【摘要】：在血液系統(tǒng)中,轉(zhuǎn)錄因子Tcfec主要在造血干細(xì)胞(Hematopoietic stem cells,HSCs)和多能祖細(xì)胞(Multipotent progenitors,MPPs)中高表達(dá)。目前為止,關(guān)于Tcfec在HSCs的功能影響及機(jī)制尚不清楚。目的:1、證實(shí)Tcfec在HSCs和MPPs中的表達(dá)水平。2、構(gòu)建Tcfec逆轉(zhuǎn)錄病毒過表達(dá)體系。3、通過體內(nèi)、體外實(shí)驗(yàn)研究過表達(dá)Tcfec對HSCs的功能影響。方法:1、分選高純度HSCs和MPPs細(xì)胞群,MySeq測序儀進(jìn)行RNA-Seq測序并獲得原始測序數(shù)據(jù)(Raw data)。利用生物信息學(xué)技術(shù)深入分析,獲得了HSCs及MPPs細(xì)胞的轉(zhuǎn)錄因子表達(dá)差異譜。2、q-PCR分析Tcfec在HSCs和MPPs中的表達(dá)水平。3、利用逆轉(zhuǎn)錄病毒過表達(dá)系統(tǒng),將小鼠Tcfec基因的開放閱讀框(Open reading frame,ORF)構(gòu)建到過表達(dá)載體,并用細(xì)胞轉(zhuǎn)染、熒光定量PCR方法驗(yàn)證過表達(dá)體系的構(gòu)建。4、將流式分選的陽性細(xì)胞(感染過表達(dá)病毒載體的小鼠胎肝Lin-細(xì)胞)移植到經(jīng)致死劑量輻照處理的受體鼠,構(gòu)建體內(nèi)移植動(dòng)物模型。5、流式細(xì)胞儀檢測移植細(xì)胞的陽性率和對骨髓造血譜系分化影響的相關(guān)檢測。6、流式細(xì)胞儀檢測Tcfec對HSPC細(xì)胞周期的影響。7、利用體外克隆形成實(shí)驗(yàn),研究Tcfec對體外克隆形成的影響效果。結(jié)果:1、Tcfec在HSCs相對MPPs高表達(dá)。2、成功構(gòu)建Tcfec過表達(dá)載體。3、移植16周后,Tcfec組中陽性細(xì)胞比例占有優(yōu)勢,血細(xì)胞各譜系分化完全正常。4、過表達(dá)Tcfec的HSCs及MPPs細(xì)胞庫總體數(shù)量增加,但對CMP、GMP、MEP祖細(xì)胞庫無影響。5、Tcfec對于HSPC的細(xì)胞周期沒有影響。6、Tcfec組體外形成的混合細(xì)胞型克隆(GEMM)較對照組增多。7、Tcfec發(fā)揮功能是依賴HSCs自身。結(jié)論:1、本實(shí)驗(yàn)初步揭示Tcfec促進(jìn)HScs植入,同時(shí)不影響HSCs的正常生理功能。2、二次移植實(shí)驗(yàn)證明過表達(dá)Tcfec的HSCs保持自我更新及多向分化能力。3、本研究鑒定了Tcfec是調(diào)控HSCs植入能力的重要功能分子。
[Abstract]:Transcription factor Tcfec is highly expressed in hematopoietic stem cells (Hematopoietic stem cells,HSCs) and pluripotent progenitor cells (Multipotent progenitors,MPPs) in the blood system. Up to now, it is unclear about the function and mechanism of Tcfec in HSCs. Objective: 1. To confirm the expression level of Tcfec in HSCs and MPPs. To construct the Tcfec retrovirus overexpression system. 3. To investigate the effect of overexpression of Tcfec on the function of HSCs in vitro and in vivo. Methods: 1. High purity HSCs and MPPs cell populations were sorted, and MySeq sequencer was used to sequence RNA-Seq and obtain the original (Raw data). Sequence data. 2. The differential expression profiles of transcription factors in HSCs and MPPs cells were obtained by using bioinformatics. 2The expression level of Tcfec in HSCs and MPPs was analyzed by q-PCR. The open reading frame (Open reading frame,ORF) of mouse Tcfec gene was constructed into the overexpression vector. Flow-sorting positive cells (Lin- cells of fetal liver of mice infected with virus vector) were transplanted into recipient mice treated with lethal dose irradiation to construct an animal model of transplantation in vivo. Flow cytometry (FCM) was used to detect the positive rate of transplanted cells and the effect on hematopoietic lineage differentiation of bone marrow. 6. Flow cytometry was used to detect the effect of Tcfec on HSPC cell cycle. To study the effect of Tcfec on clone formation in vitro. Results: 1 the expression of Tcfec in HSCs was higher than that in MPPs. 2. The overexpression vector of Tcfec was successfully constructed. After 16 weeks of transplantation, the percentage of positive cells in Tcfec group was dominant, and the differentiation of blood cell lineages was completely normal. The total number of HSCs and MPPs cell banks overexpression of Tcfec was increased, but there was no effect on CMP,GMP,MEP progenitor cell bank. 5 Tcfec had no effect on the cell cycle of HSPC. Tcfec functions by relying on HSCs itself. Conclusion: 1. This experiment preliminarily revealed that Tcfec promoted HScs implantation without affecting the normal physiological function of HSCs. 2. The second transplant experiment proved that HSCs expressing Tcfec could maintain self-renewal and multi-differentiation ability. In this study, Tcfec was identified as an important functional molecule regulating the implantation ability of HSCs.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R457.7

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1 閆洪敏;劉靜;薛梅;王志東;朱玲;丁麗;王恒湘;;造血干細(xì)胞移植與非移植治療重型再生障礙性貧血的比較[J];中國組織工程研究與臨床康復(fù);2011年01期

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