逆轉H3K9me2保護耳蝸毛細胞的基礎研究
發(fā)布時間：2018-09-14 09:02
【摘要】：感音神經性聾(SNHL)是耳鼻咽喉科的常見病和多發(fā)病,近年來發(fā)病呈上升趨勢,它不僅給患者及其家庭帶來生理上和心理上的痛苦,而且給社會和經濟發(fā)展造成嚴重影響。我國衛(wèi)生部2000年公布的調查結果顯示,感音神經性聾約占耳聾患者的63%,絕對數量超過8千萬。感音神經性聾是一個醫(yī)學難題,由于內耳毛細胞和螺旋神經的損傷修復和再生能力的缺乏,感音神經性聾的防治工作仍無突破性進展。目前感音神經性聾的研究主要集中在兩個方面,一是提高毛細胞對致聾因素損害的耐受能力,阻斷毛細胞凋亡(抗凋亡途徑)；二是重建毛細胞和神經節(jié)神經元到達結構和功能修復(再生途徑)。本研究將從毛細胞保護的角度探討感音神經性聾的防治策略。 既往研究提示組蛋白H3第9位賴氨酸二甲基化(H3K9me2)在細胞發(fā)育和分化成熟中都有重要作用。我們在前期研究中發(fā)現,斑馬魚神經丘H3K9me2的富集程度與細胞所處的狀態(tài)密切相關。耳蝸毛細胞是一類高耗能、易損傷的不可再生的細胞。關于H3K9me2在毛細胞中的作用尚無研究報道,我們通過描述耳蝸基底膜上H3K9me2的區(qū)域性分布,并觀察H3K9me2在不同損傷條件下的表達變化規(guī)律,有助于深入理解H3K9me2表達與內耳毛細胞的關系。我們進一步建立體內外毛細胞損傷模型,研究H3K9me2表達水平變化對耳蝸毛細胞的保護作用,并探討其可能的機制。 我們以小鼠作為動物模型,實驗方法包括離體實驗部分和在體試驗部分。 1.離體實驗部分：本實驗采用新生小鼠體外耳蝸培養(yǎng)模型,研究降低H3K9me2在保護耳蝸毛細胞中的作用和機制。首先采用免疫熒光組織化學技術和western blot技術檢測H3K9me2在耳蝸基底膜上H3K9me2的區(qū)域性分布,并觀察H3K9me2在不同損傷條件下的表達變化規(guī)律。然后根據BIX-01294和/或新霉素給藥情況不同,將實驗對象分為四組,即對照組、早期處理組、中期處理組和后期處理組,觀察不同組別耳蝸組織中毛細胞存活情況和凋亡情況,并檢測毛細胞對GTTR和FM1-43FX的攝取能力。最后采用免疫熒光組織化學技術、線粒體膜電位標記法和western blot等方法對其保護機制進行研究。 2.在體實驗部分：為了進一步研究體內降低H3K9me2在保護耳蝸毛細胞中的作用,本實驗采用C57小鼠作為體內動物模型,采用自身對照設計,左耳為實驗組,右耳為對照組,利用免疫熒光組織化學技術,觀察各組耳蝸毛細胞存活情況；利用掃描電鏡技術,觀察各組耳蝸毛細胞纖毛損害情況；利用聽性腦干反應(ABR)檢測各組小鼠聽力損害情況。 我們所得到的結果如下： 1. H3K9me2表達：正常小鼠耳蝸基底膜組蛋白H3K9me2均呈陽性表達,小上皮嵴表達較強,內毛細胞表達相對較弱；小鼠耳蝸基底膜H3K9me2在新霉素、順鉑、銅及紫外損傷處理后表達強度增強；小鼠耳蝸基底膜H3K9me2在BIX-01294處理后表達水平降低,2μmBIX-01294處理未見明顯細胞缺失,10μnmBIX-01294處理后毛細胞排列紊亂,可見明顯細胞缺失。 2.離體實驗結果：早期處理組的中圈和底圈存活毛細胞數量明顯高于對照組,凋亡細胞數量明顯低于對照組,差異均具有統計學意義(P0.01)。后期處理組的存活毛細胞數量明顯低于對照組,差異具有統計學意義(P0.01)。 3.