本文關(guān)鍵詞： 年齡相關(guān)性聽力損失 耳蝸血管紋邊緣細(xì)胞 D-半乳糖 mtDNA4834bp缺失突變 年齡相關(guān)性聽力損失 耳蝸血管紋邊緣細(xì)胞 Tom40 DNA聚合酶γ mtDNA普遍性缺失 D-半乳糖 年焍相關(guān)性聽力損失 耳蝸血管紋邊緣細(xì)胞 Tom20 Tom70 mt　出處：《華中科技大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分建立大鼠耳蝸血管紋邊緣細(xì)胞mtDNA4834bp缺失突變早衰模型 目的：用大鼠耳蝸血管紋邊緣細(xì)胞建立線粒體DNA4834bp缺失突變早衰模型,為進一步研究mtDNA4834bp缺失突變在年齡相關(guān)性聽力損失中的作用機制提供易獲取的研究對象。 方法：選取出生3天左右的大鼠,顯微鏡下解剖后分離耳蝸血管紋。消化法接種細(xì)胞。經(jīng)多次純化后獲取比較均一的血管紋邊緣細(xì)胞,CK-18的免疫熒光化學(xué)方法鑒定。細(xì)胞經(jīng)不同濃度梯度(0,2,4,6,8,10,12,14,16g/1)的D-半乳糖和D-甘露醇滲透壓組處理3,7天和11天后,采用CCK-8檢測不同濃度D-半乳糖對細(xì)胞活力的影響。濃度梯度D-半乳糖處理不同時間(3天和7天)和12g/1D-半乳糖處理細(xì)胞不同天數(shù)(1,3,5,7,9,11,13,15天)后,Taqman探針實時定量PCR技術(shù)檢測細(xì)胞經(jīng)D-半乳糖誘導(dǎo)的濃度和時間依賴性的mtDNA4834bp缺失突變率的累積。細(xì)胞經(jīng)12g/1D-半乳糖處理不同天數(shù)(1,5,11天)后,采用p-半乳糖苷酶染色檢測細(xì)胞衰老的程度。12g/1D-半乳糖處理細(xì)胞1天和15天后,行TUNEL原位顯色法檢測邊緣細(xì)胞的凋亡狀況。 結(jié)果：細(xì)胞經(jīng)CK-18的免疫熒光染色后,胞漿呈現(xiàn)較強特異性熒光。邊緣細(xì)胞經(jīng)濃度梯度D-半乳糖和滲透壓匹配的D-甘露醇處理后,細(xì)胞活力變化在D-半乳糖組和滲透壓對照組之間無顯著差異,14g/1和16g/1時細(xì)胞活力呈現(xiàn)濃度依賴性下降,以此為依據(jù)選取12g/1作為后續(xù)處理細(xì)胞的最佳濃度。D-半乳糖能誘導(dǎo)邊緣細(xì)胞出現(xiàn)濃度和時間依賴性的mtDNA4834bp缺失突變的累積,在處理5,7,9,11天時,與對照組相比有顯著差異。β-半乳糖苷酶染色結(jié)果顯示,與對照組相比D-半乳糖組藍(lán)染細(xì)胞呈現(xiàn)時間依賴性的增多,表示細(xì)胞的明顯衰老。細(xì)胞經(jīng)長時間的D-半乳糖處理后,TUNEL染色結(jié)果顯示陽性細(xì)胞較對照組明顯增加。 結(jié)論：成功培養(yǎng)了原代耳蝸血管紋邊緣細(xì)胞；D-半乳糖引致的細(xì)胞培養(yǎng)環(huán)境滲透壓的變化是導(dǎo)致細(xì)胞活力下降的主要原因；D-半乳糖能成功誘導(dǎo)邊緣細(xì)胞出現(xiàn)mtDNA4834bp缺失突變,且呈現(xiàn)濃度和時間依賴性的累積;D-半乳糖能誘導(dǎo)細(xì)胞出現(xiàn)早衰的特點；細(xì)胞經(jīng)D-半乳糖處理較長時間后才出現(xiàn)明顯的細(xì)胞凋亡。故成功建立了大鼠mtDNA4834bp缺失突變細(xì)胞早衰模型。 第二部分線粒體外膜轉(zhuǎn)位酶Tom40的功能狀態(tài)與DNA聚合酶γ的線粒體輸送改變致mtDNA4834bp缺失突變機制的研究 目的：探討掌管線粒體前體蛋白輸送的線粒體外膜轉(zhuǎn)位酶Tom40在mtDNA4834bp缺失突變的累積過程中的功能變化,與線粒體DNA修復(fù)酶DNA聚合酶γ的線粒體輸送的關(guān)系,以揭示線粒體前體蛋白輸入的功能變化在年齡相關(guān)性聽力損失發(fā)病機制中的作用。 方法：取正常成年大鼠耳蝸,石(?)包埋后制成石蠟切片,行組織的免疫熒光染色技術(shù)檢測Tom40在正常大鼠耳蝸中的表達(dá)。原代培養(yǎng)的耳蝸邊緣細(xì)胞經(jīng)12g/1D-半乳糖處理11天后,行細(xì)胞的免疫熒光化學(xué)染色檢測細(xì)胞內(nèi)Tom40的表達(dá)情況。邊緣細(xì)胞經(jīng)濃度梯度(0,2,4,6,8,10,12g/1)D-半乳糖處理3天和7天后,12g/1D-半乳糖處理不同時間(0,13,5,7,9,11天)后納入實驗,分別提取細(xì)胞總蛋白以及線粒體蛋白,經(jīng)western blot方法檢測細(xì)胞內(nèi)和線粒體的Tom40,DNA聚合酶丫的表達(dá)變化。 