retinal Muller cells and other cells. On the other hand, it may also aggravate the retinal inflammation and cause nerve damage, which needs further study.
【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】：2013
【分類號】：R595.6

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本文編號：2076590