hESCs和hiPSCs向红细胞分化的DNA甲基化谱分析及OCT4诱导hHFMSCs向红细胞分化

发布时间：2018-05-21 01:01

本文选题：红细胞生成 + 胚胎干细胞　；参考：《吉林大学》2015年博士论文

【摘要】：红细胞生成过程受表观遗传学和遗传学双重调控，其中DNA甲基化作为表观遗传学的重要机制，通过调控基因组表达动力学及红细胞生成相关转录因子诱导干细胞向红细胞分化。本论文利用DNA甲基化芯片和全基因组表达谱芯片技术，分析hESCs和iPSCs向红细胞分化过程中DNA甲基化谱和基因表达谱的改变，并通过负相关基因的分析探讨多潜能干细胞向红细胞分化中DNA甲基化的作用机制；同时研究OCT4诱导hHFMSCs直接向红细胞分化的作用。一、 hESCs向红细胞分化中DNA甲基化谱与基因表达谱的负相关分析利用DNA甲基化芯片和基因表达谱芯片技术，对hESC（人胚胎干细胞）、hES-EB（由hESC获得的中胚层祖细胞群）以及hES-BL（由hES-EB分化的红系细胞）三种细胞样本进行分析，筛选差异甲基化CpG（differentially methylatedCpG，DMC）位点及差异表达基因，联合分析DNA甲基化与基因表达呈负相关的基因，利用GO分析富集基因功能，用KEGG分析富集信号通路，用GeneSpring软件构建调控网络，最后用real-time MSP和RT-qPCR验证芯片结果。实验结果如下： 1. hESC向红细胞分化中DNA甲基化谱的分析（1）hES-EB与hESC相比共有639个DMC位点；hES-BL与hESC和hES-EB相比，DMC位点分别为100991个和134736个。高甲基化CpG位点主要富集于CpG岛岸和CpG岛及启动子区，低甲基化CpG位点主要富集于CpG岛shelf和open sea及转录区，二者在编码RNA序列的分布比例没有明显差别。（2）GO分析显示，hESC向hES-EB分化中，DMC位点所在基因主要参与GTP酶介导的信号转导、细胞形态的调节及特异性基因转录；hES-EB向hES-BL分化中，DMC位点所在基因主要参与心血管发育、造血和淋巴器官发育等。（3）KEGG分析显示，hESC向hES-EB分化中，DMC位点所在基因参与黏着斑、II型糖尿病和肌动蛋白细胞骨架的调节等通路；hES-EB向hES-BL分化中，，DMC位点所在基因主要参与黏着斑、细胞粘附分子和钙离子信号通路等。 2. hESC向红细胞分化中基因表达谱的分析（1）hES-EB和hESC比较，共有810个差异表达基因，hES-BL与hESC和hES-EB相比，差异表达基因分别为5264个和5843个。（2）GO分析显示，hESC向hES-EB分化中上调基因与循环系统发育相关；下调基因参与细胞粘附和神经发育。hES-EB向hES-BL分化中上调基因参与造血、红细胞发育与成熟过程；下调基因参与神经系统和胚胎发育。（3）KEGG分析显示，hESC向hES-EB分化中上调基因参与TGF-β、癌症和凝血等相关信号通路；下调基因未富集到相关信号通路。hES-EB向hES-BL分化中上调基因参与造血细胞谱系、趋化因子和JAK-STAT信号通路等；下调基因参与紧密连接和细胞周期等通路。 3. DNA甲基化与mRNA表达呈负相关基因的分析（1）负相关基因的筛选及其DMC位点的分布：hESC向hES-EB分化中负相关基因有15个，hES-EB向hES-BL分化中为2624个，负相关基因的DMC位点主要位于启动子区和转录区。（2）GO分析与功能特异性基因的DMC位点分布：上调的负相关基因涉及红细胞特异的细胞组分、分子功能及生物过程；下调的负相关基因参与细胞连接、胚胎和神经发育过程；造血相关基因DNA去甲基化并表达上调，其DMC位点富集于CpG岛shelf和open sea，其转录区去甲基化与增强子和DNase1高敏感位点（DHS）有关。（3）KEGG分析与调控网络：共富集53条信号通路，趋化因子通路和造血细胞谱系通路富集度最高，其中上调基因参与38条通路，下调基因参与15条通路；hESC向红细胞分化中OCT4发生DNA甲基化并表达下调，101个OCT4靶基因表达改变，其中与红细胞生成相关基因表达上调，胚胎和神经系统发育的靶基因表达下调。 4．芯片结果的验证（1）real-time MSP结果显示，hESC向红细胞分化中GATA2和GYPB的启动子区DNA发生部分去甲基化，SOX2的启动子区DNA发生部分甲基化，与DNA甲基化芯片结果相符。（2）RT-qPCR结果显示，hESC向红细胞分化中造血相关基因GATA2、TAL1、LMO2、CD34和GYPB表达上调，多潜能相关基因OCT4和SOX2表达下调，与表达谱芯片结果一致。二、hiPSC向红细胞分化中DNA甲基化谱与基因表达谱的负相关分析用与hESC相同的方法检测并分析hiPSC向红系细胞分化中iPSC、中胚层祖细胞iPS-EB和红系细胞iPS-BL的DNA甲基化谱和基因表达谱，并联合分析DNA甲基化与基因表达呈负相关的基因。