TOM复合体和DNA聚合酶γ的线粒体运输在早衰边缘细胞模型中的作用与机制

发布时间：2018-02-11 00:15

本文关键词：年龄相关性听力损失耳蜗血管纹边缘细胞 D-半乳糖 mtDNA4834bp缺失突变年龄相关性听力损失耳蜗血管纹边缘细胞 Tom40 DNA聚合酶γ mtDNA普遍性缺失 D-半乳糖年焍相关性听力损失耳蜗血管纹边缘细胞 Tom20 Tom70 mt　出处：《华中科技大学》2012年博士论文　论文类型：学位论文

【摘要】：第一部分建立大鼠耳蜗血管纹边缘细胞mtDNA4834bp缺失突变早衰模型目的：用大鼠耳蜗血管纹边缘细胞建立线粒体DNA4834bp缺失突变早衰模型,为进一步研究mtDNA4834bp缺失突变在年龄相关性听力损失中的作用机制提供易获取的研究对象。方法：选取出生3天左右的大鼠,显微镜下解剖后分离耳蜗血管纹。消化法接种细胞。经多次纯化后获取比较均一的血管纹边缘细胞,CK-18的免疫荧光化学方法鉴定。细胞经不同浓度梯度(0,2,4,6,8,10,12,14,16g/1)的D-半乳糖和D-甘露醇渗透压组处理3,7天和11天后,采用CCK-8检测不同浓度D-半乳糖对细胞活力的影响。浓度梯度D-半乳糖处理不同时间(3天和7天)和12g/1D-半乳糖处理细胞不同天数(1,3,5,7,9,11,13,15天)后,Taqman探针实时定量PCR技术检测细胞经D-半乳糖诱导的浓度和时间依赖性的mtDNA4834bp缺失突变率的累积。细胞经12g/1D-半乳糖处理不同天数(1,5,11天)后,采用p-半乳糖苷酶染色检测细胞衰老的程度。12g/1D-半乳糖处理细胞1天和15天后,行TUNEL原位显色法检测边缘细胞的凋亡状况。结果：细胞经CK-18的免疫荧光染色后,胞浆呈现较强特异性荧光。边缘细胞经浓度梯度D-半乳糖和渗透压匹配的D-甘露醇处理后,细胞活力变化在D-半乳糖组和渗透压对照组之间无显著差异,14g/1和16g/1时细胞活力呈现浓度依赖性下降,以此为依据选取12g/1作为后续处理细胞的最佳浓度。D-半乳糖能诱导边缘细胞出现浓度和时间依赖性的mtDNA4834bp缺失突变的累积,在处理5,7,9,11天时,与对照组相比有显著差异。β-半乳糖苷酶染色结果显示,与对照组相比D-半乳糖组蓝染细胞呈现时间依赖性的增多,表示细胞的明显衰老。细胞经长时间的D-半乳糖处理后,TUNEL染色结果显示阳性细胞较对照组明显增加。结论：成功培养了原代耳蜗血管纹边缘细胞；D-半乳糖引致的细胞培养环境渗透压的变化是导致细胞活力下降的主要原因；D-半乳糖能成功诱导边缘细胞出现mtDNA4834bp缺失突变,且呈现浓度和时间依赖性的累积;D-半乳糖能诱导细胞出现早衰的特点；细胞经D-半乳糖处理较长时间后才出现明显的细胞凋亡。故成功建立了大鼠mtDNA4834bp缺失突变细胞早衰模型。第二部分线粒体外膜转位酶Tom40的功能状态与DNA聚合酶γ的线粒体输送改变致mtDNA4834bp缺失突变机制的研究目的：探讨掌管线粒体前体蛋白输送的线粒体外膜转位酶Tom40在mtDNA4834bp缺失突变的累积过程中的功能变化,与线粒体DNA修复酶DNA聚合酶γ的线粒体输送的关系,以揭示线粒体前体蛋白输入的功能变化在年龄相关性听力损失发病机制中的作用。方法：取正常成年大鼠耳蜗,石(?)包埋后制成石蜡切片,行组织的免疫荧光染色技术检测Tom40在正常大鼠耳蜗中的表达。原代培养的耳蜗边缘细胞经12g/1D-半乳糖处理11天后,行细胞的免疫荧光化学染色检测细胞内Tom40的表达情况。边缘细胞经浓度梯度(0,2,4,6,8,10,12g/1)D-半乳糖处理3天和7天后,12g/1D-半乳糖处理不同时间(0,13,5,7,9,11天)后纳入实验,分别提取细胞总蛋白以及线粒体蛋白,经western blot方法检测细胞内和线粒体的Tom40,DNA聚合酶丫的表达变化。结果：(1)耳蜗的组织免疫荧光染色结果显示,Tom40在耳蜗组织中表达丰富, Corti氏器,耳蜗外侧壁血管纹以及螺旋神经节区域均有特异性表达。