【作者】 徐静；

【导师】 狄国虎；

【作者基本信息】 青岛大学 ， 特种医学， 2022， 硕士

【摘要】 目的 利用聚己内酰胺-聚乙酸乙烯酯-聚乙二醇共聚物(Polyvinyl caprolactam–Polyvinyl acetate–Polyethylene glycol,PVCL-PVA-PEG),构建褪黑素纳米胶束溶液(褪黑素-胶束,Melatonin-Micelle,Mel-Mic),探究其在干眼病(Dry eye disease,DED)中的治疗作用及分子机制。方法 利用透射电镜,粒径及电位分析,药物吸收,包封率等对褪黑素-胶束进行鉴定和评估。细胞实验中,利用人角膜上皮细胞系(Human corneal epithelial cells,HCECs)建立经典细胞高渗模型,分别添加褪黑素(Melatonin,Mel)和褪黑素-胶束进行干预。采用TUNEL染色,流式细胞术(Flow cytometry,FCM)检测褪黑素-胶束对细胞凋亡的影响;免疫荧光染色和蛋白免疫印迹实验(Western Blot,WB)检测褪黑素-胶束对氧化应激和线粒体自噬的影响。利用小干扰RNA(Small interfering RNA,si RNA)在HCECs中转染,验证下游关键自噬调控通路。体内实验中,利用荧光素钠染色、泪液分泌试验、过碘酸雪夫(Periodic Acid-Schiff,PAS)染色观察褪黑素-胶束是否改善DED的临床症状。利用免疫荧光染色、TUNEL染色、电镜等检测褪黑素-胶束对DED小鼠角膜中自噬的改善情况。此外,利用褪黑素受体的小分子拮抗剂Luzindole和4-P-PDOT,以研究褪黑素1型或2型受体(MT1/MT2)是否介导褪黑素-胶束在干眼中调控的线粒体自噬。结果 褪黑素-胶束提高了褪黑素在水溶液中的溶解度和生物活性,并且具有良好的稳定性和生物利用度。褪黑素-胶束处理后可降低高渗环境下HCECs的凋亡。此外,褪黑素-胶束的应用抑制了HCECs中活性氧自由基(Reactive Oxygen Species,ROS)的产生,改善线粒体功能,降低了氧化应激相关标志物(COX-2和4-HNE)的表达。机制方面,在高渗环境下,褪黑素-胶束处理后的HCECs线粒体自噬增加。利用si RNA敲低PINK1之后,褪黑素-胶束的保护作用显著被抑制。在体内,与对照组相比,局部褪黑素-胶束溶液点眼的干眼小鼠的临床症状显著改善,泪液分泌显著增加,杯状细胞密度显著增加,且呈剂量依赖性。透射电镜检测显示褪黑素-胶束治疗组的干眼小鼠角膜上皮细胞自噬体数量增加。非选择性MT1/MT2拮抗剂Luzindole可明显阻断褪黑素-胶束的保护作用,而选择性MT2拮抗剂4-P-PDOT没有显著效果,提示MT1受体介导褪黑素-胶束在实验性干眼中的保护作用。结论 我们的研究结果表明,褪黑素-胶束对实验性干眼有显著的保护作用。褪黑素-胶束通过调控PINK1介导的线粒体自噬改善干眼眼表损伤。MT1受体介导褪黑素-胶束在实验性干眼的保护作用。更多还原

【Abstract】 Purpose To investigate the role and molecular mechanism of melatonin(Mel)-loaded polymer polyvinyl caprolactam-polyvinyl acetate-polyethyleneglycol graft copolymer(PVCLPVA-PEG)micelles(melatonin-micelle,Mel-Mic)in dry eye disease(DED).Methods Mel-Mic was identified and evaluated by transmission electron microscope(TEM),particle size analysis,drug absorption and encapsulation rate.In vitro,TUNEL staining and flow cytometry(FCM)were used to detect the effect of Mel-Mic on cell apoptosis.The effects of Mel-Mic on oxidative stress and mitochondrial autophagy were detected by immunofluorescence staining and Western Blot(WB).PINK1 knockdown was analyzed by small interfering RNA(si RNA).In vivo,fluorescein sodium staining,tear secretion test and periodate schiff(PAS)staining were used to observe whether Mel-Mic could improve the clinical symptoms of DED.Immunofluorescence staining,TUNEL staining and transmission electron microscopy were used to detect the cell apoptosis and autophagy in the cornea.In addition,Luzindole and 4-P-PDOT,small molecule antagonists of melatonin receptors,were used to investigate whether melatonin type 1 and/or type 2 receptors(MT1/MT2)mediate Mel-Mic mediated mitochondrial autophagy in DED.Results Mel-Mic improved the solubility and biological activity of Mel in aqueous solution,and had good stability and bioavailability.Pretreatment with Mel-Mic can reduce the apoptosis of human corneal epithelial cells(HCECs)in hypertonic environment.In addition,the application of Mel-Mic inhibited ROS production in HCECs,improved mitochondrial function,and reduced the expression of oxidative stress related markers(COX-2 and 4-HNE).In addition,mitochondrial autophagy of HCECs increased after Mel-Mic treatment under hypertonic conditions.However,after PINK1 was knocked down in HCECs,mitochondrial autophagy was significantly weakened.In vivo,compared with the control group,clinical parameters of mice treated with local Mel-Mic solution were significantly improved,with increased tear secretion and reduced goblet cell loss in a dose-dependent manner.TEM showed that the number of autophagosomes in corneal epithelial cells of dry eye mice in Mel-Mic treatment group increased.Luzindole,a non-selective MT1/MT2 antagonist,significantly blocked the protective effect of Mel-Mic,while 4-P-PDOT,a selective MT2 antagonist,had no significant effect,suggesting that MT1 receptor mediated the protective effect of Mel-Mic on experimental dry eye.Conclusion Our findings demonstrated that Mel-Mic ameliorate ocular surface damage in DED through regulating PINK1 mediated mitophagy,and Mel-Mic has a significant protective effect against experimental dry eye mediated by MT1 receptor.更多还原