DNA去甲基化對(duì)絨山羊脂肪間充質(zhì)干細(xì)胞特性和三胚層分化的影響
發(fā)布時(shí)間：2018-08-19 17:43
【摘要】：表觀遺傳標(biāo)記的變化是決定干細(xì)胞命運(yùn)和分化的重要調(diào)控因素。在過去的幾年里,表觀遺傳調(diào)控干細(xì)胞生物學(xué)的研究主要集中在胚胎干細(xì)胞(Embryonic stem cells,ESCs)。然而,對(duì)表觀遺傳調(diào)控成體干細(xì)胞生物學(xué)過程的理解卻很有限。間充質(zhì)干細(xì)胞(Mensenchymal stem cells,MSCs)是研究最多的成體干細(xì)胞群體,但是對(duì)調(diào)節(jié)間充質(zhì)干細(xì)胞的分化狀態(tài)和特性的分子機(jī)制的研究仍不成熟。由于脂肪間充質(zhì)干細(xì)胞(Adipose-derived stem cells,ADSCs)取材容易、來源廣泛,成為近些年理論研究和臨床應(yīng)用的首選材料。本研究以特色優(yōu)質(zhì)阿爾巴斯絨山羊的脂肪間充質(zhì)干細(xì)胞作為研究對(duì)象,使用表觀遺傳試劑氮胞苷(5-azacytidine,5-Aza)和地西他濱(5-aza-2'-deoxycytidine,5-Aza-dC)體外改變脂肪間充質(zhì)干細(xì)胞的表觀遺傳修飾,研究DNA去甲基化對(duì)脂肪間充質(zhì)干細(xì)胞的特性和功能的調(diào)控作用,以便豐富表觀遺傳調(diào)控干細(xì)胞分化的內(nèi)容,為理解干細(xì)胞特性和功能的分子機(jī)制提供實(shí)驗(yàn)依據(jù)。一、表觀遺傳試劑對(duì)阿爾巴斯絨山羊脂肪間充質(zhì)干細(xì)胞生長(zhǎng)的影響本研究通過細(xì)胞計(jì)數(shù)法、MTT法和流式細(xì)胞周期和凋亡檢測(cè)的結(jié)合發(fā)現(xiàn),以半抑制濃度培養(yǎng)細(xì)胞24h時(shí),雖然細(xì)胞周期阻斷在G0/G1期,但細(xì)胞凋亡數(shù)目較少,活性最強(qiáng)。二、表觀遺傳試劑對(duì)阿爾巴斯絨山羊脂肪間充質(zhì)干細(xì)胞去甲基化作用我們使用半抑制濃度的5-Aza和5-Aza-dC分別培養(yǎng)細(xì)胞24h,提取5-Aza和5-Aza-dC處理后的gADSCs基因組,檢測(cè)發(fā)現(xiàn)DNA甲基化水平降低,然而羥甲基化水平升高。5-Aza和5-Aza-dC對(duì)基因轉(zhuǎn)錄水平的影響是一致的,都引起DNMT1和DNMT3B基因轉(zhuǎn)錄抑制,DNMT3A、TET1、TET2和TET3基因轉(zhuǎn)錄升高。蛋白質(zhì)水平上,gADSCs在5-Aza處理下,DNMT1表達(dá)量下降,TET1和TET3表達(dá)升高;5-Aza-dC處理后,雖然沒有抑制DNMT1表達(dá),但是TET1、TET2和TET3蛋白表達(dá)升高。結(jié)果表明,雖然5-Aza和5-Aza-dC的作用機(jī)制略有不同,但是都引起了 TET家族促進(jìn)5-甲基胞嘧啶向5-羥甲基胞嘧啶發(fā)生轉(zhuǎn)換,基因組DNA發(fā)生很大程度的去甲基化。三、DNA去甲基化對(duì)細(xì)胞增殖、凋亡和多能性相關(guān)基因表達(dá)的影響細(xì)胞免疫熒光、實(shí)時(shí)定量PCR和蛋白質(zhì)免疫印跡檢測(cè)5-Aza和5-Aza-dC處理前后的gADSCs中,增殖相關(guān)基因TERT和PCNA,凋亡相關(guān)基因P53和BAX和多能性相關(guān)基因Nanog、Oct4和Sox2的轉(zhuǎn)錄和表達(dá)的情況。研究發(fā)現(xiàn),5-Aza和5-Aza-dC處理后的細(xì)胞中,Oct4、Sox2、TERT、PCNA和P53的轉(zhuǎn)錄水平均降低;在5-Aza處理后的細(xì)胞中,Nanog的轉(zhuǎn)錄水平升高,BAX的轉(zhuǎn)錄水平降低;在5-Aza-dC處理后的細(xì)胞中,Nanog的轉(zhuǎn)錄水平降低,BAX的轉(zhuǎn)錄水平升高。進(jìn)一步地,5-Aza和5-Aza-dC處理提高了 Sox2的表達(dá),降低了 PCNA的表達(dá),而BAX的表達(dá)升高。Nanog、Oct4、TERT和P53基因表達(dá)沒有變化。結(jié)果表明,基因組去甲基化改變了增殖、凋亡和多能性相關(guān)基因的轉(zhuǎn)錄水平。干細(xì)胞多能性的提高依賴于Sox2表達(dá)提高。四、DNA去甲基化對(duì)gADSCs向三胚層分化的影響通過檢測(cè)處理前后誘導(dǎo)形成的脂肪細(xì)胞分泌的脂滴含量和脂肪細(xì)胞特異性因子PPARG、Adipod、Fabp4和Leptin轉(zhuǎn)錄水平,發(fā)現(xiàn)處理后脂肪細(xì)胞的脂滴產(chǎn)量增加,PPARG轉(zhuǎn)錄水平降低,Adipod、Fabp4和Leptin轉(zhuǎn)錄水平升高。ELISA檢測(cè)5-Aza和5-Aza-dC處理前后gADSCs分化形成的神經(jīng)細(xì)胞中NGF含量,發(fā)現(xiàn)其含量增加。實(shí)時(shí)定量PCR結(jié)果顯示,處理后分化的神經(jīng)細(xì)胞中EN02和RBFOX3轉(zhuǎn)錄水平升高。通過檢測(cè)5-Aza和5-Aza-dC處理前后gADSCs分化形成的肝臟細(xì)胞中ALB和尿素含量、AFP轉(zhuǎn)錄水平,發(fā)現(xiàn)處理后ALB和尿素含量增加、AFP轉(zhuǎn)錄水平升高。這些結(jié)果表明,5-Aza和5-Aza-dC處理促進(jìn)了gADSCs向脂肪細(xì)胞、神經(jīng)細(xì)胞和肝臟細(xì)胞分化。五、DNA去甲基化對(duì)PPARG、RBFOX3和HNF4A啟動(dòng)子甲基化水平的影響實(shí)時(shí)定量PCR檢測(cè)發(fā)現(xiàn),分化前5-Aza和5-Aza-dC改變了脂肪細(xì)胞、神經(jīng)細(xì)胞、肝臟細(xì)胞分化相關(guān)的轉(zhuǎn)錄因子PPARG、RBOXF3和HNF4A的轉(zhuǎn)錄水平。亞硫酸氫鹽測(cè)序檢測(cè)PPARG、RBOXF3和HNF4A啟動(dòng)子區(qū)域的甲基化水平發(fā)生改變。PPARG啟動(dòng)子區(qū)域第81位CpG去甲基化位點(diǎn)、RBFOX3啟動(dòng)子區(qū)域第8、20、44、70、174和181位CpG甲基化位點(diǎn)和HNF4A啟動(dòng)子區(qū)域的4個(gè)CpG去甲基化位點(diǎn)對(duì)PPARG、RBFOX3和HNF4A的轉(zhuǎn)錄調(diào)控具有重要作用。綜上研究得出,5-Aza和5-Aza-dC引起TET家族高表達(dá)促進(jìn)5-甲基胞嘧啶向5-羥甲基胞嘧啶發(fā)生轉(zhuǎn)換,基因組DNA發(fā)生去甲基化;去甲基化使得不同基因的轉(zhuǎn)錄水平發(fā)生改變,但是卻依賴Sox2基因調(diào)控網(wǎng)絡(luò)促進(jìn)gADSCs干細(xì)胞特性;基因組去甲基化改變了脂肪細(xì)胞、神經(jīng)細(xì)胞、肝臟細(xì)胞分化相關(guān)的轉(zhuǎn)錄因子PPARG、RBOXF3和HNF4A的啟動(dòng)子區(qū)域部分CpG位點(diǎn)的甲基化狀態(tài);去甲基化促進(jìn)gADSCs向脂肪細(xì)胞、神經(jīng)細(xì)胞和肝臟細(xì)胞體外分化。
