Brn-3a标记大鼠视网膜神经节细胞的实验研究
发布时间：2019-03-30 12:08
【摘要】：目的 对比Brn-3a与Thy-1.1标记大鼠视网膜神经节细胞(RGCs)的特点及计数差异，评估Brn-3a是否可作为一种可靠的内源性标记物，标记并计数大鼠RGCs。 方法 20只SD大鼠，随机选取大鼠一眼为Thy-1.1组，另一眼作为Brn-3a组,过量麻醉处死，行3点钟位缝线标记,立即摘除眼球，浸泡于4%多聚甲醛的固定液中。48h后沿角巩膜缘切除眼前节，剥离晶状体，余下部分逐节脱水、透明、石蜡包埋，，于视乳头颞侧旁开1mm，做4μm切片，烘干备用，做视网膜石蜡切片，常规苏木素-伊红染色镜下观察RGCs的形态，运用免疫组织化学法检测Brn-3a与Thy-1.1的表达情况，每只眼球取1张切片，在每张待测视网膜切片上，以视网膜后极部中点为起点，至近角膜缘视网膜止点处，将视网膜分为三等份，即中央区、中间区及周边区，每个区域视网膜随机取5个高倍视野，分别计数Thy-1.1、Brn-3a阳性细胞均数。 结果 光镜下正常大鼠的视网膜从内向外依次为神经节细胞层(GCL)、内核层(INL)、外核层(ONL)。GCL的细胞呈单层排列，较为整齐，胞核清楚数目相对较少，INL和ONL呈多层排列，且细胞排列紧密，数目相对较多。两种标记物染色都仅仅存在于视网膜的GCL，但在大鼠RGCs中分布呈明显差异：Brn-3a主要在胞核中着色，Thy-1.1主要在胞浆中着色；运用此两种标记物标记计数RGCs,中央区Thy-1.1组(n=20)和Brn-3a组(n=20)分别是29.75±2.02（个/高倍视野）和29.46±1.25（个/高倍视野），中间区Thy-1.1组(n=20)和Brn-3a组(n=20)分别是24.72±1.10（个/高倍视野）和24.43±1.66（个/高倍视野），周边区Thy-1.1组(n=20)和Brn-3a组(n=20)分别是21.79±1.25（个/高倍视野）和21.84±1.37（个/高倍视野）,各个相同区域内Thy-1.1和Brn-3a阳性细胞计数比较，差异均无统计学意义（P0.05)。Thy-1.1组中央区、中间区和周边区两两比较，差异均有统计学意义（P 0.05），Brn-3a组中央区、中间区和周边区两两比较，差异均有统计学意义（P 0.05）。 结论 在大鼠视网膜中Brn-3a阳性细胞仅存在于视网膜的GCL，主要在胞核中着色。与Thy-1.1标记大鼠RGCs比较, Brn-3a标记的数量基本相当。Brn-3a可以作为一种比较可靠的内源性标记物，标记并计数大鼠RGCs。
[Abstract]:Objective to compare the characteristics and counts of (RGCs) labeled by Brn-3a and Thy-1.1 in rat retinal ganglion cells, and to evaluate whether Brn-3a can be used as a reliable endogenous marker for labeling and counting rat RGCs.. Methods Twenty SD rats were randomly selected as Thy-1.1 group and Brn-3a group respectively. The rats were killed under excessive anesthesia and were labeled with suture at 3 o'clock, and the eyeball was removed immediately. 48 hours later, the anterior segment of the eye was removed along the border of the cornea and sclera, and the lens was stripped. The rest was dehydrated, transparent, and paraffin-embedded, and opened at the side of the temporal side of the optic papilla for 1 mm, then sliced 4 渭 m and dried for reserve. The morphology of RGCs was observed under routine hematoxylin-eosin staining. The expression of Brn-3a and Thy-1.1 was detected by immunohistochemical method. One slice was taken from each eye and on each retinal section to be tested. Taking the middle point of the posterior pole of retina as the starting point, to the end point of the retina near the limbus of cornea, the retina is divided into three parts, namely, the central area, the intermediate area and the peripheral area. The retina of each region is randomly taken out of 5 high magnification visual fields, and the Thy-1.1, is counted respectively. Mean number of Brn-3a positive cells. Results under light microscope, the retina of normal rats was composed of ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL) in turn from inside to outside. The cells of GCL were arranged in a monolayer, and the number of clear nuclei was relatively small. INL and ONL were arranged in multiple layers, and the cells were arranged closely, and the number of cells was relatively large. The staining of the two markers only existed in the retina of GCL, but the distribution of the two markers in the rat RGCs was significantly different: Brn-3a was mainly stained in the nucleus and Thy-1.1 was mainly in the cytoplasm; These two markers were used to count the Thy- 1.1 group (n = 20) and the Brn-3a group (n = 20) in the central area of RGCs, which were 29.75 卤2.02 (/ HVA) and 29.46 卤1.25 (HPF), respectively. In the middle region, Thy- 1.1 (n = 20) and Brn-3a (n = 20) were 24.72 卤1.10 (/ high visual field) and 24.43 卤1.66 (/ high power visual field), respectively. In peripheral area, Thy- 1.1 group (n = 20) and Brn-3a group (n = 20) were 21.79 卤1.25 and 21.84 卤1.37, respectively. The number of Thy-1.1 and Brn-3a positive cells in the same area was significantly higher than that in control group (P < 0.05), and the number of positive cells in the same area was significantly higher than that in control group (P < 0.05). There was no statistically significant difference (P0.05) in the central, middle and peripheral regions of the Brn-3a group (P0.05), and there was a significant difference in the central, intermediate and peripheral regions of the THEY group (P0.05), and there was no significant difference in the central area, middle area and peripheral area between the two groups (P0.05). The difference was statistically significant (P 0.05). Conclusion in rat retina, Brn-3a-positive cells are mainly stained in the nucleus of the retina, but only in the retina. Compared with Thy-1.1 labeled rat RGCs, the number of Brn-3a labeled rats was about the same. Brn-3a could be used as a reliable endogenous marker to mark and count rat RGCs..
