DNA去甲基化对绒山羊脂肪间充质干细胞特性和三胚层分化的影响

发布时间：2018-08-19 17:43

【摘要】：表观遗传标记的变化是决定干细胞命运和分化的重要调控因素。在过去的几年里,表观遗传调控干细胞生物学的研究主要集中在胚胎干细胞(Embryonic stem cells,ESCs)。然而,对表观遗传调控成体干细胞生物学过程的理解却很有限。间充质干细胞(Mensenchymal stem cells,MSCs)是研究最多的成体干细胞群体,但是对调节间充质干细胞的分化状态和特性的分子机制的研究仍不成熟。由于脂肪间充质干细胞(Adipose-derived stem cells,ADSCs)取材容易、来源广泛,成为近些年理论研究和临床应用的首选材料。本研究以特色优质阿尔巴斯绒山羊的脂肪间充质干细胞作为研究对象,使用表观遗传试剂氮胞苷(5-azacytidine,5-Aza)和地西他滨(5-aza-2'-deoxycytidine,5-Aza-dC)体外改变脂肪间充质干细胞的表观遗传修饰,研究DNA去甲基化对脂肪间充质干细胞的特性和功能的调控作用,以便丰富表观遗传调控干细胞分化的内容,为理解干细胞特性和功能的分子机制提供实验依据。一、表观遗传试剂对阿尔巴斯绒山羊脂肪间充质干细胞生长的影响本研究通过细胞计数法、MTT法和流式细胞周期和凋亡检测的结合发现,以半抑制浓度培养细胞24h时,虽然细胞周期阻断在G0/G1期,但细胞凋亡数目较少,活性最强。二、表观遗传试剂对阿尔巴斯绒山羊脂肪间充质干细胞去甲基化作用我们使用半抑制浓度的5-Aza和5-Aza-dC分别培养细胞24h,提取5-Aza和5-Aza-dC处理后的gADSCs基因组,检测发现DNA甲基化水平降低,然而羟甲基化水平升高。5-Aza和5-Aza-dC对基因转录水平的影响是一致的,都引起DNMT1和DNMT3B基因转录抑制,DNMT3A、TET1、TET2和TET3基因转录升高。蛋白质水平上,gADSCs在5-Aza处理下,DNMT1表达量下降,TET1和TET3表达升高;5-Aza-dC处理后,虽然没有抑制DNMT1表达,但是TET1、TET2和TET3蛋白表达升高。结果表明,虽然5-Aza和5-Aza-dC的作用机制略有不同,但是都引起了 TET家族促进5-甲基胞嘧啶向5-羟甲基胞嘧啶发生转换,基因组DNA发生很大程度的去甲基化。三、DNA去甲基化对细胞增殖、凋亡和多能性相关基因表达的影响细胞免疫荧光、实时定量PCR和蛋白质免疫印迹检测5-Aza和5-Aza-dC处理前后的gADSCs中,增殖相关基因TERT和PCNA,凋亡相关基因P53和BAX和多能性相关基因Nanog、Oct4和Sox2的转录和表达的情况。研究发现,5-Aza和5-Aza-dC处理后的细胞中,Oct4、Sox2、TERT、PCNA和P53的转录水平均降低;在5-Aza处理后的细胞中,Nanog的转录水平升高,BAX的转录水平降低;在5-Aza-dC处理后的细胞中,Nanog的转录水平降低,BAX的转录水平升高。进一步地,5-Aza和5-Aza-dC处理提高了 Sox2的表达,降低了 PCNA的表达,而BAX的表达升高。Nanog、Oct4、TERT和P53基因表达没有变化。结果表明,基因组去甲基化改变了增殖、凋亡和多能性相关基因的转录水平。干细胞多能性的提高依赖于Sox2表达提高。四、DNA去甲基化对gADSCs向三胚层分化的影响通过检测处理前后诱导形成的脂肪细胞分泌的脂滴含量和脂肪细胞特异性因子PPARG、Adipod、Fabp4和Leptin转录水平,发现处理后脂肪细胞的脂滴产量增加,PPARG转录水平降低,Adipod、Fabp4和Leptin转录水平升高。ELISA检测5-Aza和5-Aza-dC处理前后gADSCs分化形成的神经细胞中NGF含量,发现其含量增加。实时定量PCR结果显示,处理后分化的神经细胞中EN02和RBFOX3转录水平升高。通过检测5-Aza和5-Aza-dC处理前后gADSCs分化形成的肝脏细胞中ALB和尿素含量、AFP转录水平,发现处理后ALB和尿素含量增加、AFP转录水平升高。这些结果表明,5-Aza和5-Aza-dC处理促进了gADSCs向脂肪细胞、神经细胞和肝脏细胞分化。五、DNA去甲基化对PPARG、RBFOX3和HNF4A启动子甲基化水平的影响实时定量PCR检测发现,分化前5-Aza和5-Aza-dC改变了脂肪细胞、神经细胞、肝脏细胞分化相关的转录因子PPARG、RBOXF3和HNF4A的转录水平。亚硫酸氢盐测序检测PPARG、RBOXF3和HNF4A启动子区域的甲基化水平发生改变。PPARG启动子区域第81位CpG去甲基化位点、RBFOX3启动子区域第8、20、44、70、174和181位CpG甲基化位点和HNF4A启动子区域的4个CpG去甲基化位点对PPARG、RBFOX3和HNF4A的转录调控具有重要作用。综上研究得出,5-Aza和5-Aza-dC引起TET家族高表达促进5-甲基胞嘧啶向5-羟甲基胞嘧啶发生转换,基因组DNA发生去甲基化;去甲基化使得不同基因的转录水平发生改变,但是却依赖Sox2基因调控网络促进gADSCs干细胞特性;基因组去甲基化改变了脂肪细胞、神经细胞、肝脏细胞分化相关的转录因子PPARG、RBOXF3和HNF4A的启动子区域部分CpG位点的甲基化状态;去甲基化促进gADSCs向脂肪细胞、神经细胞和肝脏细胞体外分化。
[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.
【学位授予单位】：内蒙古大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S827

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