發(fā)布時(shí)間：2018-06-20 07:18

  本文選題：瓊玉膏 + 延緩衰老　； 參考：《廣州中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：目的:通過(guò)對(duì)老年大鼠與青年大鼠下丘腦衰老相關(guān)基因GnRH1、Sirt1、DNMT1、DNMT3a mRNA和蛋白表達(dá)水平以及GnRH1基因甲基化情況進(jìn)行分析,從表觀遺傳水平探討大鼠自然衰老進(jìn)程中DNA甲基轉(zhuǎn)移酶活性及GnRH1基因甲基化的改變;通過(guò)分離新生大鼠下丘腦神經(jīng)元干細(xì)胞,建立D-半乳糖誘導(dǎo)的下丘腦神經(jīng)元干細(xì)胞衰老模型,比較正常神經(jīng)元干細(xì)胞組、D-半乳糖誘導(dǎo)衰老組與不同濃度瓊玉膏干預(yù)組之間細(xì)胞成球能力、炎性因子及NF-κB表達(dá)的差異,為揭示瓊玉膏延緩神經(jīng)元干細(xì)胞衰老的功效及其可能分子機(jī)制提供實(shí)驗(yàn)參考和數(shù)據(jù)支持。方法:觀察比較6只24月齡自然衰老SD大鼠和6只2月齡青年SD大鼠下丘腦衰老相關(guān)基因 mRNA 和蛋白表達(dá)水平:Real-Time PCR 檢測(cè) GnRH1、Sirt1、DNMT1、DNMT3a基因 mRNA 表達(dá)水平;Western Blot 檢測(cè) GnRH1、Sirt1、DNMT1、DNMT3a 蛋白表達(dá)水平;使用SPSS19.0統(tǒng)計(jì)軟件做統(tǒng)計(jì)學(xué)分析。MSP法檢測(cè)老年大鼠和青年大鼠GnRH1基因甲基化情況。分離培養(yǎng)8只新生SD大鼠(出生24h內(nèi))下丘腦神經(jīng)元干細(xì)胞,免疫熒光法檢測(cè)神經(jīng)元烯醇化酶(NSE),鑒定神經(jīng)元干細(xì)胞;CCK8法檢測(cè)瓊玉膏對(duì)神經(jīng)元干細(xì)胞增殖的影響(0D值),使用SPSS19.0統(tǒng)計(jì)軟件計(jì)算IC50,為后續(xù)分組實(shí)驗(yàn)提供依據(jù)。取神經(jīng)元干細(xì)胞分成A、B、C、D四組,采用NSCs完全培養(yǎng)基分別對(duì)四組進(jìn)行原代培養(yǎng)至第8天;從第9天起,A組繼續(xù)采用培養(yǎng)基培養(yǎng)24h;B組依據(jù)參考文獻(xiàn)加入10mg/mL D-半乳糖致衰24h;C組先加9μg/mL(1/2 IC50)瓊玉膏干預(yù)6h,再加10mg/mL D-半乳糖致衰24h;D組先加45μg/mL(1/4 IC50)瓊玉膏干預(yù)6h,再加10mg/mL D-半乳糖致衰24h。倒置熒光顯微鏡下觀察各組神經(jīng)元干細(xì)胞形態(tài)及成球能力(SFE);Real-Time PCR檢測(cè)各組腫瘤壞死因子-α(TNF-α)及白細(xì)胞介素1β(IL-1β)mRNA表達(dá)水平;Western Blot檢測(cè)核轉(zhuǎn)錄因子-kappaB(NF-κB)蛋白表達(dá)水平;Elisa檢測(cè)腫瘤壞死因子-α(TNF-α)及白細(xì)胞介素1β(IL-1β)蛋白表達(dá)水平;使用SPSS19.0統(tǒng)計(jì)軟件做統(tǒng)計(jì)學(xué)分析。結(jié)果:1.GnRH1、Sirt1、DNMT1基因mRNA及蛋白表達(dá)水平在老年鼠中明顯降低,與青年鼠比較有顯著性差異(p0.05);DNMT3a基因mRNA及蛋白表達(dá)水平在老年鼠和青年鼠之間無(wú)顯著性差異(p0.05);老年鼠GnRH1基因出現(xiàn)部分甲基化,青年鼠GnRHl基因未出現(xiàn)甲基化。2.