老年大鼠GnRH1表观变化及琼玉膏延缓衰老的分子机制探讨

发布时间：2018-06-20 07:18

本文选题：琼玉膏 + 延缓衰老　；参考：《广州中医药大学》2017年博士论文

【摘要】：目的:通过对老年大鼠与青年大鼠下丘脑衰老相关基因GnRH1、Sirt1、DNMT1、DNMT3a mRNA和蛋白表达水平以及GnRH1基因甲基化情况进行分析,从表观遗传水平探讨大鼠自然衰老进程中DNA甲基转移酶活性及GnRH1基因甲基化的改变;通过分离新生大鼠下丘脑神经元干细胞,建立D-半乳糖诱导的下丘脑神经元干细胞衰老模型,比较正常神经元干细胞组、D-半乳糖诱导衰老组与不同浓度琼玉膏干预组之间细胞成球能力、炎性因子及NF-κB表达的差异,为揭示琼玉膏延缓神经元干细胞衰老的功效及其可能分子机制提供实验参考和数据支持。方法:观察比较6只24月龄自然衰老SD大鼠和6只2月龄青年SD大鼠下丘脑衰老相关基因 mRNA 和蛋白表达水平:Real-Time PCR 检测 GnRH1、Sirt1、DNMT1、DNMT3a基因 mRNA 表达水平;Western Blot 检测 GnRH1、Sirt1、DNMT1、DNMT3a 蛋白表达水平;使用SPSS19.0统计软件做统计学分析。MSP法检测老年大鼠和青年大鼠GnRH1基因甲基化情况。分离培养8只新生SD大鼠(出生24h内)下丘脑神经元干细胞,免疫荧光法检测神经元烯醇化酶(NSE),鉴定神经元干细胞;CCK8法检测琼玉膏对神经元干细胞增殖的影响(0D值),使用SPSS19.0统计软件计算IC50,为后续分组实验提供依据。取神经元干细胞分成A、B、C、D四组,采用NSCs完全培养基分别对四组进行原代培养至第8天;从第9天起,A组继续采用培养基培养24h;B组依据参考文献加入10mg/mL D-半乳糖致衰24h;C组先加9μg/mL(1/2 IC50)琼玉膏干预6h,再加10mg/mL D-半乳糖致衰24h;D组先加45μg/mL(1/4 IC50)琼玉膏干预6h,再加10mg/mL D-半乳糖致衰24h。倒置荧光显微镜下观察各组神经元干细胞形态及成球能力(SFE);Real-Time PCR检测各组肿瘤坏死因子-α(TNF-α)及白细胞介素1β(IL-1β)mRNA表达水平;Western Blot检测核转录因子-kappaB(NF-κB)蛋白表达水平;Elisa检测肿瘤坏死因子-α(TNF-α)及白细胞介素1β(IL-1β)蛋白表达水平;使用SPSS19.0统计软件做统计学分析。结果:1.GnRH1、Sirt1、DNMT1基因mRNA及蛋白表达水平在老年鼠中明显降低,与青年鼠比较有显著性差异(p0.05);DNMT3a基因mRNA及蛋白表达水平在老年鼠和青年鼠之间无显著性差异(p0.05);老年鼠GnRH1基因出现部分甲基化,青年鼠GnRHl基因未出现甲基化。2.从新生鼠下丘脑成功分离神经元干细胞,倒置显微镜下观察细胞成球状生长;免疫荧光法检测到细胞球体有NSE清晰的红色荧光信号,即NSE在神经元干细胞中阳性表达。3.CCK8法检测到琼玉膏对神经元干细胞的增殖作用(0D值)与时间和剂量存在一定的依赖关系,24h内琼玉膏浓度变化对细胞的影响较小;根据24h对应的IC50值(185.91μg/mL),选取1/2IC50(90μg/mL)和1/4 I050(45μg/mL)作为后续分组实验时琼玉膏的浓度值。4.成功建立D-半乳糖诱导的下丘脑神经元干细胞衰老模型,呈现典型的炎症样变化:细胞成球能力降低,肿瘤坏死因子-α(TNF-α)及白细胞介素1β(IL-1β)mRNA和蛋白表达水平显著增加,核转录因子-kappaB(NF-κB)信号通路被激活,cytoplasm P65表达水平下降,nuclear P65表达水平上升。5.90μg/mL(1/2IC50)琼玉膏干预组与D-半乳糖衰老模型组比较细胞成球能力显著恢复(P0.05),肿瘤坏死因子-α(TNF-α)及白细胞介素1β(IL-1β)mRNA和蛋白表达水平显著降低(p0.05),核转录因子-kappaB(NF-κB)转录活性被抑制,cytoplasm P65表达水平上升,nuclear P65表达水平下降。结论:1.GnRH1、Sirt1基因mRNA及蛋白表达水平在老年鼠和青年鼠之间有显著性差异,说明GnRH1、Sirt1的表达水平与增龄呈负相关。2.DNMT1基因mRNA及蛋白表达水平在老年鼠中显著降低,DNMT3a基因mRNA及蛋白表达水平在老年鼠和青年鼠之间无显著性差异,说明DNMT1的表达水平与增龄呈负相关,DNMT3a的表达水平不随增龄而变化。3.GnRH1基因在老年鼠中出现部分甲基化,在青年鼠中未出现甲基化,说明GnRH1基因的表达水平受到其启动子区甲基化的调控,大鼠机体发生自然衰老与GnRH1基因启动子甲基化从而抑制GnRH1的表达水平有关。4.琼玉膏能预防D-半乳糖诱导的神经元干细胞衰老模型的炎症反应:恢复细胞成球能力、抑制炎性指标,且其作用强度呈浓度依赖性,随着琼玉膏浓度从45μg/mL增加到9μg/mL,其延缓衰老作用也相应增强。5.由于NF-κ B具有负调控GnRH1基因mRNA表达的功能,因此琼玉膏延缓衰老作用的可能分子机制在于抑制下丘脑神经元干细胞炎性细胞因子TNF-α、IL-1βP的表达以及NF-κ B的转录活性,进而逆转自然衰老进程中GnRH1基因甲基化及其mRNA表达水平的下降。
[Abstract]:Objective: to analyze the expression level of GnRH1, Sirt1, DNMT1, DNMT3a mRNA and protein and the methylation of GnRH1 gene in the hypothalamus of old rats and young rats, and to explore the changes of the activity of DNA methyltransferase and the methylation of GnRH1 gene from the epigenetic level in the process of natural aging in rats. In the rat hypothalamus neuron stem cells, the aging model of D- galactose induced hypothalamic neuron stem cells was established. Compared with normal neural stem cells, the cell formation ability, inflammatory factors and NF- kappa B expression between D- galactose induced senescence group and different concentration of Qiong ointment intervention group were found to reveal that