發(fā)布時(shí)間：2018-02-27 17:01

  本文關(guān)鍵詞： 造血干細(xì)胞 microRNA 非編碼RNA 基因芯片 干細(xì)胞功能 基因表達(dá)調(diào)控 生物信息學(xué) 發(fā)育生物學(xué)　出處：《第三軍醫(yī)大學(xué)》2007年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 造血干細(xì)胞(hematopoietic stem cell,HSCs)是目前研究最為深入的成體干細(xì)胞,其具有向髓系、淋巴系、巨核系祖細(xì)胞的多向分化的功能,是極富應(yīng)用前景的研究領(lǐng)域,很可能給人類(lèi)帶來(lái)革命性的變化。然而,造血干細(xì)胞研究尚存在許多亟待解決的困惑,比如,造血干細(xì)胞特異性分子標(biāo)志、多向分化潛能分子機(jī)制、造血干細(xì)胞在未分化狀態(tài)下長(zhǎng)期增殖的關(guān)鍵因子、造血干細(xì)胞受損相關(guān)疾病(白血病、再生障礙性貧血、骨髓增生異常綜合征)發(fā)生機(jī)制等,既是造血干細(xì)胞研究的熱點(diǎn),也是造血干細(xì)胞研究的難點(diǎn)。近年來(lái),一類(lèi)叫做microRNAs(miRNAs)的非編碼小RNA分子,在監(jiān)控個(gè)體發(fā)育時(shí)相轉(zhuǎn)變、調(diào)控特定細(xì)胞增殖、分化進(jìn)程等方面起著重要作用,其在維持干細(xì)胞定向分化和自我更新功能維持中的作用,逐漸被科學(xué)家們發(fā)現(xiàn),目前已經(jīng)掀起miRNAs在干細(xì)胞研究中的熱潮。HSCs中miRNAs表達(dá)狀況尚未見(jiàn)報(bào)道,miRNAs在HSCs發(fā)育中的作用尚知之甚少。建立高通量篩選miRNAs的技術(shù)平臺(tái),獲得并鑒定造血干細(xì)胞發(fā)育相關(guān)的miRNAs,是研究造血干細(xì)胞相關(guān)miRNAs的起點(diǎn)。因此,本課題擬研制新型哺乳動(dòng)物miRNAs檢測(cè)基因芯片,篩選造血干細(xì)胞發(fā)育相關(guān)miRNAs,生物信息學(xué)分析及驗(yàn)證其作用靶點(diǎn),并探討其在促進(jìn)造血干細(xì)胞發(fā)育中的作用,研究結(jié)果為探明miRNAs調(diào)控造血干細(xì)胞發(fā)育的分子機(jī)制、探索誘導(dǎo)造血干細(xì)胞定向分化的新手段提供理論指導(dǎo)和實(shí)驗(yàn)依據(jù)。 研究目的 1、研制新型哺乳動(dòng)物miRNAs檢測(cè)芯片。 2、通過(guò)miRNAs芯片雜交,獲取CD34+CD38-造血干細(xì)胞發(fā)育相關(guān)miRNAs表達(dá)譜。 3、生物信息學(xué)分析預(yù)測(cè)并驗(yàn)證miRNAs作用靶點(diǎn)。 4、探索miRNAs在造血干細(xì)胞發(fā)育中的作用。 研究?jī)?nèi)容和方法 1、研制新型哺乳動(dòng)物miRNAs檢測(cè)芯片 以已知哺乳動(dòng)物基因組miRNAs序列為依據(jù),合成相應(yīng)DNA探針,制作新型哺乳動(dòng)物miRNAs檢測(cè)基因芯片。 2、CD34+CD38-造血干細(xì)胞發(fā)育相關(guān)miRNAs表達(dá)譜的篩選 流式細(xì)胞儀細(xì)胞分選人臍血CD34+CD38-造血干細(xì)胞及CD34+造血祖細(xì)胞,提取其總RNA,分離純化18-100nt小片段RNA,經(jīng)擴(kuò)增及熒光標(biāo)記后,與基因芯片進(jìn)行分子雜交,獲得CD34+CD38-造血干細(xì)胞miRNAs表達(dá)譜。 3、實(shí)時(shí)熒光定量RT-PCR(Q-RT-PCR)對(duì)miRNAs芯片結(jié)果的驗(yàn)證 選取6個(gè)在CD34+CD38-造血干細(xì)胞中差異表達(dá)的miRNAs,以Q-RT-PCR加以驗(yàn)證,對(duì)miRNAs芯片結(jié)果進(jìn)行初步驗(yàn)證,對(duì)結(jié)果一致的miRNAs行3次重復(fù)實(shí)驗(yàn)以確證Q-RT-PCR結(jié)果的可靠性。 4、生物信息學(xué)分析及miRNAs作用靶點(diǎn)的驗(yàn)證 生物信息學(xué)分析miR-129、miR-526b*和miR-520h的染色體定位、保守性分析等,利用網(wǎng)絡(luò)共享軟件picTar、miRanda 3.0和targetscan3.1,分析其可能作用的靶基因。選擇在CD34+CD38-差異較為顯著的miR-129、miR-520h的進(jìn)行作用靶基因的驗(yàn)證。