实验结果如下： 1. hiPSC向红细胞分化中DNA甲基化谱的分析 iPS-EB与iPSC相比，共有759个DMC位点，高甲基化位点多于低甲基化位点；iPS-BL分别与iPS-EB和iPSC相比，DMC位点分别为66922个和59003个，且低甲基化位点多于高甲基化位点；DMC位点所在基因主要参与代谢、生物调节及发育过程。 2. hiPSC向红细胞分化中基因表达谱的分析（1）iPS-EB与iPSC相比，共有1473个差异表达基因，iPS-BL与iPS-EB和iPSC相比，差异表达基因分别为2776个和5137个。（2）iPSC向iPS-EB分化中，上调基因参与脉管系统和心脏发育过程，下调基因参与应答反应及循环系统发育；iPS-EB向iPS-BL分化中，上调基因参与造血和红细胞生成，下调基因参与神经发育等；iPS-BL与iPSC相比，上调基因涉及血红素代谢和细胞凋亡的调节，而下调基因参与细胞周期及细胞间连接。 3. DNA甲基化与mRNA表达水平呈负相关基因的分析（1）负相关基因的筛选及其DMC位点的分布：iPSC向iPS-EB分化中负相关基因为15个，iPS-EB向iPS-BL分化中为2185个；负相关基因的DMC位点主要位于启动子区和转录区。（2）负相关基因的GO分析显示，iPS-EB向iPS-BL分化中，负相关基因参与血管、血液循环和细胞粘附等生物过程，但未参与红系细胞相关的生物过程。 4．hiPSC向红细胞分化中OCT4及其靶基因的表达（1）iPSC向红细胞分化中，OCT4表达水平在iPSC向iPS-EB分化中上调，差异表达的OCT4靶基因为36个；OCT4表达水平在iPS-EB向iPS-BL分化中未改变，差异表达的OCT4靶基因为52个。（2）上调和下调靶基因均参与胚胎、神经和中胚层发育，少数造血相关靶基因表达上调。三、OCT4诱导hHFMSCs向红细胞分化利用携带OCT4的慢病毒载体转导hHFMSCs，选取悬浮的hHFMSCOCT4，经造血因子诱导、红系分化及脱核培养。在分化过程中，利用Wright-Giemsa染色观察细胞形态改变，用细胞免疫荧光和流式细胞术检测红细胞特异性标志物，最后利用RT-qPCR检测OCT4靶基因。实验结果如下： 1．OCT4转导后hHFMSCs的细胞形态和造血标志物表达（1）转导后的hHFMSCs中EGFP阳性细胞占88.92%，OCT4表达水平明显上调。（2）转导后细胞由大变小，由长梭形变成类圆形，14d后出现易悬浮的细胞亚群，呈克隆样生长。（3）OCT4转导后hHFMSCs中CD45阳性细胞占1.5%，悬浮的hHFMSCs中CD34阳性细胞占2.19%。 3.转导OCT4的hHFMSCs向红细胞分化（1）光镜下观察，造血诱导第7d开始出现造血集落，10-20d先后出现红系爆炸式集落及红系集落形成单位；Wright-Giemsa染色显示，第7d开始先后出现早幼红、中幼红和晚幼红细胞，第20d晚幼红细胞数量不再增加。（2）造血诱导第7-20d的细胞表达红系祖细胞标志CD71和CD235a，同时表达血型蛋白A和B；诱导第23d红细胞完成脱核，80%以上红细胞表达成人型β血红蛋白；hHFMSC向红细胞分化中细胞直径及核质比逐渐减小，血红蛋白化逐渐增加。 4．hHFMSCs向红细胞分化中OCT4靶基因的表达 OCT4转导后，多潜能相关的靶基因OCT4、NANOG、SEMA3A和LEFTY2和造血相关的靶基因FLI1、TAL1、HBG1和CA2表达均上调。造血诱导21d后，多潜能相关的靶基因LEFTY2及OCT4显著下调，而造血相关的靶基因TAL1、HBG1和RHD表达显著上调。综上所述，本研究发现DNA甲基化谱改变可调控红细胞生成过程中发育阶段特异性的基因表达谱，从而为研究红细胞生成的分子机制提供一个表观遗传学机制，而利用OCT4诱导hHFMSCs向红细胞分化的方法，可为患者个体化细胞治疗提供新途径。
[Abstract]:DNA methylation was used as an important mechanism for epigenetics . The DNA methylation patterns and gene expression profiles were analyzed by regulating genomic expression kinetics and erythroid production related transcription factors . The mechanism of DNA methylation in the differentiation of human erythrocytes into red blood cells was investigated by means of the analysis of negative correlation gene .
At the same time , the effect of OCT4 on the differentiation of hHFMSCs into red blood cells was investigated .