結(jié)果：(1)耳蝸的組織免疫熒光染色結(jié)果顯示,Tom40在耳蝸組織中表達(dá)豐富, Corti氏器,耳蝸外側(cè)壁血管紋以及螺旋神經(jīng)節(jié)區(qū)域均有特異性表達(dá)。(2)邊緣細(xì)胞的免疫熒光結(jié)果顯示,細(xì)胞含有豐富的線粒體,胞漿和胞核均有Tom40的強表達(dá),且經(jīng)D-半乳糖處理后,胞漿內(nèi)Tom40的表達(dá)減弱。(3)濃度梯度D-半乳糖誘導(dǎo)細(xì)胞內(nèi)總Tom40的表達(dá)先緩慢增加然后降低,且與處理時間無明顯關(guān)系。(4)D-半乳糖誘導(dǎo)細(xì)胞內(nèi)Tom40表現(xiàn)出時間依賴性的動態(tài)變化；線粒體的Tom40的表達(dá)則明顯增強。(5)細(xì)胞內(nèi)和線粒體的DNA聚合酶γ的表達(dá)呈現(xiàn)先增高再減低的趨勢,在mtDNA4834bp缺失突變顯著累積時則表現(xiàn)為明顯下降。 結(jié)論：Tom40在大鼠內(nèi)耳血管紋區(qū)域以及原代培養(yǎng)的邊緣細(xì)胞中表達(dá)豐富,它的功能變化可能會影響到耳蝸細(xì)胞的正常功能,而導(dǎo)致疾病的發(fā)生；D-Gal誘導(dǎo)Tom40和DNA聚合酶γ的動態(tài)變化反應(yīng)了細(xì)胞對氧化應(yīng)激的代償與失代償；細(xì)胞內(nèi)Tom40和DNA聚合酶γ的D-Gal時間累積性變化趨勢吻合,表明Tom40的功能變化影響DNA聚合酶γ的線粒體輸送；細(xì)胞內(nèi)Tom40和DNA聚合酶γ的D-Gal濃度依賴性變化趨勢不一致,尤其是短期誘導(dǎo)后,提示可能有其他因素參與到DNA聚合酶γ的線粒體轉(zhuǎn)運過程中；線粒體的Tom40在mtDNA4834bp缺失累積的過程中,其功能增強；相反,DNA聚合酶Y的線粒體輸送減少；細(xì)胞內(nèi)和線粒體內(nèi)Tom40蛋白表達(dá)的變化趨勢不一致,提示Tom40蛋白的表達(dá)可能受到轉(zhuǎn)錄后機制的調(diào)控。 第三部分線粒體外膜轉(zhuǎn)位酶Tom20和Tom70的功能狀態(tài)與mtDNA4834bp缺失突變機制的研究 目的：進一步探討掌管線粒體前體蛋白輸送的線粒體外膜轉(zhuǎn)位酶的識別受體Tom20和Tom70在mtDNA4834bp缺失突變的累積過程中的功能變化,以揭示在線粒體前體蛋白輸入線粒體內(nèi)部之前,蛋白的識別功能變化在年齡相關(guān)性聽力損失發(fā)病機制中的作用。 方法：原代培養(yǎng)的耳蝸邊緣細(xì)胞純化后,行細(xì)胞的免疫熒光化學(xué)染色檢測細(xì)胞內(nèi)Tom20和Tom70的表達(dá)情況。邊緣細(xì)胞經(jīng)濃度梯度(0,2,4,6,8,10,12g/1)D-半乳糖處理3天和7天后,12g/1D-半乳糖處理不同時間(0,1,3,5,7,911天)后納入實驗,分別提取細(xì)胞總蛋白以及線粒體蛋白,經(jīng)western blot方法檢測細(xì)胞內(nèi)和線粒體的Tom20和Tom70的表達(dá)變化。 結(jié)果：邊緣細(xì)胞的免疫熒光結(jié)果顯示,細(xì)胞胞漿內(nèi)含有豐富的Tom70和Tom20,Tom70沿核膜分布明顯；Tom20和Tom70的D-半乳糖濃度依賴性的變化不一致,Tom70經(jīng)低濃度D-半乳糖處理后表達(dá)即升至最高,濃度梯度D-Gal誘導(dǎo)的Tom20早期(3天)表達(dá)就逐漸增加；線粒體Tom70的表達(dá)變化與總蛋白變化一致,表現(xiàn)為先快速增加后緩慢降低。 結(jié)論：Tom20和Tom70在原代培養(yǎng)的邊緣細(xì)胞中表達(dá)豐富；Tom20和Tom70對低濃度D-Gal誘導(dǎo)的氧化應(yīng)激反應(yīng)更為敏感；Tom20和Tom70的識別功能變化在DNA聚合酶γ的線粒體輸送中有重要作用,前體蛋白識別能力的下降導(dǎo)致突變相關(guān)修復(fù)酶的轉(zhuǎn)運障礙而最終出現(xiàn)mtDNA缺失突變的累積,引致老年性聾的發(fā)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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R764

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本文編號：1501785