逆转H3K9me2保护耳蜗毛细胞的基础研究
发布时间：2018-09-14 09:02
【摘要】：感音神经性聋(SNHL)是耳鼻咽喉科的常见病和多发病,近年来发病呈上升趋势,它不仅给患者及其家庭带来生理上和心理上的痛苦,而且给社会和经济发展造成严重影响。我国卫生部2000年公布的调查结果显示,感音神经性聋约占耳聋患者的63%,绝对数量超过8千万。感音神经性聋是一个医学难题,由于内耳毛细胞和螺旋神经的损伤修复和再生能力的缺乏,感音神经性聋的防治工作仍无突破性进展。目前感音神经性聋的研究主要集中在两个方面,一是提高毛细胞对致聋因素损害的耐受能力,阻断毛细胞凋亡(抗凋亡途径)；二是重建毛细胞和神经节神经元到达结构和功能修复(再生途径)。本研究将从毛细胞保护的角度探讨感音神经性聋的防治策略。 既往研究提示组蛋白H3第9位赖氨酸二甲基化(H3K9me2)在细胞发育和分化成熟中都有重要作用。我们在前期研究中发现,斑马鱼神经丘H3K9me2的富集程度与细胞所处的状态密切相关。耳蜗毛细胞是一类高耗能、易损伤的不可再生的细胞。关于H3K9me2在毛细胞中的作用尚无研究报道,我们通过描述耳蜗基底膜上H3K9me2的区域性分布,并观察H3K9me2在不同损伤条件下的表达变化规律,有助于深入理解H3K9me2表达与内耳毛细胞的关系。我们进一步建立体内外毛细胞损伤模型,研究H3K9me2表达水平变化对耳蜗毛细胞的保护作用,并探讨其可能的机制。 我们以小鼠作为动物模型,实验方法包括离体实验部分和在体试验部分。 1.离体实验部分：本实验采用新生小鼠体外耳蜗培养模型,研究降低H3K9me2在保护耳蜗毛细胞中的作用和机制。首先采用免疫荧光组织化学技术和western blot技术检测H3K9me2在耳蜗基底膜上H3K9me2的区域性分布,并观察H3K9me2在不同损伤条件下的表达变化规律。然后根据BIX-01294和/或新霉素给药情况不同,将实验对象分为四组,即对照组、早期处理组、中期处理组和后期处理组,观察不同组别耳蜗组织中毛细胞存活情况和凋亡情况,并检测毛细胞对GTTR和FM1-43FX的摄取能力。最后采用免疫荧光组织化学技术、线粒体膜电位标记法和western blot等方法对其保护机制进行研究。 2.在体实验部分：为了进一步研究体内降低H3K9me2在保护耳蜗毛细胞中的作用,本实验采用C57小鼠作为体内动物模型,采用自身对照设计,左耳为实验组,右耳为对照组,利用免疫荧光组织化学技术,观察各组耳蜗毛细胞存活情况；利用扫描电镜技术,观察各组耳蜗毛细胞纤毛损害情况；利用听性脑干反应(ABR)检测各组小鼠听力损害情况。 我们所得到的结果如下： 1. H3K9me2表达：正常小鼠耳蜗基底膜组蛋白H3K9me2均呈阳性表达,小上皮嵴表达较强,内毛细胞表达相对较弱；小鼠耳蜗基底膜H3K9me2在新霉素、顺铂、铜及紫外损伤处理后表达强度增强；小鼠耳蜗基底膜H3K9me2在BIX-01294处理后表达水平降低,2μmBIX-01294处理未见明显细胞缺失,10μnmBIX-01294处理后毛细胞排列紊乱,可见明显细胞缺失。 2.离体实验结果：早期处理组的中圈和底圈存活毛细胞数量明显高于对照组,凋亡细胞数量明显低于对照组,差异均具有统计学意义(P0.01)。后期处理组的存活毛细胞数量明显低于对照组,差异具有统计学意义(P0.01)。 3.毛细胞对GTTR和FM1-43FX摄取情况：BIX-01294处理组和对照组的内、外毛细胞均呈GTTR和FM1-43FX阳性表达,主要见细胞浆和/或细胞膜着色,未见明显细胞核着色。 4.在目前认为的细胞死亡的三种途径中——即凋亡、自噬和坏死,TUNEL阳性细胞常见于毛细胞区,主要位于基底膜的外毛细胞区。LC3阳性细胞和PI阳性细胞偶见于毛细胞区。 5.线粒体膜电位检测结果发现,对照组在新霉素损伤后线粒体膜电位(TMRM)水平明显下调。BIX-01294预处理组TMRM下调不明显,荧光强度定量分析发现,实验组线粒体膜电位明显高于对照组,差异具有统计学意义(P0.01)。 6. Caspase-3染色结果发现,实验组和对照组均出现cleaved caspase-3染色阳性细胞。Western blot及其灰度分析结果显示,实验组和对照组均有cleaved caspase-3表达,实验组cleaved caspase-3表达明显低于对照组,差异有统计学意义(P0.01)。 7.在体实验结果：从扫描电镜来看,对照组顶圈毛细胞纤毛出现明显倒伏和融合；而实验组顶圈纤毛形态基本正常；对照组中圈毛细胞纤毛明显缺失,而实验组中圈毛细胞纤毛偶见缺失；对照组底圈毛细胞纤毛基本完全缺失；实验组底圈毛细胞纤毛缺失严重。从免疫荧光染色来看,对照组Myosin7a阳性细胞数量明显少于实验组,差距具有统计学意义(P0.01)。从ABR听力学检测来看,对照组听阈明显高于实验组,听阈差距平均超过15dB,其中8kHz和16kHz处听阈差距有显著性差异(P0.01)。 我们所得到的结论如下： 1.小鼠耳蜗基底膜均有H3K9me2表达,提示H3K9me2可能在耳蜗的发育过程中起到一定作用；但H3K9me2在耳蜗基底膜表达强度呈区域性,表明H3K9me2表达强度与细胞类型相关,提示H3K9me2可能参与耳蜗细胞的分化过程。 2.小鼠耳蜗基底膜组蛋白H3K9me2在损伤处理后表达增强,在BIX-01294处理后表达减弱,提示H3K9me2表达是可以调控的生理病理过程,其表达改变可能参与毛细胞损伤过程。 3.BIX-01294预处理组对GTTR和FM1-43FX摄取功能试验表明BIX-01294预处理不影响毛细胞的摄取功能。 4.凋亡、自噬和坏死三种方式均参与了氨基糖苷类药物介导的毛细胞损伤和死亡,其中凋亡是毛细胞死亡的主要方式。 5.离体实验和在体实验结果均证实了下调组蛋白H3K9me2可以从功能和形态上对氨基糖苷类药物造成的毛细胞损伤起到一定的保护作用。 6.组蛋白H3K9me2下调介导毛细胞保护作用的机制可能是通过强化线粒体膜电位的稳定性、抑制caspase-3活化来实现的。
