本文選題：SIRT1 + 耳蝸�。� 參考：《華中科技大學(xué)》2010年博士論文

【摘要】： 目的：研究SIRT1基因在大鼠耳蝸組織中的表達特點,為離體研究奠定理論依據(jù)。 方法：分別選取新生0-3天大鼠、正常成年大鼠及5%D—半乳糖頸部皮下注射8周誘導(dǎo)老化的大鼠各五只分離出雙側(cè)耳蝸組織,用OCT包埋后行耳蝸冰凍中軸切片,應(yīng)用免疫熒光染色技術(shù)檢測SIRT1在各組大鼠中的表達。 結(jié)果：新生O-3天大鼠組、正常成年大鼠組及5%D—半乳糖頸部皮下注射8周誘導(dǎo)老化大鼠組耳蝸冰凍切片免疫熒光染色后均可見廣泛、明亮的綠色熒光,在血管紋、螺旋神經(jīng)節(jié)、Corti器區(qū)域,熒光增強尤為顯著。各組間熒光強度無明顯差異。 結(jié)論：SIRT1在大鼠耳蝸組織中廣泛表達,其表達在血管紋、螺旋神經(jīng)節(jié)、螺旋韌帶、Corti器尤為突出。SIRT1在新生O-3天大鼠組、正常成年大鼠組及5%D—半乳糖頸部皮下注射8周誘導(dǎo)老化大鼠耳蝸組織中的表達無免疫熒光檢測范圍內(nèi)可見明顯差異。 目的：建立離體大鼠耳蝸血管紋邊緣細胞(Marginal cells, MCs)和螺旋神經(jīng)節(jié)細胞(Spiral ganglion cells, SGCs)線粒體常見缺失突變模型,為老年性聾的機制研究提供合適的研究對象。 方法：選取新生0-3天大鼠,剝離雙側(cè)耳蝸組織,分離出血管紋組織和螺旋神經(jīng)節(jié)組織塊,分別行組織塊貼壁法和消化法培養(yǎng)獲取較均一的血管紋邊緣細胞及螺旋神經(jīng)節(jié)細胞。向體外培養(yǎng)的耳蝸血管紋細胞和螺旋神經(jīng)節(jié)細胞中分別加入不同梯度濃度的D—半乳糖,采用MTT法檢測0、2、4、6、8、10、12、14、16g/L D—半乳糖作用48h對血管紋邊緣細胞及螺旋神經(jīng)細胞活性的影響,篩選出最適誘導(dǎo)濃度；應(yīng)用Taqman熒光探針實時定量PCR方法檢測最適濃度D—半乳糖分別作用血管紋邊緣細胞和螺旋神經(jīng)細胞24h、72 h、120 h、168 h、216 h后mtDNA4834bp缺失突變的檢出率；應(yīng)用β—半乳糖苷酶(senescence-associatedβ-galactosidase, SA-β-gal)染色法檢測細胞衰老狀態(tài)；應(yīng)用碘化丙錠(Propidium Iodide, PI)染色流式細胞儀測定細胞周期；采用AnnexinⅤ-PI雙染流式細胞儀及TUNEL法測定細胞凋亡率；采用JC-1熒光探針染色流式細胞儀及熒光顯微鏡計數(shù)法測定細胞線粒體膜電位變化。 結(jié)果：血管紋邊緣細胞和螺旋神經(jīng)節(jié)細胞分別在14g/L和10g/L D—半乳糖作用點出現(xiàn)濃度依賴性活性降低,故12g/L和8g/LD—半乳糖分別為MCs和SGCs的最適誘導(dǎo)濃度。12g/L D—半乳糖連續(xù)作用血管紋邊緣細胞168h后可誘導(dǎo)出較顯著的mtDNA4834bp缺失突變；細胞β—半乳糖苷酶表達明顯升高；細胞分裂緩慢,細胞周期停滯于G0／G1期,S細胞及G2／M期細胞趨于消失；細胞凋亡率明顯升高；線粒體膜電位明顯降低。8g/L D—半乳糖連續(xù)作用螺旋神經(jīng)節(jié)細胞120h后也可誘導(dǎo)出有統(tǒng)計學(xué)意義的mtDNA4834bp缺失突變,TUNEL檢測陽性率顯著增加,線粒體膜電位降低。 結(jié)論：D—半乳糖可離體誘導(dǎo)細胞mtDNA4834bp缺失突變,可模擬耳蝸血管紋邊緣細胞和螺旋神經(jīng)節(jié)細胞的“早老性”衰老,成功建立血管紋邊緣細胞和螺旋神經(jīng)節(jié)細胞mtDNA4834bp缺失突變擬老化細胞模型。 目的：研究SIRT1在mtDNA4834bp缺失突變形成中的作用,對細胞衰老(p-半乳糖苷酶和凋亡)和線粒體功能(線粒體膜電位)的影響,與線粒體相關(guān)因子核呼吸因子—1(NRF-1)、線粒體轉(zhuǎn)錄因子A (mitochondrial transcription factor A, mtTFA)、過氧化物酶增值子激活受體-γ亞單位共激活因子1α(peroxisome proliferator-activated receptor y coactivator 1 a, PGC-1 a )、線粒體細胞色素C氧化酶亞單位Ⅲ(cytochrome oxidase cytochrome oxidase subunit III, COXIII)和線粒體內(nèi)膜質(zhì)子轉(zhuǎn)運相關(guān)蛋白解偶聯(lián)蛋白2(uncoupling protein 2, UCP-2)之間的關(guān)系,以及其對自由基清道夫超氧化物歧化酶的作用,探討SIRT1影響mtDNA4834bp缺失突變及細胞衰老的機制。 