【作者】 刘敏；

【导师】 王雨生；

【作者基本信息】 第四军医大学 ， 眼科学， 2008， 硕士

【摘要】 研究背景缺血缺氧是众多视网膜脉络膜疾病的关键始发因素。缺氧不仅诱导视网膜色素上皮（retinal pigment epithelium, RPE）细胞的增生,而且通过影响相关细胞因子（如VEGF、PEDF等）的分泌水平间接参与了眼底新生血管性疾病的发生发展。近十年来,缺氧下RPE细胞的相关研究已逐步成为学术界热点之一。有研究表明,在多种眼底疾病中（年龄相关性黄斑变性（age related macular degeneration, AMD）、遗传性外层视网膜营养不良和增生性玻璃体视网膜病变（proliferative vitreoretinopathy, PVR）等）均可观察到RPE细胞凋亡,而且部分证实了凋亡参与了疾病的发生发展。但人们对缺氧下RPE细胞凋亡的发生机制和特征却了解甚少。JNK和p38属于丝裂素活化蛋白激酶（mitogen activated protein kinasea, MAPKs）家族,两者通常由紫外线、渗透压变化、细胞因子和生理激活因素所启动,又被称作MAPK应激信号通路。已有证据表明ERK通路参与了RPE细胞的增生。对于大多数细胞来说,ERK1/2和JNK、p38在对细胞凋亡的调控中作用相反。因此有理由推测p38和JNK亦可能参与了缺氧导致的RPE细胞凋亡。本研究旨在探讨JNK和p38通路在缺氧诱导的RPE凋亡中的调控作用,为临床防治缺血缺氧性眼内疾病提供实验依据。目的观察p38和JNK信号通路在缺氧诱导的体外培养人RPE细胞凋亡中可能的调控作用。方法（1）将培养的人RPE细胞置于含体积分数为1%O₂、5%CO₂和94%N2的培养箱内建立缺氧模型。于缺氧后1、3、6、12和24h,用光镜、扫描电镜、Annexin-V-FITC/PI染色流式细胞术（FCM）及TUNEL法检测凋亡,于缺氧后1、2、3和4d后用四甲基偶氮唑蓝（MTT）比色法检测RPE细胞的增生;（2）于缺氧后1,3,6,12和24h,用Western blot检测RPE细胞内p38蛋白的表达;以及用免疫组化及免疫荧光观察p38和p-p38蛋白在RPE细胞内的定位。用p38途径抑制剂预处理RPE细胞30分钟后置于缺氧孵箱内培养3小时。Western blot检测p38信号阻断前后RPE细胞内p38蛋白的表达。（3）用p38通路抑制剂SB203580和JNK通路抑制剂SP600125预处理RPE细胞30分钟,将RPE细胞置于缺氧孵箱内培养3小时。用光镜、透射电镜、FCM及TUNEL法检测对细胞凋亡的影响;用MTT比色法检测抑制剂对细胞增生的影响。结果（1）缺氧可致RPE细胞凋亡,凋亡细胞数随缺氧时间延长而增加（ P< 0. 01）,在3小时达高峰;MTT显示,缺氧处理后的RPE细胞增生较快,缺氧后2、3和4d时,缺氧组较对照组A值显著增大（P< 0.01）;（2） Western blot显示缺氧1、3、6、12和24h时,均有磷酸化p38蛋白表达,3h水平最高,持续至24h。经p38抑制剂处理后细胞中磷酸化p38蛋白水平降低,表明SB203580能有效抑制p38的磷酸化;免疫组化显示,常氧状态下p38蛋白在RPE细胞胞浆和胞核中均有分布。缺氧后,细胞浆内棕色强度减弱,胞核颜色明显加深,提示p38蛋白由胞浆向胞核内转位。免疫荧光证实,常氧条件下p-p38蛋白荧光仅在RPE细胞胞浆内有微量表达,胞核内无表达。随缺氧时间的延长,p38蛋白的磷酸化水平逐渐增高,细胞核荧光强度明显增强,至3h表达最强,24h开始下降。（3）缺氧3小时时RPE细胞凋亡明显;经p38和JNK抑制剂处理的细胞凋亡水平明显下降（P < 0.01）;MTT未显示抑制剂对细胞增生有影响（P > 0.05）。结论缺氧不仅能诱导RPE细胞增生,亦能诱导凋亡。p38和JNK转导通路参与了缺氧诱导人RPE细胞的凋亡。通过本课题的初步研究,有理由推测缺氧通过激活p38和JNK转导途径调控体外培养的人RPE细胞的凋亡。本研究为下一步通过信号转导通路调控缺氧下RPE凋亡提供了试验证据,亦为临床防治缺血缺氧性眼内疾病提供了新思路。更多还原

