發(fā)布時(shí)間：2018-05-21 01:01

  本文選題：紅細(xì)胞生成 + 胚胎干細(xì)胞��； 參考：《吉林大學(xué)》2015年博士論文

【摘要】：紅細(xì)胞生成過(guò)程受表觀遺傳學(xué)和遺傳學(xué)雙重調(diào)控，其中DNA甲基化作為表觀遺傳學(xué)的重要機(jī)制，通過(guò)調(diào)控基因組表達(dá)動(dòng)力學(xué)及紅細(xì)胞生成相關(guān)轉(zhuǎn)錄因子誘導(dǎo)干細(xì)胞向紅細(xì)胞分化。本論文利用DNA甲基化芯片和全基因組表達(dá)譜芯片技術(shù)，分析hESCs和iPSCs向紅細(xì)胞分化過(guò)程中DNA甲基化譜和基因表達(dá)譜的改變，并通過(guò)負(fù)相關(guān)基因的分析探討多潛能干細(xì)胞向紅細(xì)胞分化中DNA甲基化的作用機(jī)制；同時(shí)研究OCT4誘導(dǎo)hHFMSCs直接向紅細(xì)胞分化的作用。 一、 hESCs向紅細(xì)胞分化中DNA甲基化譜與基因表達(dá)譜的負(fù)相關(guān)分析 利用DNA甲基化芯片和基因表達(dá)譜芯片技術(shù)，對(duì)hESC（人胚胎干細(xì)胞）、hES-EB（由hESC獲得的中胚層祖細(xì)胞群）以及hES-BL（由hES-EB分化的紅系細(xì)胞）三種細(xì)胞樣本進(jìn)行分析，篩選差異甲基化CpG（differentially methylatedCpG，DMC）位點(diǎn)及差異表達(dá)基因，聯(lián)合分析DNA甲基化與基因表達(dá)呈負(fù)相關(guān)的基因，利用GO分析富集基因功能，用KEGG分析富集信號(hào)通路，用GeneSpring軟件構(gòu)建調(diào)控網(wǎng)絡(luò)，最后用real-time MSP和RT-qPCR驗(yàn)證芯片結(jié)果。實(shí)驗(yàn)結(jié)果如下： 1. hESC向紅細(xì)胞分化中DNA甲基化譜的分析 （1）hES-EB與hESC相比共有639個(gè)DMC位點(diǎn)；hES-BL與hESC和hES-EB相比，DMC位點(diǎn)分別為100991個(gè)和134736個(gè)。高甲基化CpG位點(diǎn)主要富集于CpG島岸和CpG島及啟動(dòng)子區(qū)，低甲基化CpG位點(diǎn)主要富集于CpG島shelf和open sea及轉(zhuǎn)錄區(qū)，二者在編碼RNA序列的分布比例沒(méi)有明顯差別。 （2）GO分析顯示，hESC向hES-EB分化中，DMC位點(diǎn)所在基因主要參與GTP酶介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞形態(tài)的調(diào)節(jié)及特異性基因轉(zhuǎn)錄；hES-EB向hES-BL分化中，DMC位點(diǎn)所在基因主要參與心血管發(fā)育、造血和淋巴器官發(fā)育等。 （3）KEGG分析顯示，hESC向hES-EB分化中，DMC位點(diǎn)所在基因參與黏著斑、II型糖尿病和肌動(dòng)蛋白細(xì)胞骨架的調(diào)節(jié)等通路；hES-EB向hES-BL分化中，，DMC位點(diǎn)所在基因主要參與黏著斑、細(xì)胞粘附分子和鈣離子信號(hào)通路等。 2. hESC向紅細(xì)胞分化中基因表達(dá)譜的分析 （1）hES-EB和hESC比較，共有810個(gè)差異表達(dá)基因，hES-BL與hESC和hES-EB相比，差異表達(dá)基因分別為5264個(gè)和5843個(gè)。 （2）GO分析顯示，hESC向hES-EB分化中上調(diào)基因與循環(huán)系統(tǒng)發(fā)育相關(guān)；下調(diào)基因參與細(xì)胞粘附和神經(jīng)發(fā)育。hES-EB向hES-BL分化中上調(diào)基因參與造血、紅細(xì)胞發(fā)育與成熟過(guò)程；下調(diào)基因參與神經(jīng)系統(tǒng)和胚胎發(fā)育。 （3）KEGG分析顯示，hESC向hES-EB分化中上調(diào)基因參與TGF-β、癌癥和凝血等相關(guān)信號(hào)通路；下調(diào)基因未富集到相關(guān)信號(hào)通路。hES-EB向hES-BL分化中上調(diào)基因參與造血細(xì)胞譜系、趨化因子和JAK-STAT信號(hào)通路等；下調(diào)基因參與緊密連接和細(xì)胞周期等通路。 