Qiong jade ointment delayed neuronal stem cell failure. The old efficacy and its possible molecular mechanism provide experimental reference and data support. Methods: To observe and compare the mRNA and protein expression levels of the hypothalamic senescence related genes in 6 24 month old natural aging SD rats and 6 2 month old young SD rats: Real-Time PCR detection of GnRH1, Sirt1, DNMT1, DNMT3a gene mRNA expression level; Western Blot test The expression level of Sirt1, DNMT1, DNMT3a protein, SPSS19.0 statistical software was used to analyze the methylation of GnRH1 gene in old rats and young rats by statistical analysis. 8 newborn SD rats were isolated and cultured in the hypothalamus neurons of the newborn SD, and the immunofluorescence assay was used to detect the nenolase (NSE), and to identify the neuron stem cells; CCK8. The effect of agar ointment on the proliferation of neural stem cells (0D value) was detected by using the SPSS19.0 statistical software to calculate IC50, which provided the basis for the follow-up group experiment. The neural stem cells were divided into four groups, A, B, C, D, and the four groups were cultured to eighth days by NSCs complete medium. From the ninth day, the A Group continued to cultivate 24h with the culture medium; B group depends on it. According to the reference literature, 10mg/mL D- galactose was added to 24h, and group C was first added 9 mu g/mL (1/2 IC50) ointment to intervene 6h, and then 10mg/mL D- galactose to induce senescence, and D group first added 45 Mu to observe the morphology and ball forming ability of neurons in each group. Me PCR detection of tumor necrosis factor - alpha (TNF- a) and interleukin 1 beta (IL-1 beta) mRNA expression level; Western Blot detection of nuclear transcription factor -kappaB (NF- kappa B) protein expression level; Elisa detection of tumor necrosis factor - alpha (TNF- alpha) and interleukin 1 beta protein expression level; results of statistical analysis using statistical software. 1.GnRH1, Sirt1, DNMT1 gene mRNA and protein expression level decreased significantly in old rats, compared with young rats (P0.05); DNMT3a gene mRNA and protein expression level had no significant difference between old rats and young rats (P0.05); the GnRH1 base of old rats was partially methylation and GnRHl genes of young rats did not appear to be methylated.2.. The stem cells were successfully isolated from the hypothalamus of the newborn rats, and the cells were spheroidal growth under the inverted microscope. The immunofluorescence method was used to detect the NSE clear red fluorescence signal of the cell sphere. That is, the positive expression of NSE in the neural stem cells was detected by the.3.CCK8 method of the proliferation (0D value) and time and dosage of the Qiong ointment on the neural stem cells. There is a certain dependence. The effect of the concentration change of the 24h ointment on the cell is small. According to the corresponding IC50 value of 24h (185.91 u g/mL), 1/2IC50 (90 g/mL) and 1/4 I050 (45 u g/mL) are selected as the concentration value of the Qiong ointment during the subsequent grouping experiment, and.4. successfully establishes the senescence model of the hypothalamic neuron stem cells induced by D- galactose, and presents a typical example. Inflammation like changes: cell forming ability decreased, tumor necrosis factor - alpha (TNF- alpha) and interleukin 1 beta (IL-1 beta) mRNA and protein expression level increased significantly, nuclear transcription factor -kappaB (NF- kappa B) signal pathway was activated, cytoplasm P65 expression level decreased, nuclear P65 expression level increased.5.90 micronux ointment intervention group and half In the lactose senescence model group, the cell forming ability was significantly recovered (P0.05), tumor necrosis factor - alpha (TNF- alpha) and interleukin 1 beta (IL-1 beta) mRNA and protein expression level were significantly decreased (P0.05), the transcriptional activity of nuclear transcription factor -kappaB (NF- kappa B) was inhibited, cytoplasm P65 expression level increased, nuclear P65 expression level decreased. Conclusion: The expression level of mRNA and protein of RT1 gene was significantly different between old and young rats. It indicated that the expression level of GnRH1 and Sirt1 was negatively correlated with the.2.DNMT1 gene mRNA and protein expression level in old rats. There was no significant difference between the mRNA and protein expression level of the DNMT3a gene between the old and the young rats, indicating that DNMT. The expression level of 1 was negatively correlated with age increasing. The expression level of DNMT3a did not change with the age of aging, the.3.GnRH1 gene was partially methylated in the old rats, and the methylation was not appeared in the young rats. It indicated that the expression level of the GnRH1 gene was regulated by the methylation of the promoter region, and the natural senescence of the rat and the methylation of the GnRH1 gene promoter occurred in the rat. The inhibition of the expression level of GnRH1 on.4. Qiong ointment can prevent the inflammatory response of D- galactose induced neuronal stem cell aging model: restore the cell forming ability and inhibit the inflammatory index, and its action intensity is concentration dependent, with the increase of the density from 45 to 9 mu g/mL with the concentration of the ointment, and its anti-aging effect is also correspondingly enhanced by.5.. NF- kappa B has the function of negative regulation of GnRH1 gene mRNA expression, so the possible molecular mechanism of Joan ointment to delay senescence is to inhibit the expression of TNF- alpha, IL-1 beta P and the transcription activity of NF- kappa B in the hypothalamic neuron stem cells, and then reverse the methylation of GnRH1 gene and the mRNA expression level of GnRH1 gene in the process of natural aging. Drop.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5

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