毛細胞對GTTR和FM1-43FX攝取情況：BIX-01294處理組和對照組的內、外毛細胞均呈GTTR和FM1-43FX陽性表達,主要見細胞漿和/或細胞膜著色,未見明顯細胞核著色。 4.在目前認為的細胞死亡的三種途徑中——即凋亡、自噬和壞死,TUNEL陽性細胞常見于毛細胞區(qū),主要位于基底膜的外毛細胞區(qū)。LC3陽性細胞和PI陽性細胞偶見于毛細胞區(qū)。 5.線粒體膜電位檢測結果發(fā)現,對照組在新霉素損傷后線粒體膜電位(TMRM)水平明顯下調。BIX-01294預處理組TMRM下調不明顯,熒光強度定量分析發(fā)現,實驗組線粒體膜電位明顯高于對照組,差異具有統計學意義(P0.01)。 6. Caspase-3染色結果發(fā)現,實驗組和對照組均出現cleaved caspase-3染色陽性細胞。Western blot及其灰度分析結果顯示,實驗組和對照組均有cleaved caspase-3表達,實驗組cleaved caspase-3表達明顯低于對照組,差異有統計學意義(P0.01)。 7.在體實驗結果：從掃描電鏡來看,對照組頂圈毛細胞纖毛出現明顯倒伏和融合；而實驗組頂圈纖毛形態(tài)基本正常；對照組中圈毛細胞纖毛明顯缺失,而實驗組中圈毛細胞纖毛偶見缺失；對照組底圈毛細胞纖毛基本完全缺失；實驗組底圈毛細胞纖毛缺失嚴重。從免疫熒光染色來看,對照組Myosin7a陽性細胞數量明顯少于實驗組,差距具有統計學意義(P0.01)。從ABR聽力學檢測來看,對照組聽閾明顯高于實驗組,聽閾差距平均超過15dB,其中8kHz和16kHz處聽閾差距有顯著性差異(P0.01)。 我們所得到的結論如下： 1.小鼠耳蝸基底膜均有H3K9me2表達,提示H3K9me2可能在耳蝸的發(fā)育過程中起到一定作用；但H3K9me2在耳蝸基底膜表達強度呈區(qū)域性,表明H3K9me2表達強度與細胞類型相關,提示H3K9me2可能參與耳蝸細胞的分化過程。 2.小鼠耳蝸基底膜組蛋白H3K9me2在損傷處理后表達增強,在BIX-01294處理后表達減弱,提示H3K9me2表達是可以調控的生理病理過程,其表達改變可能參與毛細胞損傷過程。 3.BIX-01294預處理組對GTTR和FM1-43FX攝取功能試驗表明BIX-01294預處理不影響毛細胞的攝取功能。 4.凋亡、自噬和壞死三種方式均參與了氨基糖苷類藥物介導的毛細胞損傷和死亡,其中凋亡是毛細胞死亡的主要方式。 5.離體實驗和在體實驗結果均證實了下調組蛋白H3K9me2可以從功能和形態(tài)上對氨基糖苷類藥物造成的毛細胞損傷起到一定的保護作用。 6.組蛋白H3K9me2下調介導毛細胞保護作用的機制可能是通過強化線粒體膜電位的穩(wěn)定性、抑制caspase-3活化來實現的。
[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.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R764.431
本文編號：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.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R764.431
【參考文獻】
相關期刊論文 前1條
1 劉建平;戴春富;王正敏;遲放魯;田潔;笪翠弟;;慶大霉素鼓室內注射后在內耳細胞中的分布[J];中華耳鼻咽喉頭頸外科雜志;2006年11期
，本文編號：2242220
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