方法：體外培養(yǎng)血管紋邊緣細胞,分為sirtinol抑制劑組(SIR組)、白藜蘆醇激動劑組(RSV組)、DMSO介質(zhì)對照組(DMSO組)及對照組(CONT組)。當(dāng)原代血管紋邊緣細胞純化融合成單層后將細胞傳代分為以上四組,按以下方法繼續(xù)培養(yǎng)168h：(1)SIR組：60μM sirtinol(無菌DMSO配制)+12g/L D—半乳糖+完全培養(yǎng)基(DMSO終濃度為0.125‰)；(2)RSV組：20μM白藜蘆醇(無菌DMSO配制)+12g/LD—半乳糖+完全培養(yǎng)基(DMSO終濃度為0.125‰)；(3)DMSO組：DMSO+12g/L D—半乳糖+完全培養(yǎng)基(DMSO終濃度為0.125‰)；(4)CONT組：12g/L D—半乳糖+完全培養(yǎng)基。應(yīng)用Taqman熒光探針實時定量PCR相對定量方法檢測比較各組mtDNA4834bp缺失突變率；β—半乳糖苷酶染色法檢測各組細胞衰老狀態(tài)；AnnexinⅤ-PI雙染以及JC-1熒光探針染色流式細胞儀分別檢測各組細胞凋亡及線粒體膜電位變化情況；實時定量PCR (real-time quantitative PCR, RTqPCRs)檢測mtDNA相對拷貝數(shù)及各組細胞SIRT1、NRF-1、mtTFA、PGC-1α、COXIII和UCP-2的：mRNA水平；蛋白免疫印記(western blot)檢測各組SIRT1、乙酰化組蛋白3(Ac-H3)、UCP-2蛋白水平；免疫沉淀+蛋白免疫印記檢測PGC-1α蛋白水平及PGC-1a乙�；�(Ac-PGC-la);WST-1法檢測各組MnSOD、Cu/ZnSOD活性。 結(jié)果：(1)SIRT1激動劑白藜蘆醇能有效降低D—半乳糖糖誘導(dǎo)的mtDNA大片段缺失,能顯著性降低D—半乳糖誘導(dǎo)的血管紋邊緣細胞的“早老性”細胞表型：p—半乳糖苷酶染色陽性率顯著性降低,細胞凋亡減少,線粒體膜電位補救性回升。SIRT1抑制劑sirtino1表現(xiàn)為拮抗效應(yīng)。(2)RSV組線粒體DNA相對拷貝數(shù)增加,SIR組降低。(3)RSV組SIRT1、NRF-1、mtTFA、PGC-1α、COXⅢ的mRNA水平較DMSO組及CONT組上調(diào),有統(tǒng)計學(xué)差異(P0.05)；SIR組SIRT1 mRNA水平相對DMSO組及CONT組無明顯差異,NRF-1、mtTFA、PGC-1α、COXⅢmRNA水平有統(tǒng)計學(xué)意義的降低(P0.05)；DMSO組與CONT組無統(tǒng)計學(xué)差異；各組UCP-2 mRNA水平無統(tǒng)計學(xué)差異。(4)RSV組SIRT1、PGC-1α蛋白水平相對DMSO組及CONT組上調(diào),Ac-H3, Ac-PGC-1α及UCP-2蛋白水平下降,差異有顯著性(P0.05)；SIR組SIRT1蛋白表達與DMSO組及CONT組比較無顯著統(tǒng)計學(xué)差異,相對于DMSO組及CONT組PGC-1α蛋白水平下調(diào),Ac-H3,乙�；疨GC-1α及UCP-2蛋白水平上調(diào),差異有統(tǒng)計學(xué)意義(P0.05)；DMSO組與CONT組之間蛋白表達量無明顯差異。(5)RSV組MnSOD活性較DMSO組及CONT組增高,差異有顯著性(P0.05)；SIR組MnSOD活性降低,差異有統(tǒng)計學(xué)意義(P0.05)；各組Cu/ZnSOD活性無明顯差異。 結(jié)論：SIRT1通過對線粒體相關(guān)基因、抗氧化防御系統(tǒng)以及線粒體內(nèi)膜質(zhì)子轉(zhuǎn)運系統(tǒng)的調(diào)控,調(diào)節(jié)線粒體缺陷及細胞衰老過程。
[Abstract]:Objective: To study the expression characteristics of SIRT1 gene in rat cochlea, and to lay a theoretical foundation for in vitro study.
