本文選題：神經(jīng)變性疾病 + 小膠質(zhì)細(xì)胞��； 參考：《河北醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的:青光眼是一組以視神經(jīng)凹陷性萎縮和視野缺損為共同特征的疾病,它是多種因素共同作用的結(jié)果。研究表明,青光眼患者外側(cè)膝狀體、視皮質(zhì)等中樞視路也存在相應(yīng)損傷,提示青光眼可能是一種神經(jīng)退變性疾病。傳統(tǒng)的機(jī)械學(xué)說(shuō)和缺血學(xué)說(shuō)并不能完全解釋青光眼神經(jīng)退變機(jī)理。近年來(lái),從免疫學(xué)角度研究青光眼的發(fā)病機(jī)制和治療成為熱點(diǎn)。 越來(lái)越多的證據(jù)表明,小膠質(zhì)細(xì)胞參與了一些神經(jīng)變性疾病的病理過(guò)程,小膠質(zhì)細(xì)胞是視網(wǎng)膜固有的免疫活性細(xì)胞,在青光眼視網(wǎng)膜神經(jīng)節(jié)細(xì)胞凋亡中發(fā)揮了重要的免疫調(diào)節(jié)作用。當(dāng)青光眼損害程度增加時(shí),小膠質(zhì)細(xì)胞及其分泌的基質(zhì)金屬蛋白酶(MMPS)參與了神經(jīng)元凋亡的過(guò)程。MMPS是小膠質(zhì)細(xì)胞分泌的主要蛋白酶類,與多種細(xì)胞外間質(zhì)重塑的正常及病理過(guò)程相關(guān)。其中基質(zhì)金屬蛋白酶2(MMP-2),基質(zhì)金屬蛋白酶9(MMP-9)可以降解多種細(xì)胞外基質(zhì)成份,最終會(huì)導(dǎo)致神經(jīng)元的變性、壞死。本實(shí)驗(yàn)通過(guò)制備大鼠青光眼高眼壓模型,對(duì)視網(wǎng)膜中小膠質(zhì)細(xì)胞及其分泌的MMP-2,9通過(guò)免疫組織化學(xué)染色后分析其表達(dá)情況,旨在對(duì)小膠質(zhì)細(xì)胞及其分泌的MMP-2,MMP-9在青光眼病變發(fā)生發(fā)展中的作用及其作用機(jī)制進(jìn)行探討,以期為今后治療及預(yù)防青光眼提供實(shí)驗(yàn)依據(jù)。 方法:清潔級(jí)健康SD大鼠30只,體重200-300g,雌雄不限,將實(shí)驗(yàn)動(dòng)物隨機(jī)分為2組,一組(正常組)6只,二組(高眼壓組)24只,二組又依據(jù)灌注時(shí)間不同,隨機(jī)分為2小時(shí),48小時(shí),72小時(shí)3小組,每組8只,最終棄去試驗(yàn)中突然猝死及不合標(biāo)準(zhǔn)眼球,每小組及正常組各保留眼球6只,二組制備完高眼壓模型[1]后,10%水合氯醛過(guò)量麻醉處死大鼠,完整取出眼球及眶內(nèi)神經(jīng),用顯微剪,顯微鑷去除結(jié)膜,肌肉完整取出視神經(jīng),PBS(磷酸鹽緩沖液)進(jìn)行清洗,取小于0.5cm×0.5cm×0.1cm組織塊,1/3經(jīng)4%多聚甲醛固定液固定用于HE染色,2/3經(jīng)上述固定液固定后行免疫組化分析MMP-2,MMP-9的和小膠質(zhì)細(xì)胞在視網(wǎng)膜各層的表達(dá)情況,采用多功能真彩色細(xì)胞圖象分析管理系統(tǒng)(Image-Pro Plus 6.0)對(duì)圖象中的陽(yáng)性反應(yīng)部位進(jìn)行平均光密度分析。所得實(shí)驗(yàn)數(shù)據(jù)采用SPSS13. 0統(tǒng)計(jì)軟件分析,組間比較用方差分析,若有顯著性差異,兩組間比較用q檢驗(yàn)。 結(jié)果:高眼壓組與正常組大鼠相比: 1.形態(tài)學(xué)比較:⑴高眼壓組與正常組相比,各個(gè)時(shí)間段各層厚度較正常組未見(jiàn)明顯變化。⑵2小時(shí)組視網(wǎng)膜神經(jīng)節(jié)細(xì)胞細(xì)胞大小一致,輪廓規(guī)則,核清晰,細(xì)胞邊界清楚;48小時(shí)組視網(wǎng)膜神經(jīng)節(jié)細(xì)胞細(xì)胞大小一致,輪廓稍顯不規(guī)則,核深染,細(xì)胞邊界清晰;72小時(shí)組除有上述變化外,外核層可見(jiàn)空泡樣改變。 2.視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層小膠質(zhì)細(xì)胞與MMP-2,MMP-9的免疫組織化學(xué)分析: (1)小膠質(zhì)細(xì)胞在視網(wǎng)膜中的表達(dá): 正常組與高眼壓組的視網(wǎng)膜神經(jīng)纖維層、內(nèi)、外叢狀層及外核層均有少量表達(dá),外核層的陽(yáng)性細(xì)胞分布占主體。正常組視網(wǎng)膜中小膠質(zhì)細(xì)胞平均光密度值為0.1293±0.0021,高眼壓2小時(shí)組,48小時(shí)組,72小時(shí)組的平均光密度值分別為0.1672±0.0042 , 0.3393±0.0077 ,0.4824±0.0038。組間比較:各組間均有顯著性差異,有統(tǒng)計(jì)學(xué)意義(P0.01)。高眼壓組與正常組相比:隨著高眼壓時(shí)間的延長(zhǎng),小膠質(zhì)細(xì)胞在視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層的表達(dá)逐漸增多。 (2)MMP-2在視網(wǎng)膜中的表達(dá): 正常組MMP-2表達(dá)較少,高眼壓組MMP-2的陽(yáng)性顆粒被染成黃色或接近棕黃色,主要散布于視網(wǎng)膜內(nèi)、外叢狀層及外核層,其他各層無(wú)明顯陽(yáng)性表達(dá)。正常組視網(wǎng)膜中MMP-2的平均光密度值為0.1857±0.0035,高眼壓2小時(shí),48小時(shí)組,72小時(shí)組的平均光密度值分別為0.2848±0.0034,0.6464±0.6696,0.8854±0.8069。組間比較:各組間均有顯著性差異,有統(tǒng)計(jì)學(xué)意義(P0.01)。高眼壓組與正常組相比:隨著高眼壓時(shí)間的延長(zhǎng),MMP-2在視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層的表達(dá)逐漸增多。 (3)MMP-9在視網(wǎng)膜中的表達(dá): 正常組MMP-9表達(dá)較少,高眼壓組MMP-9的陽(yáng)性顆粒被染成黃色或接近棕黃色,主要散布于視網(wǎng)膜內(nèi)、外叢狀層及外核層,其他各層無(wú)明顯陽(yáng)性表達(dá)。正常組視網(wǎng)膜中MMP-9的平均光密度值為0.1473±0.037,高眼壓2小時(shí),48小時(shí)組,72小時(shí)組的平均光密度值分別為0.1647±0.0035,0.6692±0.0075,0.8771±0.1333。組間比較:各組間均有顯著性差異,有統(tǒng)計(jì)學(xué)意義(P0.01)。高眼壓組與正常組相比:隨著高眼壓時(shí)間的延長(zhǎng),MMP-9在視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層的表達(dá)逐漸增多。 結(jié)論:1.高眼壓大鼠的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層,輪廓不規(guī)則,核深染,可見(jiàn)空泡樣改變。 2.高眼壓大鼠的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層中,小膠質(zhì)細(xì)胞表達(dá)較正常眼壓時(shí)增高。 3.高眼壓大鼠的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層中,基質(zhì)金屬蛋白酶-2表達(dá)較正常眼壓時(shí)增加。 4.高眼壓大鼠的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層中,基質(zhì)金屬蛋白酶-9表達(dá)較正常眼壓時(shí)增加. 5.小膠質(zhì)細(xì)胞及MMP-2,MMP-9參與了高眼壓大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層的損傷修復(fù)過(guò)程。 6.小膠質(zhì)細(xì)胞及MMP-2,MMP-9后期在高眼壓大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層的高表達(dá)可以增強(qiáng)高眼壓對(duì)視神經(jīng)的損害。
[Abstract]:Objective: glaucoma is a group of diseases with the common characteristics of the atrophy of the optic nerve and the defect of the visual field. It is the result of a variety of factors. The study shows that the lateral geniculate body and the visual cortex of the glaucoma patients also have corresponding injuries, suggesting that glaucoma may be a neurodegenerative disease. The theory of ischemia does not fully explain the mechanism of nerve degeneration in glaucoma. In recent years, the pathogenesis and treatment of glaucoma have been a hot topic from an immunological perspective.
More and more evidence shows that microglia is involved in the pathological process of some neurodegenerative diseases. Microglia is an inherent immune active cell of the retina. It plays an important role in the immunoregulation of retinal ganglion cell apoptosis in glaucoma. When the degree of glaucoma damage is increased, microglia and its secreted matrix Metalloproteinase (MMPS) is involved in the process of neuronal apoptosis..MMPS is a major protease secreted by microglia, which is related to the normal and pathological processes of a variety of extracellular matrix remodeling. Matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) can degrade a variety of extracellular matrix components and eventually lead to neurons The purpose of this study was to investigate the expression of the retinal glial cells and their secreted MMP-2,9 through immunohistochemical staining, and to explore the role of microglia and its secreted MMP-2 and MMP-9 in the development of glaucoma and the mechanism of its action and mechanism. This study will provide an experimental basis for future treatment and prevention of glaucoma.