[Abstract]:Sensorineural hearing loss (SNHL) is a common and frequently-occurring disease in otolaryngology. In recent years, the incidence of SNHL is on the rise. It not only brings physical and psychological pain to patients and their families, but also seriously affects social and economic development. Sensorineural deafness is a medical problem. Due to the lack of repair and regeneration of inner ear hair cells and spiral nerve, there is still no breakthrough in the prevention and treatment of sensorineural deafness. This study will explore the prevention and treatment strategies of sensorineural hearing loss from the perspective of hair cell protection.
Previous studies have suggested that Lysine dimethylation at position 9 of histone H3 (H3K9me2) plays an important role in cell development, differentiation and maturation. In our previous studies, we found that the accumulation of H3K9me2 in the cumulus of zebrafish nerve is closely related to the state of cells. Cochlear hair cells are a kind of high-energy-consuming and easily damaged non-renewable cells. The role of H3K9me2 in hair cells has not been reported. By describing the regional distribution of H3K9me2 on the basilar membrane of the cochlea and observing the expression of H3K9me2 under different injury conditions, we can understand the relationship between H3K9me2 expression and inner ear hair cells. The protective effect of K9me2 expression on cochlear hair cells and its possible mechanism were discussed.
We used mice as animal models. The experimental methods included in vitro experiments and in vivo experiments.
1. In vitro experiment: In order to study the effect and mechanism of reducing H3K9me2 on protecting cochlear hair cells, a new model of neonatal mouse cochlea culture in vitro was used. Then, according to the different dosage of BIX-01294 and/or neomycin, the subjects were divided into four groups: control group, early treatment group, middle treatment group and late treatment group. Finally, the protective mechanism was studied by immunofluorescence histochemistry, mitochondrial membrane potential labeling and Western blot.
2. In vivo experiment: In order to further study the role of reducing H3K9me2 in protecting cochlear hair cells in vivo, C57 mice were used as animal model in vivo. The survival of cochlear hair cells in each group was observed by immunofluorescence histochemistry. Scanning electron microscopy was used to observe the cilia damage of cochlear hair cells in each group, and auditory brainstem response (ABR) was used to detect the hearing damage in each group.
