【作者】 李明；

【导师】 卢志明； 白晓卉；

【作者基本信息】 山东大学 ， 临床检验诊断学， 2021， 硕士

【摘要】 研究目的接触耳毒性药物是导致听力损失的一个重要原因,每年约有3万名的儿童会因接受耳毒性药物治疗导致听力缺陷,造成严重的身体、社会和心理障碍。氨基糖甙类药物,包括庆大霉素(Gentamicin)和新霉素(Neomycin)等,和顺铂(Cisplatin)作为临床常用的抗生素和肿瘤化疗药,有很大概率会通过引起听觉毛细胞死亡的方式产生耳毒性。在这里,我利用模式生物斑马鱼,探索黄酮类化合物柚皮甙(Naringin)在拮抗氨基糖甙类药物和顺铂耳毒性方面的潜在应用价值,并研究其作用的机制。研究方法(1)药源性毛细胞损伤模型的构建。受精后五天(five days post-fertilization,dpf)的斑马鱼幼鱼,包括野生型AB和绿色荧光蛋白标记毛细胞的转基因斑马鱼Tg(Brn3c:GFP),接受不同浓度和时间的耳毒性药物处理(庆大霉素50-150μmol/L,3h;新霉素 50-150 μmol/L,1h;顺铂 150-250 μmol/L,2h)。采用小鼠耳蜗毛细胞系HEI-OC1进行体外模型构建(庆大霉素50μmol/L,2h;新霉素50μmol/L,1h;顺铂 10 μmol/L,3h)。(2)采用预先及与耳毒性药物共孵育的方法检测柚皮甙拮抗药源性毛细胞损伤的效果:不同浓度的柚皮甙(100-400 μmol/L)预先处理斑马鱼3h,然后和不同浓度的耳毒性药物共同处理(庆大霉素100μmol/L,3h;新霉素100μmol/L,1h;顺铂200μmol/L,2h)。在斑马鱼测线毛细胞被耳毒性药物消耗殆尽后加入柚皮甙(400μmol/L,24h),检测其促进毛细胞再生的效果。体外实验中柚皮甙(25-150μmol/L)单独处理或与耳毒性药物共处理HEI-OC1细胞。(3)应用免疫荧光技术标记斑马鱼测线毛细胞(抗myosinⅦa蛋白)和神经丘增殖细胞(抗Brdu);活细胞染料FM1-43染色标记功能毛细胞;Tunel染色标记神经丘凋亡细胞;mito-SOX标记线粒体活性氧(Reactive oxygen species,ROS);共聚焦显微镜拍照,记录存活毛细胞数量,对神经丘形态进行评分,量化荧光信号强度;体外实验中使用DCFH-DA探针标记ROS,应用流式细胞术标记凋亡细胞。(4)应用纸片扩散法药敏试验检测柚皮甙对氨基糖甙类药物抑菌(大肠杆菌)效果的影响,应用CCK-8和流式细胞术检测柚皮甙对顺铂杀伤肿瘤细胞(Hela细胞和4T1细胞)效果的影响。(5)应用实时荧光定量PCR技术检测斑马鱼和HEI-OC1细胞经过不同处理后与细胞凋亡(P53,Bax,Caspase-3和Bcl-2)和焦亡(斑马鱼:caspb,caspa;小鼠:Caspase-1,NLRP3)相关基因的mRNA水平的变化。研究结果(1)成功构建药源性毛细胞损伤模型,斑马鱼测线毛细胞经过耳毒性药物处理后不同程度丢失,神经丘形态受损;HEI-OC1细胞存活率降低。(2)柚皮甙有效改善氨基糖甙类药物和顺铂引起的毛细胞丢失和神经丘形态,提高HEI-OC1细胞存活率,并促进斑马鱼测线功能性毛细胞再生。(3)柚皮甙有效减弱氨基糖甙类药物和顺铂引起毛细胞凋亡、线粒体ROS聚集,并增加神经丘中增殖期细胞的数量。(4)柚皮甙不影响氨基糖甙类药物的抑菌效果,并促进顺铂杀伤肿瘤细胞。(5)无论是体内实验时候体外实验,柚皮甙都可以显著改善耳毒性药物引起的凋亡和焦亡相关基因表达(P<0.05)。结论综上所述,本研究首次证实了柚皮甙对氨基糖甙类药物和顺铂诱发耳毒性的保护作用,并解释了可能的机制。我从抗凋亡、抗氧化、增殖和分化等方面证明了柚皮甙保障毛细胞存活的机制,并强调了其在临床应用中的潜力。更多还原