[Abstract]:In the past few years, studies on epigenetic regulation of stem cell biology have focused on embryonic stem cells (ESCs). However, the understanding of epigenetic regulation of adult stem cell biology is limited. Mensenchymal stem cells (MSCs) are the most widely studied adult stem cell populations, but the research on the molecular mechanism regulating the differentiation status and characteristics of mesenchymal stem cells is still immature. In this study, adipose-derived mesenchymal stem cells (ADMSCs) from high-quality Albanian cashmere goats were used as the research object. Apparent genetic reagents 5-azacytidine (5-Aza) and 5-aza-2'-deoxycytidine (5-Aza-dC) were used to alter the epigenetic modification of ADMSCs in vitro and to study the effect of DNA demethylation on lipids. In order to enrich the content of epigenetic regulation of stem cell differentiation and provide experimental basis for understanding the molecular mechanism of stem cell characteristics and functions, we studied the effects of epigenetic reagents on the growth of adipose-derived mesenchymal stem cells in Albas cashmere goats. Combining with flow cytometry and apoptosis detection, we found that although the cell cycle was blocked at G0/G1 phase at semiinhibitory concentration for 24 hours, the number of apoptotic cells was small and the activity was strongest. The genome of gADSCs treated with 5-Aza and 5-Aza-dC was extracted and cultured for 24 hours respectively. DNA methylation level was decreased, but the effect of 5-Aza and 5-Aza-dC on gene transcription level was consistent. Both of them resulted in the transcription inhibition of DNMT1 and DNMT3B genes, and the transcription of DNMT3A, TET1, TET2 and TET3 genes increased. Up to 5-Aza treatment, the expression of DNMT1 decreased and the expression of TET1 and TET3 increased, while the expression of TET1, TET2 and TET3 increased after 5-Aza-dC treatment. The results showed that although the mechanism of 5-Aza and 5-Aza-dC was slightly different, the TET family promoted 5-methyl cytosine to 5-hydroxymethyl cytosine. DNA demethylation affects cell proliferation, apoptosis and expression of genes related to pluripotency. Real-time quantitative PCR and Western blotting were used to detect the proliferation-related genes TERT and PCNA, apoptosis-related genes P53 and B in gADSCs before and after treatment with 5-Aza and 5-Aza-dC. Transcription and expression of AX and P53-related genes Nanog, Oct4 and Sox2 were observed. Transcription levels of Oct4, Sox2, TERT, PCNA and P53 were decreased in the cells treated with 5-Aza and 5-Aza-dC. Transcription levels of Nanog and BAX