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1
本文编号：2450063
[Abstract]:Objective to compare the characteristics and counts of (RGCs) labeled by Brn-3a and Thy-1.1 in rat retinal ganglion cells, and to evaluate whether Brn-3a can be used as a reliable endogenous marker for labeling and counting rat RGCs.. Methods Twenty SD rats were randomly selected as Thy-1.1 group and Brn-3a group respectively. The rats were killed under excessive anesthesia and were labeled with suture at 3 o'clock, and the eyeball was removed immediately. 48 hours later, the anterior segment of the eye was removed along the border of the cornea and sclera, and the lens was stripped. The rest was dehydrated, transparent, and paraffin-embedded, and opened at the side of the temporal side of the optic papilla for 1 mm, then sliced 4 渭 m and dried for reserve. The morphology of RGCs was observed under routine hematoxylin-eosin staining. The expression of Brn-3a and Thy-1.1 was detected by immunohistochemical method. One slice was taken from each eye and on each retinal section to be tested. Taking the middle point of the posterior pole of retina as the starting point, to the end point of the retina near the limbus of cornea, the retina is divided into three parts, namely, the central area, the intermediate area and the peripheral area. The retina of each region is randomly taken out of 5 high magnification visual fields, and the Thy-1.1, is counted respectively. Mean number of Brn-3a positive cells. Results under light microscope, the retina of normal rats was composed of ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL) in turn from inside to outside. The cells of GCL were arranged in a monolayer, and the number of clear nuclei was relatively small. INL and ONL were arranged in multiple layers, and the cells were arranged closely, and the number of cells was relatively large. The staining of the two markers only existed in the retina of GCL, but the distribution of the two markers in the rat RGCs was significantly different: Brn-3a was mainly stained in the nucleus and Thy-1.1 was mainly in the cytoplasm; These two markers were used to count the Thy- 1.1 group (n = 20) and the Brn-3a group (n = 20) in the central area of RGCs, which were 29.75 卤2.02 (/ HVA) and 29.46 卤1.25 (HPF), respectively. In the middle region, Thy- 1.1 (n = 20) and Brn-3a (n = 20) were 24.72 卤1.10 (/ high visual field) and 24.43 卤1.66 (/ high power visual field), respectively. In peripheral area, Thy- 1.1 group (n = 20) and Brn-3a group (n = 20) were 21.79 卤1.25 and 21.84 卤1.37, respectively. The number of Thy-1.1 and Brn-3a positive cells in the same area was significantly higher than that in control group (P < 0.05), and the number of positive cells in the same area was significantly higher than that in control group (P < 0.05). There was no statistically significant difference (P0.05) in the central, middle and peripheral regions of the Brn-3a group (P0.05), and there was a significant difference in the central, intermediate and peripheral regions of the THEY group (P0.05), and there was no significant difference in the central area, middle area and peripheral area between the two groups (P0.05). The difference was statistically significant (P 0.05). Conclusion in rat retina, Brn-3a-positive cells are mainly stained in the nucleus of the retina, but only in the retina. Compared with Thy-1.1 labeled rat RGCs, the number of Brn-3a labeled rats was about the same. Brn-3a could be used as a reliable endogenous marker to mark and count rat RGCs..
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1
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本文编号：2450063
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