【Abstract】 PurposeExposure to ototoxic drugs is an important cause of hearing loss.Every year,about 30,000 children receive ototoxic drug treatment resulting in hearing impairment,resulting in serious physical,social and psychological disorders.Aminoglycosides,including gentamicin and neomycin,and cisplatin,as commonly used antibiotics and tumor chemotherapy drugs,have a high probability of ototoxicity by causing the death of auditory hair cells.Here,we use the model organism zebrafish to explore the potential application value of flavonoid naringin in antagonizing aminoglycoside and cisplatin ototoxicity,and study its mechanism.Methods(1)Construction of drug-induced hair cell injury model.Five days post-fertilization(dpf)zebrafish juveniles,including wild-type AB and green fluorescent protein-labeled hair cell transgenic zebrafish Tg(Brn3c:GFP),received different concentrations and time of ototoxic drugs treatment(Gentamicin 50-150μmol/L,3h;Neomycin 50-150μmol/L,1h;Cisplatin 150-250μmol/L,2h).The mouse cochlear hair cell line HEI-OC1 was used for in vitro model construction(gentamicin 50μmol/L,2h;neomycin 50μmol/L,1h;cisplatin 10μmol/L,3h).(2)Use the method of pre-incubation and co-incubation with ototoxic drugs to detect the effect of naringin in antagonizing drug-induced hair cell damage:different concentrations of naringin(100-400μmol/L)are pre-treated with zebrafish for 3h,and then different Concentrations of ototoxic drugs were treated together(gentamicin 100μmol/L,3h;neomycin 100μmol/L,1h;cisplatin 200μmol/L,2h).naringin(400μmol/L,24h)was added to test the effect of naringin(400pμmol/L,24h)in promoting hair cell regeneration after the hair cells of the zebrafish line were consumed by ototoxic drugs.In in vitro experiments,naringin(25-150μmol/L)was treated alone or co-treated with ototoxic drugs on HEI-OC1 cells.(3)Use immunofluorescence technology to label zebrafish line hair cells(anti-myosinVIIa protein)and neural mound proliferating cells(anti-Brdu);live cell dye FM1-43 staining to mark functional hair cells;Tunel staining to mark neural mound apoptotic cells;Mito-SOX labels mitochondrial reactive oxygen species(ROS);confocal microscopy takes pictures,records the number of viable hair cells,scores the morphology of neural mounds,and quantifies the fluorescence signal intensity;in vitro experiments use DCFH-DA to label ROS,and use flow cytometry Mark apoptotic cells by technique.(4)Apply the paper diffusion method to detect the effect of naringin on the antibacterial effect of aminoglycoside drugs(E.coli),and use CCK-8 and flow cytometry to detect the effect of naringin on cisplatin killing tumor cells(Hela cells and 4T1 cells)effect.(5)Using real-time fluorescence quantitative PCR technology to detect the relationship between zebrafish and HEI-OC1 cells after different treatments and apoptosis(P53,Bax,Caspase-3 and Bcl-2)and pyrolysis(zebrafish:caspb,caspa;small Mouse:Caspase-1,NLRP3)changes in mRNA levels of related genesResults(1)The drug-induced hair cell injury model was successfully constructed.The hair cells of the zebrafish line were lost to varying degrees after treatment with ototoxic drugs,and the morphology of the nerve mound was damaged;the survival rate of HEI-OC1 cells was reduced(2)Naringin can effectively improve hair cell loss and nerve mound morphology caused by aminoglycoside drugs and cisplatin,increase the survival rate of HEI-OC1 cells,and promote the regeneration of functional hair cells in zebrafish line measurement.(3)Naringin effectively attenuates the hair cell apoptosis,mitochondrial ROS accumulation caused by aminoglycoside drugs and cisplatin,and increases the number of proliferating cells in the neural mound.(4)Naringin does not affect the antibacterial effect of aminoglycoside drugs,and promotes cisplatin to kill tumor cells.(5)Naringin can significantly improve the expression of apoptosis and pyrolysis-related genes caused by ototoxic drugs in both in vivo and in vitro experiments(P<0.05).ConclusionIn conclusion,this study demonstrated for the first time the oto-protective effect of naringin against the ototoxicity of cisplatin and aminoglycosides and explained the possible mechanisms.We provided evidence that naringin could support the survival of functional hair cells in terms of anti-apoptosis,antioxidation,proliferation and differentiation and emphasize its potential in clinical application.更多还原