【作者】 赵浩；

【导师】 李建峰； 白晓卉；

【作者基本信息】 山东大学 ， 临床医学（耳鼻咽喉科学）， 2022， 硕士

【摘要】 研究目的:感音神经性耳聋(Sensorineural hearing loss,SNHL)是人类常见的感觉缺陷,已构成全球性的健康问题。顺铂作为一种有效的化疗药物,广泛应用于多种肿瘤的治疗,但其副作用,尤其是产生的耳毒性可导致双侧、进行性听力损失,很大程度上限制了其临床应用。目前,顺铂所致的SNHL多归咎于内质网应激,炎症,凋亡自噬所致的听觉细胞死亡。钙离子作为真核细胞中广泛存在的第二信使,参与诸多生理过程,而钙离子稳态失衡可导致诸多疾病的发生。内质网和线粒体作为重要的钙储器,参与胞内钙离子稳态的维持。肌醇-1,4,5-三磷酸受体(Inositol 1,4,5-triphosphate receptor,IP3R)/葡萄糖调节蛋白 75(Glucose-regulated protein 75,Grp75)/电压依赖性阴离子通道 1(Voltage-dependentanionchannel 1,VDAC1)复合物作为内质网与线粒体间的重要结构,参与两者间的钙调节。有研究发现,钙离子稳态失衡可导致毛细胞凋亡。但是,其在顺铂所致的毛细胞损伤中的机制目前并不明确。本研究旨在讨论顺铂致毛细胞损伤的新机制,侧重于亚细胞结构中的钙稳态及相应通道的变化。研究方法:(1)流式细胞术及TUNEL染色分别验证30 μM顺铂对HEI-OC1细胞及乳鼠毛细胞的损伤情况。(2)免疫荧光(Immunofluorescence,IF)观察30μM顺铂作用24 h后在HEI-OC1细胞及乳鼠毛细胞中caspase-3的活化情况。(3)免疫印迹技术(Western blotting,WB)探究磷酸化蛋白激酶R样内质网激酶(Phosphorylated protein kinase R-like ER kinase,p-PERK),活化转录因子 6(Activating transcription factor 6,ATF6),Caspase-12 等内质网应激指标在 30 μM 顺铂作用 0,6,12,24 h的变化;IF观察30 μM顺铂作用24 h后于乳鼠毛细胞caspase-12的表达变化。(4)WB观察Bcl-2,Bax,cleaved-caspase 9等线粒体凋亡指标在30 μM顺铂作用0,6,12,24 h时的变化;IF观察30 μM顺铂作用24 h后于乳鼠毛细胞caspase-9的活化情况。(5)Mag-fluo-4 AM及Rhod-2 AM分别标记内质网及线粒体的钙离子,观察30 μM顺铂作用0,6,12,24 h,HEI-OC1细胞及乳鼠毛细胞中内质网及线粒体中钙离子的变化。(6)WB及IF观察30 μM顺铂作用0,6,12,24h时,HEI-OC1细胞及乳鼠毛细胞中,内质网钙通道IP3R及线粒体钙通道VDAC1的变化。(7)IF观察30 μM作用0,6,12,24 h时,HEI-OC1细胞中IP3R与beclin-1的共定位情况;WB观察30 μM顺铂作用0,6,12,24 h时,HEI-OC1细胞中自噬指标Beclin-1及轻链蛋白3β(Light chain 3β,LC3B)的表达变化;IF观察30 μM顺铂作用24 h后,乳鼠毛细胞中的表达变化。研究结果:(1)30μM顺铂处理24h后,HEI-OC1细胞凋亡比例明显增加,乳鼠毛细胞出现明显的缺失、紊乱、凋亡。(2)30 μM顺铂处理24h后,HEI-OC1细胞及乳鼠毛细胞中的caspase-3出现明显活化。(3)30μM顺铂加入后,HEI-OC1细胞中的内质网应激指标p-PERK,ATF6先升高后降低,caspase-12随时间递增,乳鼠毛细胞中的caspase-12相较于对照组表达明显增强。(4)30μM顺铂加入后,HEI-OC1细胞中的线粒体凋亡指标Bcl-2降低,Bax增加,caspase-9明显活化,同样地,乳鼠毛细胞中的caspase-9相较于对照组明显活化。(5)30μM顺铂加入后,HEI-OC1细胞及乳鼠毛细胞中,内质网的钙离子水平先降低后升高,而线粒体中的钙离子随时间累积。(6)相应地,30μM顺铂加入后,HEI-OC1细胞及乳鼠毛细胞中,IP3R蛋白水平先升高后降低,而VDAC1蛋白水平随时间递增。(7)30 μM顺铂加入后,IP3R与beclin-1逐渐解离,HEI-OC1细胞中的beclin-1及LC3B表达增加,乳鼠毛细胞中LC3B逐渐增加,且出现更多的点状聚集。结论:(1)顺铂可能通过影响HEI-OC1细胞及乳鼠毛细胞中IP3R及VDAC1的表达,导致内质网及线粒体的钙离子水平变化,激活内质网应激及线粒体途径,影响细胞结局。(2)顺铂可能通过促进HEI-OC1细胞及乳鼠毛细胞中IP3R与beclin-1的解离诱导自噬发生,参与细胞损伤。更多还原