In the differentiation of hES - BL into hES - BL , the gene of DMC locus is mainly involved in cardiovascular development , hematopoietic and lymphoid organ development .

Three cell samples of hESC ( human embryonic stem cells ) , hES - EB ( mesoderm progenitor cells obtained from hESC ) and hES - BL ( erythroid cells differentiated by hESC ) were analyzed by using DNA methylation chip and gene expression profiling chip technology .

1 . Analysis of DNA methylation patterns in erythrocyte differentiation by hESC

( 1 ) There were 639 DMC sites in hES - EB compared with hESC ;
Compared with hESC and hES - EB , hES - BL is 100991 and 134736 , respectively . Hypermethylated CpG sites are mainly enriched in CpG island and CpG island and promoter region . Low methylation CpG sites are mainly enriched in CpG island shelf and open sea and transcription region .

( 2 ) GO analysis showed that the genes involved in the differentiation of hESC into hES - EB were mainly involved in the signal transduction mediated by GTP enzyme , the regulation of cellular morphology and the transcription of specific genes .
negative correlation between DNA methylation profile and gene expression profile in erythrocyte differentiation

( 3 ) In the differentiation of hESC to hES - EB , the genes involved in the differentiation of hESC into hES - EB were involved in the regulation of adhesion spot , type II diabetes mellitus and actin cytoskeletal framework .
In the differentiation of hES - BL into hES - BL , the gene of DMC locus is mainly involved in adhesion spot , cell adhesion molecule and calcium ion signal pathway .

2 . Analysis of Gene Expression Profiles in Human Erythrocytes Differentiation by hESC

( 1 ) There were 810 differentially expressed genes compared with hES - EB and hESC , and the expression of hES - BL was 5264 and 5843 , respectively , compared with hESC and hES - EB .