從新生鼠下丘腦成功分離神經(jīng)元干細(xì)胞,倒置顯微鏡下觀察細(xì)胞成球狀生長(zhǎng);免疫熒光法檢測(cè)到細(xì)胞球體有NSE清晰的紅色熒光信號(hào),即NSE在神經(jīng)元干細(xì)胞中陽(yáng)性表達(dá)。3.CCK8法檢測(cè)到瓊玉膏對(duì)神經(jīng)元干細(xì)胞的增殖作用(0D值)與時(shí)間和劑量存在一定的依賴關(guān)系,24h內(nèi)瓊玉膏濃度變化對(duì)細(xì)胞的影響較小;根據(jù)24h對(duì)應(yīng)的IC50值(185.91μg/mL),選取1/2IC50(90μg/mL)和1/4 I050(45μg/mL)作為后續(xù)分組實(shí)驗(yàn)時(shí)瓊玉膏的濃度值。4.成功建立D-半乳糖誘導(dǎo)的下丘腦神經(jīng)元干細(xì)胞衰老模型,呈現(xiàn)典型的炎癥樣變化:細(xì)胞成球能力降低,腫瘤壞死因子-α(TNF-α)及白細(xì)胞介素1β(IL-1β)mRNA和蛋白表達(dá)水平顯著增加,核轉(zhuǎn)錄因子-kappaB(NF-κB)信號(hào)通路被激活,cytoplasm P65表達(dá)水平下降,nuclear P65表達(dá)水平上升。5.90μg/mL(1/2IC50)瓊玉膏干預(yù)組與D-半乳糖衰老模型組比較細(xì)胞成球能力顯著恢復(fù)(P0.05),腫瘤壞死因子-α(TNF-α)及白細(xì)胞介素1β(IL-1β)mRNA和蛋白表達(dá)水平顯著降低(p0.05),核轉(zhuǎn)錄因子-kappaB(NF-κB)轉(zhuǎn)錄活性被抑制,cytoplasm P65表達(dá)水平上升,nuclear P65表達(dá)水平下降。結(jié)論:1.GnRH1、Sirt1基因mRNA及蛋白表達(dá)水平在老年鼠和青年鼠之間有顯著性差異,說(shuō)明GnRH1、Sirt1的表達(dá)水平與增齡呈負(fù)相關(guān)。2.DNMT1基因mRNA及蛋白表達(dá)水平在老年鼠中顯著降低,DNMT3a基因mRNA及蛋白表達(dá)水平在老年鼠和青年鼠之間無(wú)顯著性差異,說(shuō)明DNMT1的表達(dá)水平與增齡呈負(fù)相關(guān),DNMT3a的表達(dá)水平不隨增齡而變化。3.GnRH1基因在老年鼠中出現(xiàn)部分甲基化,在青年鼠中未出現(xiàn)甲基化,說(shuō)明GnRH1基因的表達(dá)水平受到其啟動(dòng)子區(qū)甲基化的調(diào)控,大鼠機(jī)體發(fā)生自然衰老與GnRH1基因啟動(dòng)子甲基化從而抑制GnRH1的表達(dá)水平有關(guān)。4.瓊玉膏能預(yù)防D-半乳糖誘導(dǎo)的神經(jīng)元干細(xì)胞衰老模型的炎癥反應(yīng):恢復(fù)細(xì)胞成球能力、抑制炎性指標(biāo),且其作用強(qiáng)度呈濃度依賴性,隨著瓊玉膏濃度從45μg/mL增加到9μg/mL,其延緩衰老作用也相應(yīng)增強(qiáng)。5.由于NF-κ B具有負(fù)調(diào)控GnRH1基因mRNA表達(dá)的功能,因此瓊玉膏延緩衰老作用的可能分子機(jī)制在于抑制下丘腦神經(jīng)元干細(xì)胞炎性細(xì)胞因子TNF-α、IL-1βP的表達(dá)以及NF-κ B的轉(zhuǎn)錄活性,進(jìn)而逆轉(zhuǎn)自然衰老進(jìn)程中GnRH1基因甲基化及其mRNA表達(dá)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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.5

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