Methods: five newborn rats, normal adult rats and five rats with 5%D - galactose neck subcutaneous injection for 8 weeks were separated from the cochlear tissues of five rats, and the cochlear frozen middle axis was sliced by OCT embedding. The expression of SIRT1 in each group was detected by immunofluorescence staining.
Results: the newborn O-3 days rats, normal adult rats and 5%D - galactose neck subcutaneous injection for 8 weeks induced aging rat cochlear frozen section immunofluorescence staining were all visible, bright green fluorescence, in the vascular pattern, spiral ganglion, Corti area, fluorescence intensities were particularly significant. There was no significant difference in fluorescence intensity among the groups.
Conclusion: SIRT1 is widely expressed in the cochlear tissue of rats. Its expression is in the vascular pattern, spiral ganglion, spiral ligament, and Corti apparatus, especially the.SIRT1 in the new O-3 day rat group. The normal adult rat group and the 5%D - galactose neck subcutaneous injection in the cochlear tissue of 8 weeks induced aging rats can be seen within the scope of no immunofluorescence detection. Difference.
Objective: to establish a common deletion mutation model of mitochondrial Marginal cells (MCs) and spiral ganglion cells (Spiral ganglion cells, SGCs) in isolated rat cochlea, so as to provide a suitable research object for the study of the mechanism of senile deafness.
Methods: a new 0-3 day old rat was selected to peel off bilateral cochlear tissue and separate the vascular tissue and spiral ganglion tissue blocks. The tissue block adherence method and digestion method were used to obtain the homogeneous marginal cells and spiral ganglion cells, and the cochlear blood tube and spiral ganglion cells were added to the cultured cochlea. Different gradient concentration of D galactose, the effects of 0,2,4,6,8,10,12,14,16g/L D - galactose on the activity of vascular fringe cells and spiral nerve cells were detected by MTT method, and the optimum concentration was screened out. The optimal concentration of D - galactose was detected by the real-time quantitative PCR method using Taqman fluorescence probe. The detection rates of 24h, 72 h, 120 h, 168 h, 216 h after mtDNA4834bp deletion were detected, and cell senescence was detected by using beta galactosidase (senescence-associated beta -galactosidase, SA- beta -gal), and cell cycle was measured by the staining flow cytometer of ingol iodide (Propidium Iodide). The apoptosis rate of cells was measured by -PI double dye flow cytometer and TUNEL method, and the changes of mitochondrial membrane potential were measured by JC-1 fluorescent probe staining flow cytometry and fluorescence microscope counting method.
Results: the concentration dependent activity of vascular fringe cells and spiral ganglion cells in 14g/L and 10g/L D - galactose, respectively, was reduced, so 12g/L and 8g/LD - galactose were the optimal inducible concentration of MCs and SGCs, respectively,.12g/L D - galactose continuous action of vascular fringe fine cell 168h, which could induce significant mtDNA4834bp deficiency. The expression of beta galactosidase was obviously increased, cell division was slow, cell cycle stagnated at G0 / G1, S cells and G2 / M phase cells tended to disappear, cell apoptosis rate was obviously increased, and mitochondrial membrane potential obviously decreased.8g/L D - galactose after 120h of spiral God ganglion cell 120h. The positive deletion rate of mtDNA4834bp was significantly increased, and the mitochondrial membrane potential of TUNEL was significantly decreased.