首先合成與miRNAs結(jié)合的靶位點(diǎn)DNA序列及互補(bǔ)序列,并分別在兩條寡核苷酸5'端懸垂添加Hind III和Spe I酶切位點(diǎn);退火形成形成帶酶切位點(diǎn)的雙鏈插入序列,與線性化pMIR-REPORT熒光素酶(Luc)報(bào)告基因載體(pMIR-Luc)連接構(gòu)建重組載體(pMIR-Luc-miR),并進(jìn)行酶切及測(cè)序驗(yàn)證。 重組載體(pMIR-Luc-miR)、標(biāo)準(zhǔn)化對(duì)照載體pMIR-REPORT-β-gal(pMIR-β-gal)與miRNAs前體(Pre-miRNAs)共轉(zhuǎn)染HeLa細(xì)胞(實(shí)驗(yàn)組),72h后分別檢測(cè)HeLa細(xì)胞的熒光素酶(Luc)和β半乳糖苷酶(β-gal)的活性,計(jì)算二者的比值(Luc/β-gal)。同時(shí)設(shè)立三個(gè)對(duì)照組(no miRNA空白對(duì)照組、Pre-miR-negative陰性對(duì)照組和Anti-miRNAs反證對(duì)照組),比較各組的Luc/β-gal比值,驗(yàn)證miRNAs作用靶點(diǎn)的真實(shí)性。 5、miR-520h促進(jìn)造血發(fā)育功能研究 選擇在CD34+CD38-造血干細(xì)胞中高表達(dá)的miR-520h,研究其在促進(jìn)造血發(fā)育中的作用。流式細(xì)胞儀分選的CD34+細(xì)胞,miR-520h轉(zhuǎn)染CD34+細(xì)胞,轉(zhuǎn)染后24h行CFC實(shí)驗(yàn)檢測(cè)CD34+細(xì)胞集落形成能力改變,轉(zhuǎn)染后72h行FACS檢測(cè)CD34+、CD34+CD38-細(xì)胞比例變化,同時(shí)設(shè)立三個(gè)對(duì)照組(no miRNA空白對(duì)照組、Pre-miR-129陰性對(duì)照組和Anti-miR-520h反證對(duì)照組),分析miR-520h在促進(jìn)造血發(fā)育中的作用。 研究結(jié)果 1、成功研制新型哺乳動(dòng)物miRNAs檢測(cè)芯片 整個(gè)點(diǎn)陣分成4個(gè)亞陣,每個(gè)亞陣有23行,21列,點(diǎn)間距為185μm,點(diǎn)的直徑約為130μm。每條探針重復(fù)三次。為保證實(shí)驗(yàn)的可靠性,在芯片的不同部位多次設(shè)定各種對(duì)照,經(jīng)點(diǎn)樣后芯片共有1754個(gè)位點(diǎn),包含469個(gè)miRNAs序列,總共588個(gè)基因(每個(gè)基因重復(fù)3個(gè)位點(diǎn))。 2、CD34+CD38-造血干細(xì)胞發(fā)育相關(guān)miRNAs的篩選 與CD34+細(xì)胞miRNAs表達(dá)微陣列比較,HSC細(xì)胞共篩選出31個(gè)miRNAs,其中4倍低于CD34+細(xì)胞的miRNAs為22個(gè)(包括miR-129等),4倍高于CD34+細(xì)胞的miRNAs為9個(gè)(包括miR-526b*和miR-520h等) ,其中預(yù)測(cè)的miRNAs (PREDICTED_MIR)為4個(gè)。 3、實(shí)時(shí)熒光定量RT-PCR(Q-RT-PCR)對(duì)miRNAs芯片結(jié)果的驗(yàn)證 選取6個(gè)CD34+CD38-造血干細(xì)胞中差異表達(dá)的miRNAs,以Q-RT-PCR加以驗(yàn)證,與芯片結(jié)果的吻合率達(dá)到50%,兩種實(shí)驗(yàn)結(jié)果相符的miRNAs為:CD34+CD38-造血干細(xì)胞低表達(dá)的miR-129和CD34+CD38-造血干細(xì)胞中高表達(dá)的miR-526b*和miR-520h,相對(duì)于CD34+細(xì)胞的相對(duì)表達(dá)率分別為0.160±0.005、2.276±0.058和5.596±0.861。 4、生物信息學(xué)分析及miRNAs作用靶點(diǎn)的驗(yàn)證 利用在線軟件Pictar、miRanda 3.0和targetscan3.1分別預(yù)測(cè)miR-129、miR-526b*和miR-520h的作用靶基因。結(jié)果發(fā)現(xiàn),①miR-129在人、鼠、狗、猩猩、雞等動(dòng)物中保守存在33個(gè)可能的靶基因,包括EIF2C3、CAMTA1、SH3KBP1、TGIF2、ING3、DLGAP2等與miRNA加工、轉(zhuǎn)錄因子、信號(hào)轉(zhuǎn)導(dǎo)等相關(guān)的基因。②miR-526b*僅在人類(lèi)存在,其可能的作用靶點(diǎn)有771個(gè),包括ABCG2、EIF2C3、CAMK4、CSF2RA等關(guān)于信號(hào)轉(zhuǎn)導(dǎo)、轉(zhuǎn)錄因子、造血發(fā)育、凋亡調(diào)控、干細(xì)胞功能維持、miRNAs加工成熟的基因。