【Abstract】 Backgruoud: Hypoxia and ischemia are etiological factors in fundus oculi diseases. A great quantity of research prove that hypoxia induces the proliferation of retinal pigment epithelial cells（RPE） and plays a important part in the pathogenesis of fundus neovascular diseases. More and more evidence support that the apoptosis of RPE is a key facor in many funds diseases （for example: age related macular degeneration, hereditary malnutrition in outer layer of retina and proliferative vitreoretinopathy）. However, there has been few study about the character of apoptosis RPE cells induced by hypoxia so far. Accordingly, it’s important for us to investigate the information about apoptosis of RPE induced by hypoxia and its relative signal paths.AIM:（1） To establish the model of RPE cells in hypoxia status, and observe the gowth situation of them; （2） Study the role of p38 in the process of apoptosis of RPE cells induced by hypoxia; （3） Block the p38 and JNK pathway, and observe the influence in apoptosis of RPE cells in the hypoxia status.METHODS:（1） To set up the hypoxia model, human REP cells were cultured in a special incubator containing volume fraction of 1%O₂, 5% CO₂ and 94% N2 for 0h, 1h, 3h, 6h, 12h and 24h. The level of apoptosis was individually measured with scanning and transmission electron microscopy, terminal deoxynucleotidyl transferase mediated nick end labeling（TUNEL） and flow cytometry（FCM）.Meanwhile, the proliferation of RPE cells were also measured with MTT. The contrast was RPE cells cultured in a normoxic incubator（5% CO₂, 95% O₂）. （2） After hypoxia 1, 3, 6, 12 and 24 h, western blot analysis was performed to detect the expression of phosphorylated p38. The methods of immunohistochemisty and immunofluorescence were also used to fix the position of p38 and p-p38 protein in the RPE cell. blocking the p38 path with SB203580, detect the expression of phosphorylated p38 by western blot （3） After blocking the p38 and JNK path with SB203580 and SP600125, the level of apoptosis of RPE cultured in hypoxia incubator for 3 hours was individually measured with scanning and transmission electron microscopy, TUNEL and FCM.RESULTS: （1） Hypoxia can induce proliferation and apoptosis in the cultured RPE cells. Compared to the control group, the apoptosis of RPE cells was significantly activated and peaked at 3h in the cultured RPE cells under hypoxia. （2） Western blot showed that the expression of phosphorylated p38 increased gradually and peaked at 3h in the cultured RPE cells treated by hypoxia.The level of phospho-p38 of hRPE in the group treated with SB203580 was decreased. Immunohistochemistry staining proved that p38 protein located in the cytoplasm and nucleus in the cultured RPE cells under normoxic conditions. Under hypoxic conditions, the brown intensity decreased within the cytoplasm and increased in the nucleus. In normoxic surrounding, there was no expression of p-p38 in nucelus. However, the fluorescence intensity in the nucleus increased under hypoxic conditions. （3） After blocking the p38 and JNK path, the level of apoptosis of RPE induced by hypoxia decreased.CONCLUSION: Hypoxia can induce proliferation and apoptosis in the cultured RPE cells; According to this study, it was suggested that p38 and JNK path were involved in RPE apoptosis induced by hypoxia. And it’s maybe the basis of possible new therapy for intraocular diseases.更多还原