Conclusion: D - galactose can induce mtDNA4834bp deletion mutation in vitro, which can simulate the "premature senility" of the cochlear vascular fringe cells and spiral ganglion cells, and successfully establish the pattern of the mtDNA4834bp deletion mutation of the marginal and spiral ganglion cells of the spiral ganglion cells.
Objective: To study the role of SIRT1 in the formation of mtDNA4834bp deletion mutation, the effect on cell senescence (p- galactosidase and apoptosis) and mitochondrial function (mitochondrial membrane potential), mitochondrial related factor nuclear respiratory factor - 1 (NRF-1), mitochondrial transcription factor A (mitochondrial transcription factor A, mtTFA), and the increment of peroxidase The subunit co activating factor 1 alpha (peroxisome proliferator-activated receptor y coactivator 1 A, PGC-1 a), mitochondrial cytochrome C oxidase subunit III (cytochrome oxidase cytochrome) and mitochondrial membrane transport associated protein uncoupling protein 2 (2) The relationship between UCP-2 and its role in scavenger superoxide dismutase (SCOD) was discussed, and the mechanism of SIRT1 affecting mtDNA4834bp deletion mutation and cell senescence was discussed.
Methods: the vascular fringe cells were cultured in vitro, which were divided into sirtinol inhibitor group (group SIR), resveratrol agonist group (group RSV), DMSO medium control group (DMSO group) and control group (CONT group). When the primary vascular fringe cells were purified and fused into single layer, the cell passages were divided into four groups, and the following methods were continued to cultivate 168h: (1) SIR group: 60 mu M sirtinol (aseptic DMSO) +12g/L D - galactose + complete medium (DMSO terminal concentration is 0.125 per thousand); (2) RSV group: 20 mu M (aseptic DMSO) +12g/LD - galactose + complete medium (DMSO concentration is 0.125 per thousand); (3) DMSO group: galactose + complete medium (0.125 per thousand final concentration); (4) group: (4): 12g/L D - galactose + complete culture medium. The mtDNA4834bp deletion mutation rate of each group was detected by Taqman fluorescence probe real-time quantitative PCR relative quantitative method; beta galactosidase staining was used to detect the senescence of each group; Annexin V -PI double staining and JC-1 fluorescence probe staining flow cytometry were used to detect the apoptosis of each group. The change of mitochondrial membrane potential; real-time quantitative PCR (real-time quantitative PCR, RTqPCRs) to detect the relative copies of mtDNA and the levels of SIRT1, NRF-1, mtTFA, PGC-1 a, COXIII and UCP-2: protein immunoimprinting, protein 3, protein levels, immunoprecipitation + eggs The level of PGC-1 alpha protein and PGC-1a acetylation level (Ac-PGC-la) were detected by white immuno imprinting, and the activity of MnSOD and Cu/ZnSOD in each group was detected by WST-1.
Results: (1) SIRT1 agonist resveratrol can effectively reduce the D - galactosyl induced mtDNA deletion, and can significantly reduce the "early aging" cell phenotype of D - galactose induced vascular fringe cells: the positive rate of P - galactosidase staining, the decrease of apoptosis and the recovery of mitochondrial membrane potential of.SIR The T1 inhibitor sirtino1 showed antagonistic effect. (2) the relative copy number of DNA in the RSV group increased and the SIR group decreased. (3) the mRNA level of SIRT1, NRF-1, mtTFA, PGC-1 a, COX III of the RSV group was higher than that of the group and the group. There was no significant difference in the level of statistical significance (P0.05). There was no statistical difference between group DMSO and group CONT, and there was no statistical difference in the level of UCP-2 mRNA in each group. (4) the level of PGC-1 alpha protein in group RSV was higher than that in the DMSO group and CONT group, Ac-H3, the level of Ac-PGC-1 A and protein decreased. There was no significant difference in the level of PGC-1 alpha protein in group DMSO and CONT group, and the level of Ac-H3, acetylation PGC-1 A and UCP-2 protein was up regulated (P0.05), and there was no significant difference in protein expression between group DMSO and CONT group. (5) MnSOD activity in RSV group was higher than that in DMSO group and group. The activity of MnSOD in group IR was decreased, the difference was statistically significant (P0.05), and there was no significant difference in Cu/ZnSOD activity in each group.
Conclusion: SIRT1 regulates mitochondrial defects and cell aging through the regulation of mitochondrial related genes, antioxidant defense system and mitochondrial membrane proton transport system.
【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】：2010
【分類號】：R764

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