【作者】 王俊；

【导师】 姜文敏；

【作者基本信息】 中南大学 ， 眼科学， 2022， 硕士

【摘要】 目的:本研究旨在观察并证实慢性高眼压状态下线粒体-内质网结构偶联(mitochondria-associated endoplasmic reticulum membrane,MAM)在形态和功能上的改变,以及线粒体融合蛋白2(mitofusin 2,Mfn2)过表达对视网膜神经节细胞(retinal ganglion cell,RGC)凋亡的影响。方法:1.运用环角膜缘缝合的方式建立大鼠慢性高眼压模型,通过视网膜切片HE染色观察视网膜和视神经的病理学改变,Brn3b抗体特异性标记RGCs并计数,蛋白免疫印迹(western blot,WB)检测凋亡蛋白caspase3的表达,证实慢性青光眼模型的成功建立;2.通过透射电镜(transmission electron microscope,TEM)观察慢性青光眼模型中MAM形态改变,WB检测慢性青光眼模型视网膜中线粒体与内质网相关蛋白的表达情况;3.通过大鼠玻璃体腔内注射AAV-MFN2-myc进行MFN2体内转染,观察并比较各组的视网膜形态学改变、线粒体形态与功能蛋白变化,及内质网应激相关通路蛋白的改变。结果:1.环角膜缘缝合诱导大鼠眼压明显升高(p<0.05),HE染色及RGC计数证实慢性高眼压导致视网膜变薄、视神经变性和RGC凋亡等特征性青光眼性病理改变;2.环角膜缘缝合诱导的慢性高眼压模型中,线粒体与内质网距离增宽,线粒体形态碎片化,线粒体动力学紊乱,线粒体融合蛋白Mfn2、Opa1表达减少,分裂蛋白Drp1表达增多,PERK/eIF2α磷酸化增加;3.在慢性青光眼大鼠模型中,MFN2基因过表达可以缩短高眼压增宽的MAM距离,部分恢复视网膜厚度以及RGC数量,抑制视网膜中的PERK通路的激活,但仍存在线粒体动力学紊乱以及caspase3激活。结论:环角膜缘缝合可以成功建立慢性高眼压大鼠模型,通过下调Mfn2表达导致MAM形态改变、线粒体动力学紊乱和未折叠蛋白反应(unfolded protein response,UPR)激活,最终导致RGC凋亡。Mfn2过表达可以通过抑制PERK通路的激活部分抑制RGC凋亡,延缓青光眼的病程。图12幅,表3个,参考文献100篇更多还原

【Abstract】 Purpose: To observe and confirm the morphological and functional changes of mitochondrial-associated endoplasmic reticulum membrane(MAM)and investigate the effects of mitofusin2(Mfn2)on retinal ganglion cells(RGCs)apoptosis in experimental glaucoma model of rats.Methods:1.In this study,the chronic elevation of intraocular pressure(IOP)was induced by circumlimbal suture in rats.To identify the successful establishment of the chronic glaucoma model,HE staining of retinas was applied to observe the pathological changes of retina and optic nerve;RGCs were specifically labeled with Brn3 b antibody and counted;and the expression of apoptotic protein caspase3 was detected by western blot(WB).2.Transmission electron microscope(TEM)was used to observe the morphological changes of MAM in chronic glaucoma model,and WB was applied to detect the expression of mitochondrial and endoplasmic reticulum-related proteins.3.Adeno-associated virus-mediated MFN2 gene was transferred to retina by vitreous injection to interfere with the expression of Mfn2 in the retina of rats.The morphological changes of retina,the morphology and function of mitochondria and the changes of endoplasmic reticulum stress-related proteins in each group were observed to explore the role and mechanism of Mfn2 in the pathogenesis of glaucoma.Results:1.Circumlimbal suture could induce chronic elevation of intraocular pressure in rats,accompanied by characteristic glaucomatous pathological changes including decrease of retinal thickness,optic nerve degeneration and RGC apoptosis shown by HE staining and RGC counting.2.In circumlimbal suture model,distance between mitochondria and endoplasmic reticulum increased;mitochondrial morphology changed from long shape to small and round;mitochondrial dynamics were disrupted;and unfolded protein response(UPR)was activated.3.In chronic glaucoma model,MFN2 overexpression could cause the increase of retinal thickness and RGC number,decrease the expression of p-PERK and p-eIF2α,and activate caspase 3.Conclusion: In chronic glaucoma model induced by circumlimbal suture,decrease of MAM-related protein Mfn2 can cause RGC apoptosis by changing the morphology and function of MAM,which present as disordered mitochondrial dynamics and activation of UPR.And overexpression Mfn2 of can recover the retinal morphology and inhibition the apoptosis of RGCs at some extent by inhibiting the activation of PERK pathway.更多还原