③miR-520h共有226個(gè)可能的作用靶點(diǎn),在人類(lèi)、小鼠和類(lèi)人猿中保守的靶基因共有30個(gè),包括ABCG2、ID 1 (DNA結(jié)合抑制劑1)、SRP19 (一種信號(hào)識(shí)別蛋白)以及SMAD6 (一種轉(zhuǎn)錄調(diào)控因子)等與干細(xì)胞功能維持、轉(zhuǎn)錄、信號(hào)轉(zhuǎn)導(dǎo)等相關(guān)的調(diào)控蛋白。 選取miR-129可能的作用靶點(diǎn)EIF2C3和CAMTA1、miR-520h可能的作用靶點(diǎn)ABCG2和SMAD6進(jìn)行驗(yàn)證。利用pMIR-REPORT熒光素酶報(bào)告基因載體,經(jīng)酶切鑒定和測(cè)序驗(yàn)證,分別成功構(gòu)建四種重組載體: pMIR-Luc-miR129-EIF2C3、pMIR-Luc-miR129-CAMTA1、pMIR-Luc-miR520h-ABCG2和pMIR-Luc-miR520h-SMAD6。 pMIR-REPORT熒光素酶報(bào)告基因系統(tǒng)進(jìn)行作用靶點(diǎn)驗(yàn)證實(shí)驗(yàn)表明,三個(gè)對(duì)照組(no miRNAs空白對(duì)照組、Pre-miR-negative陰性對(duì)照組和Anti-miRNAs反證對(duì)照組),Luc/β-gal比值接近。實(shí)驗(yàn)組,miR-129對(duì)于EIF2C3和CAMTA1,熒光素酶活性分別下降41%和47%,表明EIF2C3和CAMTA1都是miR-129的真實(shí)作用靶點(diǎn);miR520h對(duì)于ABCG2和SMAD6,熒光素酶活性分別下降68%和27%,表明ABCG2是miR-520h的真實(shí)作用靶點(diǎn),而miR-520h對(duì)SMAD6的抑制能力作用較弱。 5、miR-520h促進(jìn)造血干細(xì)胞發(fā)育功能研究 CFC實(shí)驗(yàn):三個(gè)對(duì)照組(no miRNAs空白對(duì)照組、miR-129陰性對(duì)照組和Anti-miR-520h反證對(duì)照組),各種祖細(xì)胞集落和集落總數(shù)均相近,其中空白對(duì)照組CFU-E、BFU-E、CFU-GM和CFU-GEMM和集落總數(shù)分別為28.50±4.32、37.83±5.60、57.67±5.16、1.50±0.55和145.50±14.35,實(shí)驗(yàn)組分別為37.83±7.70、46.5±4.85、68.17±7.73、1.67±0.52和174.33±17.82。經(jīng)方差分析表明,CFU-E、BFU-E、CFU-GM和集落總數(shù)有顯著統(tǒng)計(jì)學(xué)差異(P0.05,n=6)。 FACS分析:三個(gè)對(duì)照組CD34+和CD34+CD38-細(xì)胞比例相近,其中空白對(duì)照組CD34+和CD34+CD38-細(xì)胞比例為22.91%±2.03和12.57%±0.81,而實(shí)驗(yàn)組則為30.43%±2.34和13.08%±1.08。經(jīng)方差分析表明,實(shí)驗(yàn)組CD34+細(xì)胞比例較高,有統(tǒng)計(jì)學(xué)差異(P0.05,n=6),而CD34+CD38-比例相差不大,無(wú)統(tǒng)計(jì)學(xué)差異(P0.05,n=6)。 實(shí)驗(yàn)表明miR-520h具有促進(jìn)造血干細(xì)胞發(fā)育和分化的功能。 結(jié)論 1、成功研制新型哺乳動(dòng)物miRNAs檢測(cè)芯片,表明利用miRNAs基因芯片來(lái)獲取CD34+CD38-造血干細(xì)胞相關(guān)miRNAs是可行的,也為研究其它成體干細(xì)胞miRNAs表達(dá)譜建立了技術(shù)平臺(tái)。 2、篩選到CD34+CD38-造血干細(xì)胞發(fā)育相關(guān)miRNAs表達(dá)譜,并部分經(jīng)實(shí)時(shí)定量RT-PCR驗(yàn)證,為探討miRNAs參與CD34+CD38-造血干細(xì)胞發(fā)育分子機(jī)制和生物功能研究奠定了基礎(chǔ)。 3、驗(yàn)證了miR-129和miR-520h部分作用靶點(diǎn),表明miRNAs通過(guò)調(diào)控EIF2C3、CAMTA1、ABCG2等在miRNA加工、轉(zhuǎn)錄因子、信號(hào)轉(zhuǎn)導(dǎo)、干細(xì)胞功能維持等方面的靶基因,從而參與造血干細(xì)胞的發(fā)育,并形成miRNAs基因網(wǎng)絡(luò),在造血干細(xì)胞發(fā)育進(jìn)程中起著協(xié)同或者拮抗作用。 4、miR-520h具有顯著的促進(jìn)造血干細(xì)胞發(fā)育和向祖細(xì)胞分化的作用。研究結(jié)果不僅對(duì)miRNAs作為促進(jìn)造血發(fā)育的分子靶點(diǎn)的探索具有重要的應(yīng)用前景,而且對(duì)于發(fā)育生物學(xué)、發(fā)展組織工程和再生醫(yī)學(xué)都具有重要的理論意義。
