E1A激活基因阻遏子抑制大鼠视网膜光损伤感光细胞凋亡及其机制的研究

发布时间：2018-05-12 02:36

本文选题：CREG + 光损伤　；参考：《内蒙古民族大学》2017年硕士论文

【摘要】：目的:研究CREG蛋白对抗大鼠视网膜感光细胞光损害的作用,客观评价其抗凋亡的能力,探索CREG在感光细胞光损伤中的抗凋亡机制。方法:1、造模:选取健康wistar大鼠30只,雌雄不限,将其随机分成3组,对照组、光照1周组和光照2周组,每组10只。对3组大鼠鼠眼的冰冻切片行HE染色,确立模型为光照一周组。2、提取光照1周组和对照组大鼠视网膜蛋白,为检测感光细胞凋亡的途径,用Western blot检测MAPK信号通路中关键蛋白ERK、P38、JNK及其磷酸化的表达量。3、选取30只wistar大鼠随机分成3组,制备光损伤模型,制备完毕后将4μlCREG蛋白和4μlPBS分别注入大鼠的左右眼玻璃体腔。注射后第1天、3天、7天摘取鼠眼,行冰冻切片及HE染色,提取大鼠视网膜蛋白,Western blot检测大鼠视网膜蛋白中凋亡因子casepase3、8、9的表达量。4、Western blot检测MAPK凋亡通路及PI3K/AKT通路中关键蛋白P38、JNK、AKT及它们的磷酸化形式的表达量。将P38抑制剂SB203580,JNK抑制剂SP600125,AKT抑制剂LY2940002对3天组大鼠注射CREG蛋白眼进行玻璃体腔注射,剂量为4μl,对照组注射同等剂量的PBS。Western blot检测两组中Caspase3的表达情况。结果:1、光照1周组视网膜外核层细胞较对照组变薄,光照2周视网膜外核层细胞消失。2、光照1周组的大鼠视网膜蛋白中p-P38、p-JNK的表达量较对照组的明显增加(P0.001),P38、JNK、ERK、p-ERK的表达量较对照组无明显变化。3、CREG蛋白注射3天组较对照组视网膜外核层细胞凋亡减少,而1天组和7天组无明显变化。4、CREG蛋白注射3天组大鼠的p-JNK、p-P38、较对照组明显减少(P0.001),p-AKT表达量明显增加(P0.001)。而1天组和7天组较对照组无明显变化。5、P38、JNK、AKT抑制剂注射组中的Caspase3的表达量较对照组明显增加(P0.001)。结论:1、视网膜光损伤感光细胞的凋亡可以通过MAPK信号通路中关键蛋白P38和JNK介导。2、CREG蛋白注射大鼠玻璃体腔后3天组能够抑制光损伤感光细胞的凋亡。3、CREG可以通过调节MAPK及PI3K/AKT信号通路,进而抑制光损伤细胞的凋亡。
[Abstract]:Aim: to study the effect of CREG protein on photodamage of photoreceptor cells in rat retina, evaluate its ability of anti-apoptosis and explore the mechanism of anti-apoptosis of CREG in photoreceptor cells. Methods: 30 healthy wistar rats, male and female, were randomly divided into 3 groups: control group, 1 week illumination group and 2 week illumination group, 10 rats in each group. The frozen sections of the eyes of the three groups were stained with HE, and the model was established as one week light group. The retinal protein was extracted from the rats in the first week group and the control group, which was the way to detect the apoptosis of photoreceptor cells. Western blot was used to detect the expression of ERKN P38 Western blot and its phosphorylation in MAPK signal pathway. Thirty wistar rats were randomly divided into 3 groups to prepare the model of light injury. After the preparation, 4 渭 lCREG protein and 4 渭 lPBS were injected into the vitreous cavity of the rats. Mouse eyes were removed on the 1st day and 7th day after injection. Frozen sections and HE staining were performed. The expression of apoptotic factor casepase3m8k9 in rat retina protein was detected by Western blot. 4Western blot was used to detect the expression of the key protein P38 blot and its phosphorylated form in MAPK pathway and PI3K/AKT pathway. P38 inhibitor SB203580 JNK inhibitor SP600125AKT inhibitor LY2940002 was injected into the vitreous body cavity of the rats of the 3-day group, the dose was 4 渭 l. The expression of Caspase3 in the two groups was detected by injecting the same dose of PBS.Western blot into the control group. Results the cells in the outer nuclear layer of the retina were thinner in the 1 week group than in the control group. After 2 weeks of illumination, the cells of the outer nuclear layer of the retina disappeared. 2. The expression of p-P38 + p-JNK in the retinal protein of the rats exposed to light for 1 week was significantly higher than that in the control group. There was no significant change in the expression of p-P38-P3NK ERKPERKPERK in the control group after 3 days of injection of p0.001, and the expression of the p-JNK protein in the 3-day group was significantly higher than that in the control group. Apoptosis in the outer nuclear layer of omentum decreased, However, there was no significant change in the expression of p-JNKN p-P38 in the rats in the 1-day and 7-day groups. The expression of p-JNKK p-P38 in the 3-day group was significantly decreased compared with the control group, and the expression of p-AKT in the P0.001 group was significantly increased than that in the control group (P 0.001). There was no significant change in the expression of Caspase3 in the control group compared with the control group. The expression of Caspase3 in the control group was significantly higher than that in the control group. Conclusion the apoptosis of photoreceptor cells induced by retinal light injury can be inhibited by modulating the apoptosis of photoreceptor cells in the rat vitreous cavity 3 days after injection of the critical protein P38 and JNK in the MAPK signaling pathway. Node MAPK and PI3K/AKT signaling pathway, And then inhibit the apoptosis of the cells damaged by light.
【学位授予单位】：内蒙古民族大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R774.1

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