(2)边缘细胞的免疫荧光结果显示,细胞含有丰富的线粒体,胞浆和胞核均有Tom40的强表达,且经D-半乳糖处理后,胞浆内Tom40的表达减弱。(3)浓度梯度D-半乳糖诱导细胞内总Tom40的表达先缓慢增加然后降低,且与处理时间无明显关系。(4)D-半乳糖诱导细胞内Tom40表现出时间依赖性的动态变化；线粒体的Tom40的表达则明显增强。(5)细胞内和线粒体的DNA聚合酶γ的表达呈现先增高再减低的趋势,在mtDNA4834bp缺失突变显著累积时则表现为明显下降。结论：Tom40在大鼠内耳血管纹区域以及原代培养的边缘细胞中表达丰富,它的功能变化可能会影响到耳蜗细胞的正常功能,而导致疾病的发生；D-Gal诱导Tom40和DNA聚合酶γ的动态变化反应了细胞对氧化应激的代偿与失代偿；细胞内Tom40和DNA聚合酶γ的D-Gal时间累积性变化趋势吻合,表明Tom40的功能变化影响DNA聚合酶γ的线粒体输送；细胞内Tom40和DNA聚合酶γ的D-Gal浓度依赖性变化趋势不一致,尤其是短期诱导后,提示可能有其他因素参与到DNA聚合酶γ的线粒体转运过程中；线粒体的Tom40在mtDNA4834bp缺失累积的过程中,其功能增强；相反,DNA聚合酶Y的线粒体输送减少；细胞内和线粒体内Tom40蛋白表达的变化趋势不一致,提示Tom40蛋白的表达可能受到转录后机制的调控。第三部分线粒体外膜转位酶Tom20和Tom70的功能状态与mtDNA4834bp缺失突变机制的研究目的：进一步探讨掌管线粒体前体蛋白输送的线粒体外膜转位酶的识别受体Tom20和Tom70在mtDNA4834bp缺失突变的累积过程中的功能变化,以揭示在线粒体前体蛋白输入线粒体内部之前,蛋白的识别功能变化在年龄相关性听力损失发病机制中的作用。方法：原代培养的耳蜗边缘细胞纯化后,行细胞的免疫荧光化学染色检测细胞内Tom20和Tom70的表达情况。边缘细胞经浓度梯度(0,2,4,6,8,10,12g/1)D-半乳糖处理3天和7天后,12g/1D-半乳糖处理不同时间(0,1,3,5,7,911天)后纳入实验,分别提取细胞总蛋白以及线粒体蛋白,经western blot方法检测细胞内和线粒体的Tom20和Tom70的表达变化。结果：边缘细胞的免疫荧光结果显示,细胞胞浆内含有丰富的Tom70和Tom20,Tom70沿核膜分布明显；Tom20和Tom70的D-半乳糖浓度依赖性的变化不一致,Tom70经低浓度D-半乳糖处理后表达即升至最高,浓度梯度D-Gal诱导的Tom20早期(3天)表达就逐渐增加；线粒体Tom70的表达变化与总蛋白变化一致,表现为先快速增加后缓慢降低。结论：Tom20和Tom70在原代培养的边缘细胞中表达丰富；Tom20和Tom70对低浓度D-Gal诱导的氧化应激反应更为敏感；Tom20和Tom70的识别功能变化在DNA聚合酶γ的线粒体输送中有重要作用,前体蛋白识别能力的下降导致突变相关修复酶的转运障碍而最终出现mtDNA缺失突变的累积,引致老年性聋的发生。
[Abstract]:The first part is to establish a model of premature failure of mtDNA4834bp deletion mutation in the rat cochlear vascular fringe cells
Objective: to establish a premature deletion model of mitochondrial DNA4834bp deletion in the marginal cells of cochlear stria vasculature in rats, and provide an easy access to further study of the mechanism of mtDNA4834bp deletion in age-related hearing loss.
