發(fā)布時間：2018-05-21 01:01

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

【摘要】：紅細胞生成過程受表觀遺傳學和遺傳學雙重調控，其中DNA甲基化作為表觀遺傳學的重要機制，通過調控基因組表達動力學及紅細胞生成相關轉錄因子誘導干細胞向紅細胞分化。本論文利用DNA甲基化芯片和全基因組表達譜芯片技術，分析hESCs和iPSCs向紅細胞分化過程中DNA甲基化譜和基因表達譜的改變，并通過負相關基因的分析探討多潛能干細胞向紅細胞分化中DNA甲基化的作用機制；同時研究OCT4誘導hHFMSCs直接向紅細胞分化的作用。 一、 hESCs向紅細胞分化中DNA甲基化譜與基因表達譜的負相關分析 利用DNA甲基化芯片和基因表達譜芯片技術，對hESC（人胚胎干細胞）、hES-EB（由hESC獲得的中胚層祖細胞群）以及hES-BL（由hES-EB分化的紅系細胞）三種細胞樣本進行分析，篩選差異甲基化CpG（differentially methylatedCpG，DMC）位點及差異表達基因，聯(lián)合分析DNA甲基化與基因表達呈負相關的基因，利用GO分析富集基因功能，用KEGG分析富集信號通路，用GeneSpring軟件構建調控網絡，最后用real-time MSP和RT-qPCR驗證芯片結果。實驗結果如下： 1. hESC向紅細胞分化中DNA甲基化譜的分析 （1）hES-EB與hESC相比共有639個DMC位點；hES-BL與hESC和hES-EB相比，DMC位點分別為100991個和134736個。高甲基化CpG位點主要富集于CpG島岸和CpG島及啟動子區(qū)，低甲基化CpG位點主要富集于CpG島shelf和open sea及轉錄區(qū)，二者在編碼RNA序列的分布比例沒有明顯差別。 （2）GO分析顯示，hESC向hES-EB分化中，DMC位點所在基因主要參與GTP酶介導的信號轉導、細胞形態(tài)的調節(jié)及特異性基因轉錄；hES-EB向hES-BL分化中，DMC位點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位點所在基因參與黏著斑、II型糖尿病和肌動蛋白細胞骨架的調節(jié)等通路；hES-EB向hES-BL分化中，，DMC位點所在基因主要參與黏著斑、細胞粘附分子和鈣離子信號通路等。 2. hESC向紅細胞分化中基因表達譜的分析 （1）hES-EB和hESC比較，共有810個差異表達基因，hES-BL與hESC和hES-EB相比，差異表達基因分別為5264個和5843個。 （2）GO分析顯示，hESC向hES-EB分化中上調基因與循環(huán)系統(tǒng)發(fā)育相關；下調基因參與細胞粘附和神經發(fā)育。hES-EB向hES-BL分化中上調基因參與造血、紅細胞發(fā)育與成熟過程；下調基因參與神經系統(tǒng)和胚胎發(fā)育。 （3）KEGG分析顯示，hESC向hES-EB分化中上調基因參與TGF-β、癌癥和凝血等相關信號通路；下調基因未富集到相關信號通路。hES-EB向hES-BL分化中上調基因參與造血細胞譜系、趨化因子和JAK-STAT信號通路等；下調基因參與緊密連接和細胞周期等通路。 3. DNA甲基化與mRNA表達呈負相關基因的分析 （1）負相關基因的篩選及其DMC位點的分布：hESC向hES-EB分化中負相關基因有15個，hES-EB向hES-BL分化中為2624個，負相關基因的DMC位點主要位于啟動子區(qū)和轉錄區(qū)。 （2）GO分析與功能特異性基因的DMC位點分布：上調的負相關基因涉及紅細胞特異的細胞組分、分子功能及生物過程；下調的負相關基因參與細胞連接、胚胎和神經發(fā)育過程；造血相關基因DNA去甲基化并表達上調，其DMC位點富集于CpG島shelf和open sea，其轉錄區(qū)去甲基化與增強子和DNase1高敏感位點（DHS）有關。 （3）KEGG分析與調控網絡：共富集53條信號通路，趨化因子通路和造血細胞譜系通路富集度最高，其中上調基因參與38條通路，下調基因參與15條通路；hESC向紅細胞分化中OCT4發(fā)生DNA甲基化并表達下調，101個OCT4靶基因表達改變，其中與紅細胞生成相關基因表達上調，胚胎和神經系統(tǒng)發(fā)育的靶基因表達下調。 4．芯片結果的驗證 （1）real-time MSP結果顯示，hESC向紅細胞分化中GATA2和GYPB的啟動子區(qū)DNA發(fā)生部分去甲基化，SOX2的啟動子區(qū)DNA發(fā)生部分甲基化，與DNA甲基化芯片結果相符。 （2）RT-qPCR結果顯示，hESC向紅細胞分化中造血相關基因GATA2、TAL1、LMO2、CD34和GYPB表達上調，多潛能相關基因OCT4和SOX2表達下調，與表達譜芯片結果一致。 