were increased and decreased in the cells treated with 5-Aza, respectively. Further, 5-Aza and 5-Aza-dC treatments increased the expression of Sox2 and decreased the expression of PCNA, while the expression of BAX increased. The expression of Nanog, Oct4, TERT and P53 genes remained unchanged. The results showed that genomic demethylation altered the transcriptional levels of genes related to proliferation, apoptosis and pluription. Fourthly, the effect of DNA demethylation on the differentiation of gADSCs into triploblasts was detected by detecting the lipid droplets secreted by adipocytes and the transcription levels of adipocyte-specific factors PPARG, Adipod, Fabp4 and Leptin before and after treatment. The transcriptional levels of Adipod, Fabp4 and Leptin were decreased, but the transcriptional levels of Adipod, Fabp4 and Leptin were increased. The NGF contents in the differentiated neurons of gADSCs were detected by ELISA before and after treatment with 5-Aza and 5-Aza-dC. The results of real-time quantitative PCR showed that the transcriptional levels of EN02 and RBFOX3 were increased in the differentiated neurons after treatment with 5-Aza and 5-Aza-dC. ALB and urea contents and AFP transcription levels in hepatocytes differentiated from SCs were found to increase after treatment. These results indicated that 5-Aza and 5-Aza-dC treatments promoted the differentiation of gADSCs into adipocytes, nerve cells and liver cells. Fifth, DNA demethylation methylated the promoters of PPARG, RBFOX3 and HNF4A. Levels of transcription factors PPARG, RBOXF3 and HNF4A were altered by 5-Aza and 5-Aza-dC before differentiation. Methylation levels of PPARG, RBOXF3 and HNF4A promoter regions were detected by bisulfite sequencing. CpG at position 81 of PPARG promoter region was altered by 5-Aza and 5-Aza-dC before differentiation. Demethylation sites, CpG methylation sites at sites 8, 20, 44, 70, 174 and 181 in the promoter region of RBFOX3, and four CpG demethylation sites in the promoter region of HNF4A play important roles in transcriptional regulation of PPARG, RBFOX3 and HNF4A. Overall studies have shown that 5-Aza and 5-Aza-dC induce TET family overexpression and promote 5-methyl cytosine to 5-hydroxymethyl cytosine. Genome DNA is demethylated; demethylation alters the transcriptional levels of different genes, but relies on the Sox2 gene regulatory network to promote the characteristics of gADSCs stem cells; genome demethylation alters the promoter regions of transcription factors PPARG, RBOXF3 and HNF4A associated with adipocyte, neural, and liver cell differentiation The methylation of some CpG sites in the domain promotes the differentiation of gADSCs into adipocytes, neurons and liver cells in vitro.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827
本文編號(hào)：2192363