Methods: born 3 days rats, under the microscope, separation of stria vascularis after dissection. Digestion of inoculated cells. To obtain uniform marginal cells of stria vascularis after repeated purification, immunofluorescence identification. CK-18 cells were treated with different concentration gradient (0,2,4,6,8,10,12,14,16g/1) D- galactose and D- mannitol group 3,7 and 11 days after treatment, the effect of CCK-8 to detect different concentrations of D- galactose on cell viability at different time. The concentration gradient of D- galactose treatment (3 days and 7 days) and 12g/1D- galactose treated cells on different days (1,3,5,7,9,11,13,15 days), the cumulative Taqman probe real-time quantitative PCR detection of cells induced by D- galactose the concentration and time dependence of the mtDNA4834bp deletion mutation rate. The cells of different 12g/1D- galactose treatment days (1,5,11 days) after using p- galactosidase staining for detection of cell senescence The apoptosis of peripheral cells was detected by TUNEL in situ chromogenic assay for 1 days and 15 days after.12g/1D- galactose treatment.
Results: the cells by immunofluorescence CK-18 staining, the cytoplasm is strong specific fluorescence. Marginal cells by the concentration gradient of D- galactose and osmotic pressure, D- mannitol treatment, cell viability changes in D- galactose group and no significant difference between the osmotic pressure control, 14g/1 and 16g/1 cell activity in a concentration dependent on the basis of the cumulative decline of 12g/1 is chosen as the optimal concentration of.D- galactose following treatment can induce cell edge cell concentration and time dependence of the mtDNA4834bp deletion mutant, in 5,7,9,11 days, compared with the control group were significantly different. Beta galactosidase staining showed that compared with the control group increased D- galactose group of blue cells in a time dependent manner, indicating significant senescent cells. Cells were treated with D- galactose treatment after a long time, TUNEL staining showed positive cells compared with the control group A significant increase.
Conclusion: the primary culture of marginal cells of stria vascularis; D- galactose induced cell culture environmental osmotic pressure is a major cause of decreased cell viability; D- galactose can successfully induce cell edge mtDNA4834bp deletion mutation accumulation and showed a time and concentration dependent; D- galactose induced cells senescence characteristics; cells with D- galactose treatment after a long time did not appear obvious apoptosis. We successfully established a rat model of premature cell mtDNA4834bp deletion mutation.
Study on the functional status of the second part of the mitochondrial translocation enzyme Tom40 and the mechanism of mtDNA4834bp deletion mutation induced by the change of DNA polymerase gamma mitochondria transport
Objective: To investigate the changes in mitochondrial outer membrane translocase Tom40 protein transport in accumulation of mtDNA4834bp deletion mutation in the mitochondrial function, the relationship between transport and mitochondrial DNA repair enzyme DNA polymerase gamma mitochondria, mitochondrial protein to reveal the changes of input function in the pathogenesis of age-related hearing loss in rats.
Methods: the normal adult rat cochlea, stone (?) embedded into paraffin sections, the expression of the tissue immunofluorescence staining technique for the detection of Tom40 in the cochlea of normal rat cochlea. The edge of cells with 12g/1D- galactose treatment after 11 days of primary culture, immunofluorescence cell staining for detection of cell Tom40. The concentration gradient of edge cells (0,2,4,6,8,10,12g/1) by D- galactose for 3 days and 7 days after different time of 12g/1D- galactose (0,13,5,7,9,11 days) after processing into the experiment, total cell proteins and mitochondrial proteins extracted by Western blot method for detection of intracellular and mitochondrial Tom40, expression of DNA polymerase of ya.