The results are as follows:
1. Expression of H3K9me2: Histone H3K9me2 was positively expressed in the basilar membrane of normal mice cochlea, the expression of H3K9me2 was stronger in the small epithelial ridge and weaker in the inner hair cells; the expression of H3K9me2 in the basilar membrane of mice cochlea was enhanced after neomycin, cisplatin, copper and ultraviolet irradiation; the expression of H3K9me2 in the basilar membrane of mice decreased after BIX-01294 treatment. No obvious cell deletion was found in the treatment of 2 micron BIX-01294, and the arrangement of hair cells was disordered after 10 micron BIX-01294.
2. In vitro experimental results: The number of surviving hair cells in the middle and bottom circles of the early treatment group was significantly higher than that of the control group, and the number of apoptotic cells was significantly lower than that of the control group (P 0.01). The number of surviving hair cells in the late treatment group was significantly lower than that of the control group (P 0.01).
3. The uptake of GTTR and FM1-43FX by hair cells: In BIX-01294 treatment group and control group, both outer and inner hair cells showed positive expression of GTTR and FM1-43FX, mainly cytoplasm and/or cell membrane staining, but no obvious nuclear staining.
4. Among the three pathways of cell death currently considered, apoptosis, autophagy and necrosis, TUNEL-positive cells are commonly found in the hair cells, mainly in the outer hair cells of the basement membrane. LC3-positive cells and PI-positive cells are occasionally found in the hair cells.
5. The results of mitochondrial membrane potential test showed that the level of mitochondrial membrane potential (TMRM) was significantly decreased in the control group after neomycin injury.
6. Caspase-3 staining showed cleaved caspase-3 positive cells in both experimental and control groups. Western blot and gray-scale analysis showed that cleaved caspase-3 was expressed in both experimental and control groups. The expression of cleaved caspase-3 in experimental group was significantly lower than that in control group (P 0.01).
7. In vivo experimental results: from the scanning electron microscopy, there were obvious lodging and fusion of hair cells in the apical circle of the control group, while the morphology of hair cells in the apical circle of the experimental group was basically normal, the hair cells in the control group were obviously missing, while the hair cells in the experimental group were occasionally missing, and the hair cells in the control group were completely missing. The number of Myosin 7a positive cells in the control group was significantly less than that in the experimental group (P Difference (P0.01).
Our conclusions are as follows:
1. The expression of H3K9me2 in the basement membrane of mouse cochlea suggests that H3K9me2 may play a role in the development of cochlea, but the expression of H3K9me2 in the basement membrane of cochlea is regional, indicating that the intensity of H3K9me2 expression is related to the cell type, suggesting that H3K9me2 may participate in the differentiation process of cochlea cells.
2. The expression of histone H3K9me2 in mouse cochlear basilar membrane increased after injury and decreased after BIX-01294 treatment, suggesting that the expression of histone H3K9me2 may be involved in the process of hair cell injury.
3. BIX-01294 pretreatment did not affect the uptake of GTTR and FM1-43FX.
4. Apoptosis, autophagy and necrosis are involved in aminoglycoside-mediated hair cell injury and death, and apoptosis is the main mode of hair cell death.
5. Both in vitro and in vivo experiments confirmed that down-regulation of histone H3K9me2 could protect hair cells from damage caused by aminoglycosides.
6. Histone H3K9me2 down-regulates hair cell protection by enhancing the stability of mitochondrial membrane potential and inhibiting caspase-3 activation.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R764.431
本文编号：2242220
[Abstract]:Sensorineural hearing loss (SNHL) is a common and frequently-occurring disease in otolaryngology. In recent years, the incidence of SNHL is on the rise. It not only brings physical and psychological pain to patients and their families, but also seriously affects social and economic development. Sensorineural deafness is a medical problem. Due to the lack of repair and regeneration of inner ear hair cells and spiral nerve, there is still no breakthrough in the prevention and treatment of sensorineural deafness. This study will explore the prevention and treatment strategies of sensorineural hearing loss from the perspective of hair cell protection.
Previous studies have suggested that Lysine dimethylation at position 9 of histone H3 (H3K9me2) plays an important role in cell development, differentiation and maturation. In our previous studies, we found that the accumulation of H3K9me2 in the cumulus of zebrafish nerve is closely related to the state of cells. Cochlear hair cells are a kind of high-energy-consuming and easily damaged non-renewable cells. The role of H3K9me2 in hair cells has not been reported. By describing the regional distribution of H3K9me2 on the basilar membrane of the cochlea and observing the expression of H3K9me2 under different injury conditions, we can understand the relationship between H3K9me2 expression and inner ear hair cells. The protective effect of K9me2 expression on cochlear hair cells and its possible mechanism were discussed.
