SIRT1在内耳拟老化伴线粒体DNA4834缺失突变细胞模型中的作用及其机制研究

发布时间：2018-06-05 07:48

本文选题：SIRT1 + 耳蜗　；参考：《华中科技大学》2010年博士论文

【摘要】： 目的：研究SIRT1基因在大鼠耳蜗组织中的表达特点,为离体研究奠定理论依据。方法：分别选取新生0-3天大鼠、正常成年大鼠及5%D—半乳糖颈部皮下注射8周诱导老化的大鼠各五只分离出双侧耳蜗组织,用OCT包埋后行耳蜗冰冻中轴切片,应用免疫荧光染色技术检测SIRT1在各组大鼠中的表达。结果：新生O-3天大鼠组、正常成年大鼠组及5%D—半乳糖颈部皮下注射8周诱导老化大鼠组耳蜗冰冻切片免疫荧光染色后均可见广泛、明亮的绿色荧光,在血管纹、螺旋神经节、Corti器区域,荧光增强尤为显著。各组间荧光强度无明显差异。结论：SIRT1在大鼠耳蜗组织中广泛表达,其表达在血管纹、螺旋神经节、螺旋韧带、Corti器尤为突出。SIRT1在新生O-3天大鼠组、正常成年大鼠组及5%D—半乳糖颈部皮下注射8周诱导老化大鼠耳蜗组织中的表达无免疫荧光检测范围内可见明显差异。目的：建立离体大鼠耳蜗血管纹边缘细胞(Marginal cells, MCs)和螺旋神经节细胞(Spiral ganglion cells, SGCs)线粒体常见缺失突变模型,为老年性聋的机制研究提供合适的研究对象。方法：选取新生0-3天大鼠,剥离双侧耳蜗组织,分离出血管纹组织和螺旋神经节组织块,分别行组织块贴壁法和消化法培养获取较均一的血管纹边缘细胞及螺旋神经节细胞。向体外培养的耳蜗血管纹细胞和螺旋神经节细胞中分别加入不同梯度浓度的D—半乳糖,采用MTT法检测0、2、4、6、8、10、12、14、16g/L D—半乳糖作用48h对血管纹边缘细胞及螺旋神经细胞活性的影响,筛选出最适诱导浓度；应用Taqman荧光探针实时定量PCR方法检测最适浓度D—半乳糖分别作用血管纹边缘细胞和螺旋神经细胞24h、72 h、120 h、168 h、216 h后mtDNA4834bp缺失突变的检出率；应用β—半乳糖苷酶(senescence-associatedβ-galactosidase, SA-β-gal)染色法检测细胞衰老状态；应用碘化丙锭(Propidium Iodide, PI)染色流式细胞仪测定细胞周期；采用AnnexinⅤ-PI双染流式细胞仪及TUNEL法测定细胞凋亡率；采用JC-1荧光探针染色流式细胞仪及荧光显微镜计数法测定细胞线粒体膜电位变化。结果：血管纹边缘细胞和螺旋神经节细胞分别在14g/L和10g/L D—半乳糖作用点出现浓度依赖性活性降低,故12g/L和8g/LD—半乳糖分别为MCs和SGCs的最适诱导浓度。12g/L D—半乳糖连续作用血管纹边缘细胞168h后可诱导出较显著的mtDNA4834bp缺失突变；细胞β—半乳糖苷酶表达明显升高；细胞分裂缓慢,细胞周期停滞于G0／G1期,S细胞及G2／M期细胞趋于消失；细胞凋亡率明显升高；线粒体膜电位明显降低。8g/L D—半乳糖连续作用螺旋神经节细胞120h后也可诱导出有统计学意义的mtDNA4834bp缺失突变,TUNEL检测阳性率显著增加,线粒体膜电位降低。结论：D—半乳糖可离体诱导细胞mtDNA4834bp缺失突变,可模拟耳蜗血管纹边缘细胞和螺旋神经节细胞的“早老性”衰老,成功建立血管纹边缘细胞和螺旋神经节细胞mtDNA4834bp缺失突变拟老化细胞模型。目的：研究SIRT1在mtDNA4834bp缺失突变形成中的作用,对细胞衰老(p-半乳糖苷酶和凋亡)和线粒体功能(线粒体膜电位)的影响,与线粒体相关因子核呼吸因子—1(NRF-1)、线粒体转录因子A (mitochondrial transcription factor A, mtTFA)、过氧化物酶增值子激活受体-γ亚单位共激活因子1α(peroxisome proliferator-activated receptor y coactivator 1 a, PGC-1 a )、线粒体细胞色素C氧化酶亚单位Ⅲ(cytochrome oxidase cytochrome oxidase subunit III, COXIII)和线粒体内膜质子转运相关蛋白解偶联蛋白2(uncoupling protein 2, UCP-2)之间的关系,以及其对自由基清道夫超氧化物歧化酶的作用,探讨SIRT1影响mtDNA4834bp缺失突变及细胞衰老的机制。