Methods: 30 healthy SD rats, body weight 200-300g, male and female, were randomly divided into 2 groups, one group (normal group) 6, two group (high intraocular pressure group) 24. The two groups were randomly divided into 2 hours, 48 hours, 72 hours 3 groups, each group 8, and finally abandoned the sudden sudden death and the non standard eyeball, each group. And in the normal group, 6 eyes were retained, and two groups were prepared with high intraocular pressure model [1]. 10% hydrous chloral hyperanaesthesia was executed in rats. The eyeball and the intraorbital nerve were taken out completely. The conjunctiva was removed with microscissors and microtweezers. The muscles were completely removed from the optic nerve and PBS (phosphate buffer) was cleaned, which was less than 0.5cm x 0.5cm x 0.1cm tissue block and 1/3 4% polymethylene. The aldehyde fixed solution was immobilized on HE staining. After the immobilization of 2/3, the expression of MMP-2, MMP-9 and microglia in each layer of the retina was analyzed by immunohistochemistry. The mean optical density analysis of the positive reaction sites in the image was analyzed by the multi-function true color cell image analysis management system (Image-Pro Plus 6). Data were analyzed by SPSS13. 0 statistical software. Variance analysis was used for comparison between groups. If there was significant difference, q test was used in comparison between the two groups.
Results: the high intraocular pressure group was compared with the normal group.
1. morphological comparison: 1. Compared with the normal group, the thickness of each layer did not change significantly compared with the normal group. (2) the size of the retinal ganglion cells in the 2 hour group was the same, the outline rule, the nucleus clear, the cell boundary clearly, and the size of the retinal ganglion cells in the 48 hour group was the same size, the outline was slightly irregular, the nucleus deep dyed, and fine. The cell boundary was clear; except for the above changes, the outer nuclear layer showed vacuolar changes in the 72 hour group.
Immunohistochemical analysis of microglia and MMP-2, MMP-9 in retinal ganglion cell layer 2.
(1) the expression of microglia in the retina:
The retinal nerve fiber layer, inner plexiform layer and outer nucleus of the normal group and the high intraocular pressure group had a small amount of expression, and the positive cell distribution of the outer nucleus was the main body. The average light density value of the normal retina medium and small glial cells was 0.1293 + 0.0021, the high intraocular pressure 2 hours group, the 48 hour group, and the 72 hour group were 0.1672 + 0.0042, respectively. Compared with 0.3393 + 0.0077 and 0.4824 + 0.0038. groups, there were significant differences between the groups (P0.01). The high intraocular pressure group was compared with the normal group: the expression of microglia in the retinal ganglion cell layer gradually increased with the prolongation of high intraocular pressure.
(2) the expression of MMP-2 in the retina:
The expression of MMP-2 in the normal group was less. The positive particles of MMP-2 in the high intraocular pressure group were dyed yellow or close to brown and yellow, mainly scattered in the retina, the outer plexiform layer and the outer nucleus, and there was no obvious positive expression in the other layers. The average light density value of MMP-2 in the retina of the normal group was 0.1857 + 0.0035, the high intraocular pressure was 2 hours, the 48 hour group, and the average light of the 72 hour group. The density values were 0.2848 + 0.0034,0.6464 + 0.6696,0.8854 + 0.8069. groups, respectively. There were significant differences in each group (P0.01). Compared with the normal group, the high intraocular pressure group was compared with the normal group: the expression of MMP-2 in the retinal ganglion cell layer increased gradually as the time of high intraocular pressure was prolonged.
(3) the expression of MMP-9 in the retina:
The expression of MMP-9 in the normal group was less. The positive particles of MMP-9 in the high intraocular pressure group were dyed yellow or close to brown and yellow, mainly scattered in the retina, the outer plexiform layer and the outer nucleus, and there was no obvious positive expression in the other layers. The average optical density value of MMP-9 in the retina of the normal group was 0.1473 + 0.037, the high intraocular pressure was 2 hours, the 48 hour group, and the average light density of the 72 hour group. Compared with 0.1647 + 0.0035,0.6692 + 0.0075,0.8771 + 0.1333. groups, there were significant differences among the groups (P0.01). Compared with the normal group, the high intraocular pressure group increased with the increase of the high intraocular pressure time, the expression of MMP-9 in the retinal ganglion cell layer gradually increased.
Conclusion: 1. the retinal ganglion cell layer in high intraocular pressure rats is irregular in shape, deep in nucleus and vacuolated.
2. the expression of microglia in the retinal ganglion cell layer of rats with high intraocular pressure was higher than that of normal intraocular pressure.
3. the expression of matrix metalloproteinase -2 was increased in the retinal ganglion cell layer of rats with high intraocular pressure than normal intraocular pressure.
4. the expression of matrix metalloproteinase -9 was increased in the retinal ganglion cell layer of rats with high intraocular pressure than normal intraocular pressure.
5. microglia and MMP-2 and MMP-9 are involved in the repair process of retinal ganglion cell layer in rats with ocular hypertension.
6. the high expression of microglia and MMP-2 and MMP-9 in the retinal ganglion cell layer of rats with high intraocular pressure can enhance the damage of optic nerve caused by high intraocular pressure.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R775

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