[Abstract]:Hematopoietic stem cells (hematopoietic stem cell, HSCs) is currently the most in-depth study of adult stem cells, which has to myeloid, lymphoid, megakaryocyte progenitor cell differentiation, is a field with significant application prospect, is likely to bring revolutionary changes to human life. However, such as hematopoietic there are still many problems of stem cell research, confusion, hematopoietic stem cell specific molecular markers and multilineage potential molecular mechanisms of hematopoietic stem cells in an undifferentiated state under the key factor of the long-term proliferation of hematopoietic stem cell damage related diseases (leukemia, aplastic anemia, myelodysplastic syndrome) occurrence mechanism etc. hematopoietic stem cell research is the hotspot, also is the difficulty of hematopoietic stem cell research. In recent years, a class called microRNAs (miRNAs) encoding non small RNA molecules, the phase transition in the monitoring of individual development, regulation of specific cells The proliferation plays an important role in the differentiation process and other aspects, in the maintenance of stem cell differentiation and self-renewal function in maintenance, gradually by scientists found that currently there has been miRNAs in stem cell research in the upsurge of.HSCs expression of miRNAs has not been reported. The role of miRNAs in the development of HSCs is still poorly understood. Establish a technology platform for high-throughput screening of miRNAs, and identification of hematopoietic stem cell development related to miRNAs, is the starting point of hematopoietic stem cell related miRNAs. Therefore, this paper intends to design a new gene chip for the detection of mammalian miRNAs, screening of hematopoietic stem cell development related miRNAs, bioinformatics analysis and validation of its target, and on the promotion of hematopoietic stem cells during the development of the role of research results to explore the molecular mechanism of miRNAs regulation of hematopoietic stem cell development, explore the induction of hematopoietic stem cell division The novice section provides theoretical guidance and experimental basis.
research objective
