小胶质细胞及其分泌物在急性高眼压大鼠视网膜组织中的表达

发布时间：2018-05-18 20:24

本文选题：神经变性疾病 + 小胶质细胞　；参考：《河北医科大学》2010年硕士论文

【摘要】： 目的:青光眼是一组以视神经凹陷性萎缩和视野缺损为共同特征的疾病,它是多种因素共同作用的结果。研究表明,青光眼患者外侧膝状体、视皮质等中枢视路也存在相应损伤,提示青光眼可能是一种神经退变性疾病。传统的机械学说和缺血学说并不能完全解释青光眼神经退变机理。近年来,从免疫学角度研究青光眼的发病机制和治疗成为热点。越来越多的证据表明,小胶质细胞参与了一些神经变性疾病的病理过程,小胶质细胞是视网膜固有的免疫活性细胞,在青光眼视网膜神经节细胞凋亡中发挥了重要的免疫调节作用。当青光眼损害程度增加时,小胶质细胞及其分泌的基质金属蛋白酶(MMPS)参与了神经元凋亡的过程。MMPS是小胶质细胞分泌的主要蛋白酶类,与多种细胞外间质重塑的正常及病理过程相关。其中基质金属蛋白酶2(MMP-2),基质金属蛋白酶9(MMP-9)可以降解多种细胞外基质成份,最终会导致神经元的变性、坏死。本实验通过制备大鼠青光眼高眼压模型,对视网膜中小胶质细胞及其分泌的MMP-2,9通过免疫组织化学染色后分析其表达情况,旨在对小胶质细胞及其分泌的MMP-2,MMP-9在青光眼病变发生发展中的作用及其作用机制进行探讨,以期为今后治疗及预防青光眼提供实验依据。方法:清洁级健康SD大鼠30只,体重200-300g,雌雄不限,将实验动物随机分为2组,一组(正常组)6只,二组(高眼压组)24只,二组又依据灌注时间不同,随机分为2小时,48小时,72小时3小组,每组8只,最终弃去试验中突然猝死及不合标准眼球,每小组及正常组各保留眼球6只,二组制备完高眼压模型[1]后,10%水合氯醛过量麻醉处死大鼠,完整取出眼球及眶内神经,用显微剪,显微镊去除结膜,肌肉完整取出视神经,PBS(磷酸盐缓冲液)进行清洗,取小于0.5cm×0.5cm×0.1cm组织块,1/3经4%多聚甲醛固定液固定用于HE染色,2/3经上述固定液固定后行免疫组化分析MMP-2,MMP-9的和小胶质细胞在视网膜各层的表达情况,采用多功能真彩色细胞图象分析管理系统(Image-Pro Plus 6.0)对图象中的阳性反应部位进行平均光密度分析。所得实验数据采用SPSS13. 0统计软件分析,组间比较用方差分析,若有显著性差异,两组间比较用q检验。结果:高眼压组与正常组大鼠相比: 1.形态学比较:⑴高眼压组与正常组相比,各个时间段各层厚度较正常组未见明显变化。⑵2小时组视网膜神经节细胞细胞大小一致,轮廓规则,核清晰,细胞边界清楚;48小时组视网膜神经节细胞细胞大小一致,轮廓稍显不规则,核深染,细胞边界清晰;72小时组除有上述变化外,外核层可见空泡样改变。 2.视网膜神经节细胞层小胶质细胞与MMP-2,MMP-9的免疫组织化学分析: (1)小胶质细胞在视网膜中的表达: 正常组与高眼压组的视网膜神经纤维层、内、外丛状层及外核层均有少量表达,外核层的阳性细胞分布占主体。正常组视网膜中小胶质细胞平均光密度值为0.1293±0.0021,高眼压2小时组,48小时组,72小时组的平均光密度值分别为0.1672±0.0042 , 0.3393±0.0077 ,0.4824±0.0038。组间比较:各组间均有显著性差异,有统计学意义(P0.01)。高眼压组与正常组相比:随着高眼压时间的延长,小胶质细胞在视网膜神经节细胞层的表达逐渐增多。 (2)MMP-2在视网膜中的表达: 正常组MMP-2表达较少,高眼压组MMP-2的阳性颗粒被染成黄色或接近棕黄色,主要散布于视网膜内、外丛状层及外核层,其他各层无明显阳性表达。正常组视网膜中MMP-2的平均光密度值为0.1857±0.0035,高眼压2小时,48小时组,72小时组的平均光密度值分别为0.2848±0.0034,0.6464±0.6696,0.8854±0.8069。组间比较:各组间均有显著性差异,有统计学意义(P0.01)。高眼压组与正常组相比:随着高眼压时间的延长,MMP-2在视网膜神经节细胞层的表达逐渐增多。 (3)MMP-9在视网膜中的表达: 正常组MMP-9表达较少,高眼压组MMP-9的阳性颗粒被染成黄色或接近棕黄色,主要散布于视网膜内、外丛状层及外核层,其他各层无明显阳性表达。正常组视网膜中MMP-9的平均光密度值为0.1473±0.037,高眼压2小时,48小时组,72小时组的平均光密度值分别为0.1647±0.0035,0.6692±0.0075,0.8771±0.1333。组间比较:各组间均有显著性差异,有统计学意义(P0.01)。高眼压组与正常组相比:随着高眼压时间的延长,MMP-9在视网膜神经节细胞层的表达逐渐增多。结论:1.高眼压大鼠的视网膜神经节细胞层,轮廓不规则,核深染,可见空泡样改变。 2.高眼压大鼠的视网膜神经节细胞层中,小胶质细胞表达较正常眼压时增高。 3.高眼压大鼠的视网膜神经节细胞层中,基质金属蛋白酶-2表达较正常眼压时增加。 4.高眼压大鼠的视网膜神经节细胞层中,基质金属蛋白酶-9表达较正常眼压时增加. 5.小胶质细胞及MMP-2,MMP-9参与了高眼压大鼠视网膜神经节细胞层的损伤修复过程。 6.小胶质细胞及MMP-2,MMP-9后期在高眼压大鼠视网膜神经节细胞层的高表达可以增强高眼压对视神经的损害。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R775

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