( 2 ) GO analysis showed that the up - regulated genes of hESC to hES - EB were correlated with the development of circulatory system ;
The downregulated genes involved in cell adhesion and neurodevelopment . hES - EB up - regulated genes involved in the differentiation of hES - BL in hematopoietic , erythrocyte development and maturation .
Down - regulate the gene ' s participation in the nervous system and embryonic development .

( 3 ) The analysis showed that hESC up - regulated genes involved in the differentiation of hES - EB into relevant signal pathways such as TGF - 尾 , cancer and coagulation ;
The down - regulated genes were not enriched in related signal pathways . hES - EB up - regulated genes involved in hematopoietic cell lineage , chemokine and JAK - STAT signaling pathway in differentiation of hES - BL .
Down - regulation gene is involved in tight connection and cell cycle .

3 . Analysis of negative correlation between DNA methylation and mRNA expression

( 1 ) The screening of negatively correlated genes and the distribution of DMC loci : There were 15 negative correlation genes in hESC to hES - EB , and 2624 in hES - EB to hES - BL , and the DMC sites of negatively correlated genes were mainly located in the promoter region and the transcription region .

( 2 ) GO analysis and the distribution of DMC sites of functional specific genes : up - regulated negatively correlated genes involved red cell specific cell components , molecular functions and biological processes ;
Down - regulated negatively correlated genes were involved in cell ligation , embryo and neurodevelopmental processes ;
The DNA demethylation of hematopoietic related gene is up - regulated , its DMC site is enriched in CpG island shelf and open sea , its transcription region demethylation is related to enhancer and DNase1 high sensitive site ( DHS ) .

( 3 ) KEVs analysis and control network : 53 signal pathways , chemokine pathway and hematopoietic cell lineage pathway enrichment were the highest , in which the up - regulated gene was involved in 38 pathways and downregulated genes involved in 15 pathways ;
DNA methylation and down - regulation of OCT4 in the differentiation of hESC into red blood cells resulted in a change in the expression of 101 OCT4 target genes , in which the expression of the genes associated with the formation of the red cells was up - regulated , and the expression of the target genes in the embryonic and nervous system development was down regulated .

4 . Verification of chip results

( 1 ) The results of real - time MSP showed that hESC was partially demethylated in the promoter region of GATA2 and GYPB in erythrocyte differentiation , and partial methylation of DNA in promoter region of SOX2 was partially methylated , which was consistent with the results of DNA methylation chip .

( 2 ) The expression of GATA2 , TAL1 , LMO2 , CD34 and GYPB were up - regulated by RT - qPCR , and the expression of OCT4 and SOX2 was downregulated in hESC .

Correlation analysis of DNA methylation profile and gene expression profile in erythrocyte differentiation by hiPSC

Using the same method as hESC , the DNA methylation profiles and gene expression profiles of the iPSC , mesoderm progenitor cells , IPS - EB and erythroid cells were analyzed and analyzed in the same way as hESC . The results were as follows :

1 . Analysis of DNA methylation profile in erythrocyte differentiation by hiPSC

Compared with the iPSC , there were 759 DMC sites , higher methylation sites and lower methylation sites .
Compared with the IPS - EB and iPSC , the DMC sites were 6,922 and 59003 , respectively , and the low methylation sites were more than the hypermethylation sites .
The gene of DMC locus is mainly involved in metabolism , bioregulation and development .

2 . Analysis of gene expression profile of hiPSC to red blood cell differentiation

( 1 ) Compared with the iPSC , there were 1473 differentially expressed genes , and the difference expression genes were 2776 and 5137 , respectively .

( 2 ) In the differentiation of the iPSC , the up - regulated genes involved in the vascular system and the heart development process , downregulated the genes involved in the response reaction and the development of the circulatory system ;
In the differentiation of IPS - EB , the up - regulated genes involved in hematopoietic and erythrocyte formation , down - regulation of genes involved in neurodevelopment , etc .
Compared with the iPSC , the up - regulation gene involved the regulation of heme metabolism and apoptosis , while downregulating the genes involved in cell cycle and intercellular communication .