Results: (1) immunofluorescence staining showed that the cochlea, Tom40 in cochlear tissues rich in Corti's device, the lateral wall of the cochlea stria vascularis and spiral ganglion regions have specific expression. (2) immunofluorescence edge cells showed that cells containing abundant mitochondria, cytoplasm and nucleus expression Tom40 was strong, and by D- galactose treatment, decreased expression in the cytoplasm of Tom40. (3) the total expression of Tom40 cells induced by D- galactose concentration gradient increases slowly first and then decreased, and no significant relationship with processing time. (4) D- galactose induced intracellular Tom40 showed the dynamic time the change of dependence; expression of mitochondrial Tom40 were significantly enhanced. (5) the expression of DNA in cells and the mitochondrial polymerase gamma showed increased first and then decreased, significantly accumulated in a mtDNA4834bp deletion mutant displayed decreased significantly.
Conclusion: Tom40 in rat inner ear stria region and edge cells cultured in rich expression, its function changes may affect the normal function of cochlear cells, which lead to the occurrence of the disease; dynamic changes of D-Gal induced by Tom40 and DNA polymerase gamma response of cells to oxidative stress and compensatory decompensated cells; Tom40 and DNA polymerase D-Gal time cumulative change trend, indicating that Tom40 function changes of DNA polymerase gamma mitochondrial transport; the intracellular concentration of D-Gal Tom40 and DNA polymerase gamma dependent trend is not consistent, particularly in the short term after induction, suggesting that there may be other factors involved in mitochondrial translocation of DNA polymerase gamma in the process of mitochondrial Tom40 in mtDNA4834bp; the lack of accumulation, enhance its function; on the contrary, DNA polymerase Y mitochondrial transport decreased in cells and mitochondria; The changes in the expression of Tom40 protein are not consistent, suggesting that the expression of Tom40 protein may be regulated by post transcriptional mechanism.
The functional state of the third part of mitochondrial translocation enzyme Tom20 and Tom70 and the mechanism of mtDNA4834bp deletion mutation
Objective: To investigate the changes in mitochondrial outer membrane translocase protein transporting mitochondrial recognition receptors Tom20 and Tom70 in the process of accumulation of mtDNA4834bp deletions in mitochondrial function, in order to reveal the internal body protein of mitochondrial input before identification. Protein changes in the pathogenesis of age-related hearing loss in rats.
Methods: primary cultured cochlear marginal cells after purification, immunofluorescence staining for cells to detect the expression of intracellular Tom20 and Tom70. The concentration gradient of edge cells (0,2,4,6,8,10,12g/1) by D- galactose for 3 days and 7 days after different time of 12g/1D- galactose (0,1,3,5,7911 days) after treatment in the experiment, the total cell protein and the mitochondrial protein was extracted by blot method to detect the expression of western in cells and mitochondria of Tom20 and Tom70.
Results: immunofluorescence showed marginal cells, cytoplasm rich in Tom70 and Tom20, Tom70 and Tom20 were distributed along the nuclear membrane; Tom70 D- galactose concentration dependence is not the same, Tom70 by sugar treatment of low concentration D- Gal expression after that rose to the highest in early concentration gradient induced by D-Gal Tom20 (3 days) the expression is increased gradually; change of expression of mitochondrial Tom70 and total protein were increased rapidly at first and then decreased slowly.
Conclusion: Tom20 and Tom70 in cultured marginal cells express abundant; oxidative stress reaction of Tom20 and Tom70 on D-Gal induced by low concentration is more sensitive; Tom20 and Tom70 recognition function in the DNA polymerase gamma mitochondria plays an important role in transporting, decreased precursor protein identification ability of lead transport disorder associated with mutations repair enzymes and eventually lead to the accumulation of mtDNA deletion mutation, causing presbycusis.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R764

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