重组人促红细胞生成素对眼挫伤后大鼠视网膜保护作用机制

发布时间：2018-01-16 09:02

本文关键词：重组人促红细胞生成素对眼挫伤后大鼠视网膜保护作用机制　出处：《辽宁医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的通过建造大鼠视网膜挫伤模型，观察视网膜内Bcl-2和Bax蛋白表达的变化，探讨重组人促红细胞生成素对挫伤视网膜Bcl2和Bax的调节作用，明确重组人促红细胞生成素是否对挫伤的大鼠视网膜具有保护作用。探讨重组人促红细胞生成素对眼挫伤大鼠视网膜保护作用的可能机制。方法雄性大鼠45只，两月龄，5只用于正常对照组，余下40只仿Allen重击法制作视网膜挫伤模型致左眼挫伤。将模型制备成功大鼠随机分为两组：模型组和rhEPO治疗组。每组又分12h，24h，3d，7d四个时间点，每个时间点大鼠5只。rhEPO组腹腔注射rhEPO（60IU/Kg），每日一次；模型组用同体积的生理盐水腹腔注射。各时段于给药给水停止后第2天取材，石蜡切片。应用HE染色方法观察各组视网膜各层细胞的形态改变；应用免疫组织化学方法检测视网膜组织中Bcl-2和Bax蛋白的表达变化。结果 1、视网膜各层组织病理学改变 HE染色观察可见正常对照组视网膜各层细胞数量多，排列整齐，形态较完整。而模型组与rhEPO组视网膜全层细胞数减少，视网膜厚度较正常组明显变薄，各层细胞排列紊乱，细胞水肿。 2、Bcl-2蛋白检测结果 Bcl-2免疫组织化学染色显示：光镜下Bcl-2免疫反应产物呈棕黄色。正常眼视网膜Bcl-2蛋白在神经节细胞层、内核层和外核层细胞的胞浆均有表达。视网膜挫伤后，Bcl-2蛋白表达随时间的延长逐渐下降，至伤后3d，下降到最低点，7d回升。在模型组，除伤后12h外，其余时间点Bcl-2蛋白表达明显低于正常组（P0.05）；在rhEPO组，，Bcl-2蛋白表达变化趋势与模型组相似，但在24h、3d、7d，Bcl-2蛋白表达较模型组明显增强，其蛋白阳性产物的平均光密度值(Mean optical density,MOD)明显高于模型组（P0.01）。各组大鼠的阴性对照片未见阳性反应发生。 3、Bax蛋白检测结果光镜下，Bax蛋白免疫反应产物呈棕黄色。正常组视网膜Bax蛋白在神经节细胞层、内核层和外核层及感光细胞层的胞浆均有表达。挫伤后12h，视网膜Bax蛋白的表达与正常组无显著性差异（P0.05）。但挫伤后24h的表达明显增加，3d达最高峰，7d回落。除伤后12h外，模型组Bax蛋白表达比正常对照组显著增高（P0.01）。在rhEPO组，除12h外，Bax蛋白表达在其余时间点较模型组明显降低，其蛋白阳性产物的平均光密度值(Mean optical density, MOD)明显低于模型组（P0.01）。结论 Bcl-2和Bax参与了眼挫伤视网膜病变过程；rhEPO能够调节挫伤后大鼠视网膜细胞凋亡因子Bcl-2和Bax的表达，抑制视网膜损伤后神经元的凋亡。rhEPO对眼挫伤视网膜病变有保护作用。
[Abstract]:Purpose The changes of Bcl-2 and Bax protein expression in retina were observed and the regulatory effects of recombinant human erythropoietin on Bcl2 and Bax in retina contusion were studied by establishing a rat model of retinal contusion. To determine whether recombinant human erythropoietin has protective effect on the retina of contusion rats and to explore the possible mechanism of the protective effect of recombinant human erythropoietin on the retina of eye contusion rats. Method 45 male rats and 5 male rats of two months old were used as normal control group. The other 40 rats were randomly divided into two groups: model group and rhEPO group, each group was divided into 12 hours and 24 hours. Five rats in each time point were injected intraperitoneally with rhEPO-60IUP / KG once a day. The rats in the model group were injected intraperitoneally with normal saline of the same volume. The samples were taken on the second day after the administration of water and the paraffin sections were taken. The morphological changes of the cells in each layer of retina were observed by HE staining. Immunohistochemical method was used to detect the expression of Bcl-2 and Bax in retinal tissues. Results 1. Histopathological changes in all layers of retina He staining showed that the number of retinal cells in the normal control group was more than that in the control group, and the number of cells in the whole retinal layer was decreased in the model group and rhEPO group. The thickness of retina was obviously thinner than that of normal group, the cells in each layer were disordered, and the cells were edema. 2detection of Bcl-2 protein Bcl-2 immunohistochemical staining showed that the immunoreactive product of Bcl-2 was brown under light microscope, and Bcl-2 protein of retina of normal eyes was in ganglion cell layer. After retinal contusion, the expression of Bcl 2 protein decreased gradually with the extension of time, then decreased to the lowest point at 3 days after injury, and increased at the lowest point 7 days after injury. In the model group, the expression of Bcl 2 protein decreased gradually after retinal contusion. The expression of Bcl-2 protein was significantly lower than that of normal group at 12 h after injury. The change trend of Bcl-2 protein expression in rhEPO group was similar to that in model group, but the expression of Bcl-2 protein was significantly increased at 24 h, 3 d, 7 d, compared with model group. The average optical density of its protein positive product was mean optical density. MOD was significantly higher than that in the model group (P 0.01). No positive reaction was found in the negative photos of the rats in each group. Detection of Bax protein The immunoreactive product of Bax protein was brown under light microscope. Bax protein was expressed in the cytoplasm of ganglion cell layer, nuclear layer, outer nuclear layer and photosensitive cell layer in normal group. 12 hours after contusion, Bax protein was expressed in the cytoplasm of the ganglion cell layer, the inner and outer nuclear layer and the photosensitive cell layer. The expression of Bax protein in the retina was not significantly different from that in the normal group (P 0.05). However, the expression of Bax protein increased significantly at 24 h after contusion and reached the peak on the 3rd day and decreased on the 7th day, except for 12 h after injury. The expression of Bax protein in the model group was significantly higher than that in the normal control group (P 0.01). The expression of Bax protein in the rhEPO group was significantly lower than that in the model group at other time points except 12 h. The mean optical density (mod) of the protein positive product was significantly lower than that of the model group (P 0.01). Conclusion Bcl-2 and Bax were involved in the process of ocular contusion retinopathy. RhEPO can regulate the expression of Bcl-2 and Bax in rat retinal cells after contusion. Inhibition of neuronal apoptosis after retinal injury. RhEPO has protective effect on retinal contusion.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.1

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