方法：体外培养血管纹边缘细胞,分为sirtinol抑制剂组(SIR组)、白藜芦醇激动剂组(RSV组)、DMSO介质对照组(DMSO组)及对照组(CONT组)。当原代血管纹边缘细胞纯化融合成单层后将细胞传代分为以上四组,按以下方法继续培养168h：(1)SIR组：60μM sirtinol(无菌DMSO配制)+12g/L D—半乳糖+完全培养基(DMSO终浓度为0.125‰)；(2)RSV组：20μM白藜芦醇(无菌DMSO配制)+12g/LD—半乳糖+完全培养基(DMSO终浓度为0.125‰)；(3)DMSO组：DMSO+12g/L D—半乳糖+完全培养基(DMSO终浓度为0.125‰)；(4)CONT组：12g/L D—半乳糖+完全培养基。应用Taqman荧光探针实时定量PCR相对定量方法检测比较各组mtDNA4834bp缺失突变率；β—半乳糖苷酶染色法检测各组细胞衰老状态；AnnexinⅤ-PI双染以及JC-1荧光探针染色流式细胞仪分别检测各组细胞凋亡及线粒体膜电位变化情况；实时定量PCR (real-time quantitative PCR, RTqPCRs)检测mtDNA相对拷贝数及各组细胞SIRT1、NRF-1、mtTFA、PGC-1α、COXIII和UCP-2的：mRNA水平；蛋白免疫印记(western blot)检测各组SIRT1、乙酰化组蛋白3(Ac-H3)、UCP-2蛋白水平；免疫沉淀+蛋白免疫印记检测PGC-1α蛋白水平及PGC-1a乙酰化水平(Ac-PGC-la);WST-1法检测各组MnSOD、Cu/ZnSOD活性。结果：(1)SIRT1激动剂白藜芦醇能有效降低D—半乳糖糖诱导的mtDNA大片段缺失,能显著性降低D—半乳糖诱导的血管纹边缘细胞的“早老性”细胞表型：p—半乳糖苷酶染色阳性率显著性降低,细胞凋亡减少,线粒体膜电位补救性回升。SIRT1抑制剂sirtino1表现为拮抗效应。(2)RSV组线粒体DNA相对拷贝数增加,SIR组降低。(3)RSV组SIRT1、NRF-1、mtTFA、PGC-1α、COXⅢ的mRNA水平较DMSO组及CONT组上调,有统计学差异(P0.05)；SIR组SIRT1 mRNA水平相对DMSO组及CONT组无明显差异,NRF-1、mtTFA、PGC-1α、COXⅢmRNA水平有统计学意义的降低(P0.05)；DMSO组与CONT组无统计学差异；各组UCP-2 mRNA水平无统计学差异。(4)RSV组SIRT1、PGC-1α蛋白水平相对DMSO组及CONT组上调,Ac-H3, Ac-PGC-1α及UCP-2蛋白水平下降,差异有显著性(P0.05)；SIR组SIRT1蛋白表达与DMSO组及CONT组比较无显著统计学差异,相对于DMSO组及CONT组PGC-1α蛋白水平下调,Ac-H3,乙酰化PGC-1α及UCP-2蛋白水平上调,差异有统计学意义(P0.05)；DMSO组与CONT组之间蛋白表达量无明显差异。(5)RSV组MnSOD活性较DMSO组及CONT组增高,差异有显著性(P0.05)；SIR组MnSOD活性降低,差异有统计学意义(P0.05)；各组Cu/ZnSOD活性无明显差异。结论：SIRT1通过对线粒体相关基因、抗氧化防御系统以及线粒体内膜质子转运系统的调控,调节线粒体缺陷及细胞衰老过程。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R764

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