【Abstract】 ObjectiveSensorineural hearing loss(SNHL),as a common sensory defect in humans,is a global health problem.Cisplatin,as a commonly used chemotherapeutic drug,can cause bilateral and progressive hearing loss,which limits its clinical application.At present,SNHL-induced by cisplatin is mostly attributed to the death of auditory cells caused by endoplasmic reticulum(ER)stress,inflammation,apoptosis,and autophagy.As a second messenger widely present in eukaryotic cells,calcium participates in many physiological processes,and the imbalance of calcium homeostasis can lead to many diseases.The ER and mitochondria,as important calcium reservoirs,play a role in the maintenance of intracellular calcium homeostasis.Inositol 1,4,5-triphosphate receptor(IP3R)/Glucose-regulated protein 75(Grp75)/Voltage-dependent anion channel 1(Voltage-The dependent anion channel 1,VDAC1)complex,as a vital structure between the ER and mitochondria,participates in calcium regulation between them.Studies have shown that the imbalance of calcium homeostasis caused hair cells apoptosis.However,its roles in hair cells damage caused by cisplatin are still unclear.This study aims to discuss the new mechanism of cisplatin-induced hair cells damage,focusing on the changes in calcium homeostasis and responding channels in the subcellular structure.Methods(1)Flow cytometry and TUNEL staining were used to verify the damage of 30 μM cisplatin to HEI-OC1 cells and neonatal mouse hair cells.(2)Immunofluorescence was used to observe the activated caspase-3 in HEI-OC1 cells and neonatal mouse hair cells after 30 μM cisplatin was treated for 24 h.(3)The change of p-PERK,ATF6,Caspase-12,and other endoplasmic reticulum stress indicators was exhibited by WB,after 30 μM cisplatin treatment for 0,6,12,24 h;the variation of Caspase-12 in hair cells of suckling mice after 30 μM cisplatin treatment for 24 h was shown by IF.(4)WB was used to observe the mitochondrial apoptosis indicators such as Bcl-2,Bax,cleaved-caspase 9 at 30 μM cisplatin for 0,6,12,24 h;IF was used to observe the activated caspase-9 in hair cells of suckling mice,after 30 μM cisplatin stimulus for 24 h.(5)The calcium ions of the ER and mitochondria were labeled by Mag-fluo-4 AM and Rhod2 AM respectively,to observe their variation in HEI-OC1 cells and hair cells of suckling mice after 30 μM cisplatin addition for 0,6,12,24 h.(6)WB and IF were used to observe the changes of ER calcium channel IP3R and mitochondrial calcium channel VDAC1 in HEI-OC1 cells and suckling mice’s hair cells when 30 μM cisplatin was treated for 0,6,12,24 h.(7)The interaction between IP3R and beclin-1 in HEI-OC1 cells was exhibited by IF at 30 μM cisplatin exposure for 0,6,12,24 h;The expression changes of autophagy indicators Beclin-1 and LC3B in HEI-OC1 cells after 30 μM cisplatin treatment for 0,6,12,24 h were shown by WB;IF was chosen to observe of the expression changes in neonatal mice’s hair cells after 30 μM cisplatin addition for 24 h.Results(1)After 30 μM cisplatin treatment for 24 h,the proportion of HEI-OC1 cell apoptosis increased significantly,and the hair cells of suckling mice showed an obvious loss,disorder,and apoptosis.(2)After addition with 30 μM cisplatin for 24 h,caspase-3 in HEI-OC1 cells and hair cells of suckling mice was significantly activated.(3)After 30 μM cisplatin was added,the ER stress indicators p-PERK and ATF6 in HEI-OC1 cells first increased and then decreased,and caspase-12 increased in a time-dependent way;the expression of caspase-12 in hair cells of suckling mice enhanced significantly,compared with the control group.(4)Under 30 μM cisplatin exposure,the mitochondrial apoptosis indicators Bcl-2 in HEI-OC1 cells decreased,Bax increased,and caspase-9 was significantly activated;similarly,caspase-9 in hair cells of suckling mice was activated obviously,compared with the control group.(5)After 30 μM cisplatin addition,the calcium level in the ER decreased firstly followed by increment in HEI-OC1 cells and suckling mice hair cells,while the calcium level in mitochondria accumulated with time.(6)Correspondingly,under 30 μM cisplatin elicitation,the level of IP3R protein in HEI-OC1 cells and suckling mice hair cells increased first and then decreased,while the level of VDAC1 protein increased in a time-dependent way.(7)Under 30 μM cisplatin exposure,in HEI-OC1 cells,IP3R and beclin-1 gradually dissociated,the expression of beclin-1 and LC3B increased,LC3B in suckling mice hair cells gradually increased,and more punctate aggregation appeared.Conclusions(1)Cisplatin may influence the fate of HEI-OC1 cells and neonatal mouse hair cells,via affecting the expression of IP3R and VDAC1,resulting in changes of calcium levels in the ER and mitochondria,which activates ER stress and mitochondrial pathways.(2)Cisplatin may participate in the damage of HEI-OC1 cells and neonatal mouse hair cells by promoting the dissociation of IP3R and beclin-1 to induce autophagy.更多还原