二、hiPSC向紅細胞分化中DNA甲基化譜與基因表達譜的負相關分析 用與hESC相同的方法檢測并分析hiPSC向紅系細胞分化中iPSC、中胚層祖細胞iPS-EB和紅系細胞iPS-BL的DNA甲基化譜和基因表達譜，并聯(lián)合分析DNA甲基化與基因表達呈負相關的基因。實驗結果如下： 1. hiPSC向紅細胞分化中DNA甲基化譜的分析 iPS-EB與iPSC相比，共有759個DMC位點，高甲基化位點多于低甲基化位點；iPS-BL分別與iPS-EB和iPSC相比，DMC位點分別為66922個和59003個，且低甲基化位點多于高甲基化位點；DMC位點所在基因主要參與代謝、生物調節(jié)及發(fā)育過程。 2. hiPSC向紅細胞分化中基因表達譜的分析 （1）iPS-EB與iPSC相比，共有1473個差異表達基因，iPS-BL與iPS-EB和iPSC相比，差異表達基因分別為2776個和5137個。 （2）iPSC向iPS-EB分化中，上調基因參與脈管系統(tǒng)和心臟發(fā)育過程，下調基因參與應答反應及循環(huán)系統(tǒng)發(fā)育；iPS-EB向iPS-BL分化中，上調基因參與造血和紅細胞生成，下調基因參與神經發(fā)育等；iPS-BL與iPSC相比，上調基因涉及血紅素代謝和細胞凋亡的調節(jié)，而下調基因參與細胞周期及細胞間連接。 3. DNA甲基化與mRNA表達水平呈負相關基因的分析 （1）負相關基因的篩選及其DMC位點的分布：iPSC向iPS-EB分化中負相關基因為15個，iPS-EB向iPS-BL分化中為2185個；負相關基因的DMC位點主要位于啟動子區(qū)和轉錄區(qū)。 （2）負相關基因的GO分析顯示，iPS-EB向iPS-BL分化中，負相關基因參與血管、血液循環(huán)和細胞粘附等生物過程，但未參與紅系細胞相關的生物過程。 4．hiPSC向紅細胞分化中OCT4及其靶基因的表達 （1）iPSC向紅細胞分化中，OCT4表達水平在iPSC向iPS-EB分化中上調，差異表達的OCT4靶基因為36個；OCT4表達水平在iPS-EB向iPS-BL分化中未改變，差異表達的OCT4靶基因為52個。 （2）上調和下調靶基因均參與胚胎、神經和中胚層發(fā)育，少數(shù)造血相關靶基因表達上調。 三、OCT4誘導hHFMSCs向紅細胞分化 利用攜帶OCT4的慢病毒載體轉導hHFMSCs，選取懸浮的hHFMSCOCT4，經造血因子誘導、紅系分化及脫核培養(yǎng)。在分化過程中，利用Wright-Giemsa染色觀察細胞形態(tài)改變，用細胞免疫熒光和流式細胞術檢測紅細胞特異性標志物，最后利用RT-qPCR檢測OCT4靶基因。實驗結果如下： 1．OCT4轉導后hHFMSCs的細胞形態(tài)和造血標志物表達 （1）轉導后的hHFMSCs中EGFP陽性細胞占88.92%，OCT4表達水平明顯上調。 （2）轉導后細胞由大變小，由長梭形變成類圓形，14d后出現(xiàn)易懸浮的細胞亞群，呈克隆樣生長。 （3）OCT4轉導后hHFMSCs中CD45陽性細胞占1.5%，懸浮的hHFMSCs中CD34陽性細胞占2.19%。 3.轉導OCT4的hHFMSCs向紅細胞分化 （1）光鏡下觀察，造血誘導第7d開始出現(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開始先后出現(xiàn)早幼紅、中幼紅和晚幼紅細胞，第20d晚幼紅細胞數(shù)量不再增加。 （2）造血誘導第7-20d的細胞表達紅系祖細胞標志CD71和CD235a，同時表達血型蛋白A和B；誘導第23d紅細胞完成脫核，80%以上紅細胞表達成人型β血紅蛋白；hHFMSC向紅細胞分化中細胞直徑及核質比逐漸減小，血紅蛋白化逐漸增加。 4．hHFMSCs向紅細胞分化中OCT4靶基因的表達 OCT4轉導后，多潛能相關的靶基因OCT4、NANOG、SEMA3A和LEFTY2和造血相關的靶基因FLI1、TAL1、HBG1和CA2表達均上調。造血誘導21d后，多潛能相關的靶基因LEFTY2及OCT4顯著下調，而造血相關的靶基因TAL1、HBG1和RHD表達顯著上調。 綜上所述，本研究發(fā)現(xiàn)DNA甲基化譜改變可調控紅細胞生成過程中發(fā)育階段特異性的基因表達譜，從而為研究紅細胞生成的分子機制提供一個表觀遺傳學機制，而利用OCT4誘導hHFMSCs向紅細胞分化的方法，可為患者個體化細胞治療提供新途徑。
[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 .
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R329.2

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