3 . Analysis of negative correlation between DNA methylation and mRNA expression level

( 1 ) The screening of negative correlation gene and the distribution of DMC loci : the negative correlation gene was 15 in the differentiation of the iPSC to the IPS - EB , and 2185 in the differentiation of IPS - EB to IPS - BL ;
The DMC site of the negative correlation gene is mainly located in the promoter region and the transcription region .

( 2 ) GO analysis of negative correlation gene showed that negative correlation gene was involved in the biological processes of blood vessel , blood circulation and cell adhesion , but did not participate in the biological process related to erythroid cell .

4 . Expression of OCT4 and its Target Gene in Erythrocytes Differentiation by hiPSC

( 1 ) The OCT4 expression level was up - regulated in the differentiation of the iPSC from the iPSC and the OCT4 target gene was 36 .
The OCT4 expression level did not change in the differentiation of IPS - EB to the IPS - BL , and the OCT4 target gene expressed differently was 52 .

( 2 ) Up - regulation and down - regulation of target genes are involved in the development of embryo , nerve and mesoderm , and the expression of a few hematopoietic - related target genes is up - regulated .

3 . OCT4 - induced differentiation of hHFMSCs into erythrocytes

The hHFMSCs were transduced with the slow virus vector carrying OCT4 , and the suspension hHFMSCOCT4 was selected . After differentiation , the morphological changes of the cells were observed by means of Wright - GigirI staining . The specific markers of red blood cells were detected by immunofluorescence and flow cytometry . Finally , the OCT4 target gene was detected by RT - qPCR . The experimental results were as follows :

1 . Cell morphology and hematopoietic marker expression of hHFMSCs transfected with OCT4

( 1 ) The expression of EGFP - positive cells in hHFMSCs was 88.92 % , and OCT4 expression was up - regulated .

( 2 ) After the transduced cells , the cells were changed from large to round , after 14 days , there appeared a subpopulation of cells which were easily suspended , which showed clonal growth .

( 3 ) In hHFMSCs transfected with OCT4 , the percentage of CD34 - positive cells in hHFMSCs was 1 . 5 % , and CD34 - positive cells in suspension hHFMSCs were 2 . 19 % .

3 . Differentiation of hHFMSCs transduced OCT4 into erythrocytes

( 1 ) Under light microscope , hematopoietic colonies began to appear on the 7th day of hematopoietic induction , and erythroid explosive colonies and erythroid colony forming units appeared in 10 - 20d .
At the 7th day , the red , medium and late red blood cells appeared in the 7th day , and the number of immature red cells in the 20th day was no longer increased .

( 2 ) expression of erythroid progenitor cell markers CD71 and CD235a in cells of day 7 - 20d after hematopoietic induction , and simultaneously expression of blood type proteins A and B ;
inducing the red blood cells to complete the decore , and the red blood cells of more than 80 percent express human beta hemoglobin ;
The diameter and cytoplasm ratio of hHFMSC to red blood cells were gradually decreased , and hemoglobin increased gradually .

4 . Expression of OCT4 Target Gene in Human Erythrocytes Differentiation by hHFMSCs

In OCT4 , the expression of OCT4 , NANOG , SEMA3A and LEFTY2 and hematopoietic related target genes FLI1 , TAL1 , HBG1 and CA2 were up - regulated after OCT4 transduction . The target genes LEFTY2 and OCT4 related to multipotential were down - regulated after 21 days of hematopoietic induction .

In conclusion , it is found that the DNA methylation pattern changes the gene expression profile specific to the developmental stage during the formation of red blood cells , thus providing an epigenetics mechanism for studying the molecular mechanism of red blood cell formation , and the method of inducing the differentiation of hHFMSCs into red blood cells by OCT4 can provide a new way for the treatment of individual cells in the patient .
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R329.2

【共引文献】