3. DNA甲基化與mRNA表達(dá)呈負(fù)相關(guān)基因的分析 （1）負(fù)相關(guān)基因的篩選及其DMC位點(diǎn)的分布：hESC向hES-EB分化中負(fù)相關(guān)基因有15個(gè)，hES-EB向hES-BL分化中為2624個(gè)，負(fù)相關(guān)基因的DMC位點(diǎn)主要位于啟動(dòng)子區(qū)和轉(zhuǎn)錄區(qū)。 （2）GO分析與功能特異性基因的DMC位點(diǎn)分布：上調(diào)的負(fù)相關(guān)基因涉及紅細(xì)胞特異的細(xì)胞組分、分子功能及生物過(guò)程；下調(diào)的負(fù)相關(guān)基因參與細(xì)胞連接、胚胎和神經(jīng)發(fā)育過(guò)程；造血相關(guān)基因DNA去甲基化并表達(dá)上調(diào)，其DMC位點(diǎn)富集于CpG島shelf和open sea，其轉(zhuǎn)錄區(qū)去甲基化與增強(qiáng)子和DNase1高敏感位點(diǎn)（DHS）有關(guān)。 （3）KEGG分析與調(diào)控網(wǎng)絡(luò)：共富集53條信號(hào)通路，趨化因子通路和造血細(xì)胞譜系通路富集度最高，其中上調(diào)基因參與38條通路，下調(diào)基因參與15條通路；hESC向紅細(xì)胞分化中OCT4發(fā)生DNA甲基化并表達(dá)下調(diào)，101個(gè)OCT4靶基因表達(dá)改變，其中與紅細(xì)胞生成相關(guān)基因表達(dá)上調(diào)，胚胎和神經(jīng)系統(tǒng)發(fā)育的靶基因表達(dá)下調(diào)。 4．芯片結(jié)果的驗(yàn)證 （1）real-time MSP結(jié)果顯示，hESC向紅細(xì)胞分化中GATA2和GYPB的啟動(dòng)子區(qū)DNA發(fā)生部分去甲基化，SOX2的啟動(dòng)子區(qū)DNA發(fā)生部分甲基化，與DNA甲基化芯片結(jié)果相符。 （2）RT-qPCR結(jié)果顯示，hESC向紅細(xì)胞分化中造血相關(guān)基因GATA2、TAL1、LMO2、CD34和GYPB表達(dá)上調(diào)，多潛能相關(guān)基因OCT4和SOX2表達(dá)下調(diào)，與表達(dá)譜芯片結(jié)果一致。 二、hiPSC向紅細(xì)胞分化中DNA甲基化譜與基因表達(dá)譜的負(fù)相關(guān)分析 用與hESC相同的方法檢測(cè)并分析hiPSC向紅系細(xì)胞分化中iPSC、中胚層祖細(xì)胞iPS-EB和紅系細(xì)胞iPS-BL的DNA甲基化譜和基因表達(dá)譜，并聯(lián)合分析DNA甲基化與基因表達(dá)呈負(fù)相關(guān)的基因。實(shí)驗(yàn)結(jié)果如下： 1. hiPSC向紅細(xì)胞分化中DNA甲基化譜的分析 iPS-EB與iPSC相比，共有759個(gè)DMC位點(diǎn)，高甲基化位點(diǎn)多于低甲基化位點(diǎn)；iPS-BL分別與iPS-EB和iPSC相比，DMC位點(diǎn)分別為66922個(gè)和59003個(gè)，且低甲基化位點(diǎn)多于高甲基化位點(diǎn)；DMC位點(diǎn)所在基因主要參與代謝、生物調(diào)節(jié)及發(fā)育過(guò)程。 2. hiPSC向紅細(xì)胞分化中基因表達(dá)譜的分析 （1）iPS-EB與iPSC相比，共有1473個(gè)差異表達(dá)基因，iPS-BL與iPS-EB和iPSC相比，差異表達(dá)基因分別為2776個(gè)和5137個(gè)。 （2）iPSC向iPS-EB分化中，上調(diào)基因參與脈管系統(tǒng)和心臟發(fā)育過(guò)程，下調(diào)基因參與應(yīng)答反應(yīng)及循環(huán)系統(tǒng)發(fā)育；iPS-EB向iPS-BL分化中，上調(diào)基因參與造血和紅細(xì)胞生成，下調(diào)基因參與神經(jīng)發(fā)育等；iPS-BL與iPSC相比，上調(diào)基因涉及血紅素代謝和細(xì)胞凋亡的調(diào)節(jié)，而下調(diào)基因參與細(xì)胞周期及細(xì)胞間連接。 3. DNA甲基化與mRNA表達(dá)水平呈負(fù)相關(guān)基因的分析 （1）負(fù)相關(guān)基因的篩選及其DMC位點(diǎn)的分布：iPSC向iPS-EB分化中負(fù)相關(guān)基因?yàn)?5個(gè)，iPS-EB向iPS-BL分化中為2185個(gè)；負(fù)相關(guān)基因的DMC位點(diǎn)主要位于啟動(dòng)子區(qū)和轉(zhuǎn)錄區(qū)。 （2）負(fù)相關(guān)基因的GO分析顯示，iPS-EB向iPS-BL分化中，負(fù)相關(guān)基因參與血管、血液循環(huán)和細(xì)胞粘附等生物過(guò)程，但未參與紅系細(xì)胞相關(guān)的生物過(guò)程。 4．hiPSC向紅細(xì)胞分化中OCT4及其靶基因的表達(dá) （1）iPSC向紅細(xì)胞分化中，OCT4表達(dá)水平在iPSC向iPS-EB分化中上調(diào)，差異表達(dá)的OCT4靶基因?yàn)?6個(gè)；OCT4表達(dá)水平在iPS-EB向iPS-BL分化中未改變，差異表達(dá)的OCT4靶基因?yàn)?2個(gè)。 （2）上調(diào)和下調(diào)靶基因均參與胚胎、神經(jīng)和中胚層發(fā)育，少數(shù)造血相關(guān)靶基因表達(dá)上調(diào)。 三、OCT4誘導(dǎo)hHFMSCs向紅細(xì)胞分化 利用攜帶OCT4的慢病毒載體轉(zhuǎn)導(dǎo)hHFMSCs，選取懸浮的hHFMSCOCT4，經(jīng)造血因子誘導(dǎo)、紅系分化及脫核培養(yǎng)。在分化過(guò)程中，利用Wright-Giemsa染色觀察細(xì)胞形態(tài)改變，用細(xì)胞免疫熒光和流式細(xì)胞術(shù)檢測(cè)紅細(xì)胞特異性標(biāo)志物，最后利用RT-qPCR檢測(cè)OCT4靶基因。實(shí)驗(yàn)結(jié)果如下： 1．OCT4轉(zhuǎn)導(dǎo)后hHFMSCs的細(xì)胞形態(tài)和造血標(biāo)志物表達(dá) （1）轉(zhuǎn)導(dǎo)后的hHFMSCs中EGFP陽(yáng)性細(xì)胞占88.92%，OCT4表達(dá)水平明顯上調(diào)。 （2）轉(zhuǎn)導(dǎo)后細(xì)胞由大變小，由長(zhǎng)梭形變成類(lèi)圓形，14d后出現(xiàn)易懸浮的細(xì)胞亞群，呈克隆樣生長(zhǎng)。 （3）OCT4轉(zhuǎn)導(dǎo)后hHFMSCs中CD45陽(yáng)性細(xì)胞占1.5%，懸浮的hHFMSCs中CD34陽(yáng)性細(xì)胞占2.19%。 3.轉(zhuǎn)導(dǎo)OCT4的hHFMSCs向紅細(xì)胞分化 （1）光鏡下觀察，造血誘導(dǎo)第7d開(kāi)始出現(xiàn)造血集落，10-20d先后出現(xiàn)紅系爆炸式集落及紅系集落形成單位；Wright-Giemsa染色顯示，第7d開(kāi)始先后出現(xiàn)早幼紅、中幼紅和晚幼紅細(xì)胞，第20d晚幼紅細(xì)胞數(shù)量不再增加。 （2）造血誘導(dǎo)第7-20d的細(xì)胞表達(dá)紅系祖細(xì)胞標(biāo)志CD71和CD235a，同時(shí)表達(dá)血型蛋白A和B；誘導(dǎo)第23d紅細(xì)胞完成脫核，80%以上紅細(xì)胞表達(dá)成人型β血紅蛋白；hHFMSC向紅細(xì)胞分化中細(xì)胞直徑及核質(zhì)比逐漸減小，血紅蛋白化逐漸增加。 4．hHFMSCs向紅細(xì)胞分化中OCT4靶基因的表達(dá) OCT4轉(zhuǎn)導(dǎo)后，多潛能相關(guān)的靶基因OCT4、NANOG、SEMA3A和LEFTY2和造血相關(guān)的靶基因FLI1、TAL1、HBG1和CA2表達(dá)均上調(diào)。造血誘導(dǎo)21d后，多潛能相關(guān)的靶基因LEFTY2及OCT4顯著下調(diào)，而造血相關(guān)的靶基因TAL1、HBG1和RHD表達(dá)顯著上調(diào)。 綜上所述，本研究發(fā)現(xiàn)DNA甲基化譜改變可調(diào)控紅細(xì)胞生成過(guò)程中發(fā)育階段特異性的基因表達(dá)譜，從而為研究紅細(xì)胞生成的分子機(jī)制提供一個(gè)表觀遺傳學(xué)機(jī)制，而利用OCT4誘導(dǎo)hHFMSCs向紅細(xì)胞分化的方法，可為患者個(gè)體化細(xì)胞治療提供新途徑。
[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 .
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R329.2

【共引文獻(xiàn)】

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本文編號(hào)：1917015