[Abstract]:In the past few years, studies on epigenetic regulation of stem cell biology have focused on embryonic stem cells (ESCs). However, the understanding of epigenetic regulation of adult stem cell biology is limited. Mensenchymal stem cells (MSCs) are the most widely studied adult stem cell populations, but the research on the molecular mechanism regulating the differentiation status and characteristics of mesenchymal stem cells is still immature. In this study, adipose-derived mesenchymal stem cells (ADMSCs) from high-quality Albanian cashmere goats were used as the research object. Apparent genetic reagents 5-azacytidine (5-Aza) and 5-aza-2'-deoxycytidine (5-Aza-dC) were used to alter the epigenetic modification of ADMSCs in vitro and to study the effect of DNA demethylation on lipids. In order to enrich the content of epigenetic regulation of stem cell differentiation and provide experimental basis for understanding the molecular mechanism of stem cell characteristics and functions, we studied the effects of epigenetic reagents on the growth of adipose-derived mesenchymal stem cells in Albas cashmere goats. Combining with flow cytometry and apoptosis detection, we found that although the cell cycle was blocked at G0/G1 phase at semiinhibitory concentration for 24 hours, the number of apoptotic cells was small and the activity was strongest. The genome of gADSCs treated with 5-Aza and 5-Aza-dC was extracted and cultured for 24 hours respectively. DNA methylation level was decreased, but the effect of 5-Aza and 5-Aza-dC on gene transcription level was consistent. Both of them resulted in the transcription inhibition of DNMT1 and DNMT3B genes, and the transcription of DNMT3A, TET1, TET2 and TET3 genes increased. Up to 5-Aza treatment, the expression of DNMT1 decreased and the expression of TET1 and TET3 increased, while the expression of TET1, TET2 and TET3 increased after 5-Aza-dC treatment. The results showed that although the mechanism of 5-Aza and 5-Aza-dC was slightly different, the TET family promoted 5-methyl cytosine to 5-hydroxymethyl cytosine. DNA demethylation affects cell proliferation, apoptosis and expression of genes related to pluripotency. Real-time quantitative PCR and Western blotting were used to detect the proliferation-related genes TERT and PCNA, apoptosis-related genes P53 and B in gADSCs before and after treatment with 5-Aza and 5-Aza-dC. Transcription and expression of AX and P53-related genes Nanog, Oct4 and Sox2 were observed. Transcription levels of Oct4, Sox2, TERT, PCNA and P53 were decreased in the cells treated with 5-Aza and 5-Aza-dC. Transcription levels of Nanog and BAX were increased and decreased in the cells treated with 5-Aza, respectively. Further, 5-Aza and 5-Aza-dC treatments increased the expression of Sox2 and decreased the expression of PCNA, while the expression of BAX increased. The expression of Nanog, Oct4, TERT and P53 genes remained unchanged. The results showed that genomic demethylation altered the transcriptional levels of genes related to proliferation, apoptosis and pluription. Fourthly, the effect of DNA demethylation on the differentiation of gADSCs into triploblasts was detected by detecting the lipid droplets