We used mice as animal models. The experimental methods included in vitro experiments and in vivo experiments.
1. In vitro experiment: In order to study the effect and mechanism of reducing H3K9me2 on protecting cochlear hair cells, a new model of neonatal mouse cochlea culture in vitro was used. Then, according to the different dosage of BIX-01294 and/or neomycin, the subjects were divided into four groups: control group, early treatment group, middle treatment group and late treatment group. Finally, the protective mechanism was studied by immunofluorescence histochemistry, mitochondrial membrane potential labeling and Western blot.
2. In vivo experiment: In order to further study the role of reducing H3K9me2 in protecting cochlear hair cells in vivo, C57 mice were used as animal model in vivo. The survival of cochlear hair cells in each group was observed by immunofluorescence histochemistry. Scanning electron microscopy was used to observe the cilia damage of cochlear hair cells in each group, and auditory brainstem response (ABR) was used to detect the hearing damage in each group.
The results are as follows:
1. Expression of H3K9me2: Histone H3K9me2 was positively expressed in the basilar membrane of normal mice cochlea, the expression of H3K9me2 was stronger in the small epithelial ridge and weaker in the inner hair cells; the expression of H3K9me2 in the basilar membrane of mice cochlea was enhanced after neomycin, cisplatin, copper and ultraviolet irradiation; the expression of H3K9me2 in the basilar membrane of mice decreased after BIX-01294 treatment. No obvious cell deletion was found in the treatment of 2 micron BIX-01294, and the arrangement of hair cells was disordered after 10 micron BIX-01294.
2. In vitro experimental results: The number of surviving hair cells in the middle and bottom circles of the early treatment group was significantly higher than that of the control group, and the number of apoptotic cells was significantly lower than that of the control group (P 0.01). The number of surviving hair cells in the late treatment group was significantly lower than that of the control group (P 0.01).
3. The uptake of GTTR and FM1-43FX by hair cells: In BIX-01294 treatment group and control group, both outer and inner hair cells showed positive expression of GTTR and FM1-43FX, mainly cytoplasm and/or cell membrane staining, but no obvious nuclear staining.
4. Among the three pathways of cell death currently considered, apoptosis, autophagy and necrosis, TUNEL-positive cells are commonly found in the hair cells, mainly in the outer hair cells of the basement membrane. LC3-positive cells and PI-positive cells are occasionally found in the hair cells.
5. The results of mitochondrial membrane potential test showed that the level of mitochondrial membrane potential (TMRM) was significantly decreased in the control group after neomycin injury.
6. Caspase-3 staining showed cleaved caspase-3 positive cells in both experimental and control groups. Western blot and gray-scale analysis showed that cleaved caspase-3 was expressed in both experimental and control groups. The expression of cleaved caspase-3 in experimental group was significantly lower than that in control group (P 0.01).
7. In vivo experimental results: from the scanning electron microscopy, there were obvious lodging and fusion of hair cells in the apical circle of the control group, while the morphology of hair cells in the apical circle of the experimental group was basically normal, the hair cells in the control group were obviously missing, while the hair cells in the experimental group were occasionally missing, and the hair cells in the control group were completely missing. The number of Myosin 7a positive cells in the control group was significantly less than that in the experimental group (P Difference (P0.01).
Our conclusions are as follows:
1. The expression of H3K9me2 in the basement membrane of mouse cochlea suggests that H3K9me2 may play a role in the development of cochlea, but the expression of H3K9me2 in the basement membrane of cochlea is regional, indicating that the intensity of H3K9me2 expression is related to the cell type, suggesting that H3K9me2 may participate in the differentiation process of cochlea cells.
2. The expression of histone H3K9me2 in mouse cochlear basilar membrane increased after injury and decreased after BIX-01294 treatment, suggesting that the expression of histone H3K9me2 may be involved in the process of hair cell injury.
3. BIX-01294 pretreatment did not affect the uptake of GTTR and FM1-43FX.
4. Apoptosis, autophagy and necrosis are involved in aminoglycoside-mediated hair cell injury and death, and apoptosis is the main mode of hair cell death.
5. Both in vitro and in vivo experiments confirmed that down-regulation of histone H3K9me2 could protect hair cells from damage caused by aminoglycosides.
6. Histone H3K9me2 down-regulates hair cell protection by enhancing the stability of mitochondrial membrane potential and inhibiting caspase-3 activation.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R764.431
【参考文献】
相关期刊论文 前1条
1 刘建平;戴春富;王正敏;迟放鲁;田洁;笪翠弟;;庆大霉素鼓室内注射后在内耳细胞中的分布[J];中华耳鼻咽喉头颈外科杂志;2006年11期
，本文编号：2242220
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