1, develop a new mammal miRNAs detection chip.
2, CD34+CD38- hematopoietic stem cell development related miRNAs expression profiles were obtained by miRNAs chip hybridization.
3, bioinformatics analysis predicts and validates miRNAs targets.
4, explore the role of miRNAs in the development of hematopoietic stem cells.
Research contents and methods
1, develop a new mammal miRNAs detection chip
Based on the known miRNAs sequence of the mammalian genome, the corresponding DNA probe was synthesized to produce a novel mammalian miRNAs detection gene chip.
2, screening of miRNAs expression profiles related to CD34+CD38- hematopoietic stem cell development
Flow cytometry cells were selected to select cord blood CD34+CD38- hematopoietic stem cells and CD34+ hematopoietic progenitor cells, extract their total RNA, and isolate and purify 18-100nt small fragment RNA. After amplification and fluorescence labeling, hybridized with gene chip to obtain miRNAs expression profile of CD34+CD38- hematopoietic stem cells.
3, verification of miRNAs chip results by real time fluorescence quantitative RT-PCR (Q-RT-PCR)
6 differentially expressed miRNAs in CD34+CD38- hematopoietic stem cells were selected and verified by Q-RT-PCR. The results of miRNAs chip were preliminarily verified, and 3 times repeated miRNAs experiments were performed to confirm the reliability of Q-RT-PCR results.
4, bioinformatics analysis and verification of the target of miRNAs
Analysis of miR-129 biological information, chromosomal location of miR-526b* and miR-520h, conservative analysis, sharing network by using the software picTar, miRanda 3 and targetscan3.1, and analyze the possible role of the target gene. Selection in CD34+CD38- is significant difference in miR-129, verify the miR-520h target gene. The first synthesis of target sites of DNA sequence and complementary sequence combined with miRNAs, and respectively in two oligonucleotide 5'end adding Hind III and Spe I dangling endonuclease site; annealed to form double chains are formed with restriction sites of insertion sequence, and linear pMIR-REPORT luciferase (Luc) reporter gene vector (pMIR-Luc). The recombinant vector (pMIR-Luc-miR), and enzyme digestion and sequencing.