secreted by adipocytes and the transcription levels of adipocyte-specific factors PPARG, Adipod, Fabp4 and Leptin before and after treatment. The transcriptional levels of Adipod, Fabp4 and Leptin were decreased, but the transcriptional levels of Adipod, Fabp4 and Leptin were increased. The NGF contents in the differentiated neurons of gADSCs were detected by ELISA before and after treatment with 5-Aza and 5-Aza-dC. The results of real-time quantitative PCR showed that the transcriptional levels of EN02 and RBFOX3 were increased in the differentiated neurons after treatment with 5-Aza and 5-Aza-dC. ALB and urea contents and AFP transcription levels in hepatocytes differentiated from SCs were found to increase after treatment. These results indicated that 5-Aza and 5-Aza-dC treatments promoted the differentiation of gADSCs into adipocytes, nerve cells and liver cells. Fifth, DNA demethylation methylated the promoters of PPARG, RBFOX3 and HNF4A. Levels of transcription factors PPARG, RBOXF3 and HNF4A were altered by 5-Aza and 5-Aza-dC before differentiation. Methylation levels of PPARG, RBOXF3 and HNF4A promoter regions were detected by bisulfite sequencing. CpG at position 81 of PPARG promoter region was altered by 5-Aza and 5-Aza-dC before differentiation. Demethylation sites, CpG methylation sites at sites 8, 20, 44, 70, 174 and 181 in the promoter region of RBFOX3, and four CpG demethylation sites in the promoter region of HNF4A play important roles in transcriptional regulation of PPARG, RBFOX3 and HNF4A. Overall studies have shown that 5-Aza and 5-Aza-dC induce TET family overexpression and promote 5-methyl cytosine to 5-hydroxymethyl cytosine. Genome DNA is demethylated; demethylation alters the transcriptional levels of different genes, but relies on the Sox2 gene regulatory network to promote the characteristics of gADSCs stem cells; genome demethylation alters the promoter regions of transcription factors PPARG, RBOXF3 and HNF4A associated with adipocyte, neural, and liver cell differentiation The methylation of some CpG sites in the domain promotes the differentiation of gADSCs into adipocytes, neurons and liver cells in vitro.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827
【參考文獻(xiàn)】
相關(guān)期刊論文 前6條
1 Shuchen Zhang;Wei Cui;;Sox2, a key factor in the regulation of pluripotency and neural differentiation[J];World Journal of Stem Cells;2014年03期
2 Anfei Liu;Xiya Yu;Shanrong Liu;;Pluripotency transcription factors and cancer stem cells:small genes make a big difference[J];Chinese Journal of Cancer;2013年09期
3 周紫微;趙宇;;脂肪干細(xì)胞研究進(jìn)展與應(yīng)用前景[J];安徽醫(yī)科大學(xué)學(xué)報(bào);2013年07期
4 陶冶;王勇;;DNA甲基化及其新進(jìn)展[J];國際婦產(chǎn)科學(xué)雜志;2010年05期
5 黎洪棉;柳大烈;余元龍;吳濤;;自體PRP促進(jìn)人脂肪來源干細(xì)胞成骨分化的體外實(shí)驗(yàn)研究[J];中國修復(fù)重建外科雜志;2009年06期
6 楊立業(yè),劉相名,孫兵,惠國楨,費(fèi)儉,郭禮和;Adipose tissue-derived stromal cells express neuronal phenotypes[J];Chinese Medical Journal;2004年03期
，本文編號(hào)：2192363
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2192363.html
最近更新
教材專著