The recombinant vector (pMIR-Luc-miR), the standard control vector pMIR-REPORT- -gal beta (pMIR- beta -gal) and precursor miRNAs (Pre-miRNAs) were transfected into HeLa cells (experimental group), 72h after HeLa cells were detected by luciferase (Luc) and beta galactosidase (beta -gal) activity, calculate the ratio of the two (Luc/ beta -gal). Three control groups were set up (no miRNA control group, Pre-miR-negative negative control group and Anti-miRNAs control group, Luc/ beta disproof) -gal ratio were compared, to verify the authenticity of the miRNAs target.
5, miR-520h promotes the function of hematopoietic development
In the selection of CD34+CD38- high expression of hematopoietic stem cells in miR-520h and study its role in promoting development of hematopoiesis. Flow cytometry sorting of CD34+ cells, miR-520h cells transfected with CD34+, 24h after transfection by CFC assay, CD34+ cell colony formation ability to change 72h after transfection were detected with FACS CD34+, changes in the proportion of CD34+CD38- cells. At the same time, the establishment of the three control groups (no miRNA control group, Pre-miR-129 negative control group and Anti-miR-520h control group, miR-520h analysis of disproof) role in promoting the development of hematopoiesis.
Research results
1, a successful development of a new mammal miRNAs detection chip
The matrix is divided into 4 sub arrays, each sub array with 23 rows, 21 rows, spacing of 185 m, the diameter is about 130 M. for each probe was repeated three times. In order to ensure the reliability of the experiment, repeatedly set various in different parts of the control chip, the chip samples had a total of 1754 site contains 469 miRNAs sequences, a total of 588 genes (gene duplication each 3 loci).
2, screening of CD34+CD38- hematopoietic stem cell development related miRNAs
Compared with CD34+ cells miRNAs expression microarray, HSC cells were screened in 31 miRNAs, which is 4 times lower than that of CD34+ cells for 22 miRNAs (including miR-129), 4 times higher than that of CD34+ cells of 9 miRNAs (including miR-526b* and miR-520h), the prediction of the miRNAs (PREDICTED_MIR) 4.
3, verification of miRNAs chip results by real time fluorescence quantitative RT-PCR (Q-RT-PCR)
We selected 6 differentially expressed in CD34+CD38- HSCs were miRNAs, Q-RT-PCR and verified with the microarray data rate reached 50%, two kinds of experimental results with miRNAs: CD34+CD38- low expression of hematopoietic stem cells miR-129 and CD34+CD38- hematopoietic stem cells have high expression of miR-526b* and miR-520h, the relative expression rate relative to CD34+ cells were 0.160 + 0.005,2.276 + 0.058 and 5.596 + 0.861.
4, bioinformatics analysis and verification of the target of miRNAs
Using online software Pictar, miRanda 3 and targetscan3.1 respectively predicted miR-129 target genes of miR-526b* and miR-520h. The results showed that the miR-129 in human, rat, dog, gorilla, 33 possible target genes are conserved, chicken and other animal including EIF2C3, CAMTA1, SH3KBP1, TGIF2, ING3, DLGAP2 and miRNA processing gene, transcription factor, signal transduction related. The miR-526b* only exists in human, the possible targets of 771, including ABCG2, EIF2C3, CAMK4, CSF2RA and so on signal transduction, transcription factor, hematopoietic development, apoptosis regulation, maintaining of stem cell function, gene of mature miRNAs. The processing of miR-520h there are 226 potential targets, in humans, mice and apes in the target gene for a total of 30, including ABCG2 1, ID (inhibitor of DNA binding 1), SRP19 (a signal recognition protein) and SMAD6 (a transcription factor) and stem cells The regulatory proteins related to cellular function maintenance, transcription, signal transduction and so on.
Select the potential targets of miR-129, EIF2C3 and CAMTA1, potential targets of miR-520h ABCG2 and SMAD6 to verify. By using the pMIR-REPORT luciferase reporter gene vector and identified by enzyme digestion and sequencing, the recombinant vector was successfully constructed respectively four kinds: pMIR-Luc-miR129-EIF2C3, pMIR-Luc-miR129-CAMTA1, pMIR-Luc-miR520h-ABCG2 and pMIR-Luc-miR520h-SMAD6.
The pMIR-REPORT luciferase reporter system showed that the target verification experiment, three control group (no miRNAs Pre-miR-negative blank control group, negative control group and Anti-miRNAs control group, Luc/ disproof) beta -gal ratio is close to the experimental group, miR-129 for EIF2C3 and CAMTA1, luciferase activity were 41% and 47%, indicating that EIF2C3 and CAMTA1 are the real target of miR-129; miR520h for ABCG2 and SMAD6, luciferase activity were 68% and 27%, indicating that ABCG2 is the real target of miR-520h, and the inhibition effect of miR-520h on SMAD6 is weak.
5, miR-520h to promote the development of hematopoietic stem cell development
CFC experiment: three control group (no miRNAs miR-129 blank control group, negative control group and Anti-miR-520h control group, all kinds of disproof) progenitor cell colony and colony count were similar to that of the control group CFU-E, BFU-E, CFU-GM and CFU-GEMM and the total number of colonies were 28.50 + 4.32,37.83 + 5.60,57.67 + 5.16,1.50 + 0.55 and 145.50 + 14.35, the experimental group were 37.83 + 7.70,46.5 + 4.85,68.17 + 7.73,1.67 + 0.52 and 174.33 + 17.82. by variance analysis showed that CFU-E, BFU-E, CFU-GM and the total number of colonies with significant difference (P0.05, n=6).
FACS analysis: three CD34+ control group and CD34+CD38- cells was similar to that of the control group CD34+ and the percentage of CD34+CD38- cells was 22.91% + 2.03 and 12.57% + 0.81, while the experimental group was 30.43% + 2.34 and 13.08% + 1.08. by the analysis of variance showed that the proportion of CD34+ cells in experimental group was higher, there was significant difference (P0.05, n=6), while the proportion of CD34+CD38- had little difference, no statistical difference (P0.05, n=6).
The experimental results show that miR-520h has the function of promoting the development and differentiation of hematopoietic stem cells.
conclusion
1, we successfully developed a new mammalian miRNAs detection chip. It showed that it is feasible to obtain CD34+CD38- hematopoietic stem cell related miRNAs by miRNAs gene chip, and also set up a technological platform for studying miRNAs expression profiles of other adult stem cells.
2, we screened the miRNAs expression profiles of CD34+CD38- hematopoietic stem cell development, and partially verified by real-time quantitative RT-PCR, which laid a foundation for exploring miRNAs involved in the molecular mechanism and biological function of CD34+CD38- hematopoietic stem cell development.
3, to verify the miR-129 and miR-520h part of the target, indicated that miRNAs regulated by EIF2C3, CAMTA1, ABCG2 in miRNA processing, transcription factor, signal transduction, function of stem cells to maintain target genes, and thus participate in hematopoietic stem cell development, and the formation of the miRNAs gene network plays a synergistic or antagonistic effects on hematopoiesis stem cell development process.
4, miR-520h has a significant role in promoting the development of hematopoietic stem cells and progenitor cells to differentiation. The results of miRNAs as not only has important application prospect to promote the exploration of molecular target of hematopoietic development, but also for the development of biology, has the important theory significance and development of tissue engineering and regenerative medicine.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 潘長(zhǎng)福;低溫環(huán)境下microRNA對(duì)小鼠腦外傷后海馬區(qū)神經(jīng)干細(xì)胞增殖分化的調(diào)控作用[D];南昌大學(xué);2011年

，

本文編號(hào)：1543534