过表达Tcfec对造血干细胞的影响研究
发布时间：2018-12-14 03:00
【摘要】：在血液系统中,转录因子Tcfec主要在造血干细胞(Hematopoietic stem cells,HSCs)和多能祖细胞(Multipotent progenitors,MPPs)中高表达。目前为止,关于Tcfec在HSCs的功能影响及机制尚不清楚。目的:1、证实Tcfec在HSCs和MPPs中的表达水平。2、构建Tcfec逆转录病毒过表达体系。3、通过体内、体外实验研究过表达Tcfec对HSCs的功能影响。方法:1、分选高纯度HSCs和MPPs细胞群,MySeq测序仪进行RNA-Seq测序并获得原始测序数据(Raw data)。利用生物信息学技术深入分析,获得了HSCs及MPPs细胞的转录因子表达差异谱。2、q-PCR分析Tcfec在HSCs和MPPs中的表达水平。3、利用逆转录病毒过表达系统,将小鼠Tcfec基因的开放阅读框(Open reading frame,ORF)构建到过表达载体,并用细胞转染、荧光定量PCR方法验证过表达体系的构建。4、将流式分选的阳性细胞(感染过表达病毒载体的小鼠胎肝Lin-细胞)移植到经致死剂量辐照处理的受体鼠,构建体内移植动物模型。5、流式细胞仪检测移植细胞的阳性率和对骨髓造血谱系分化影响的相关检测。6、流式细胞仪检测Tcfec对HSPC细胞周期的影响。7、利用体外克隆形成实验,研究Tcfec对体外克隆形成的影响效果。结果:1、Tcfec在HSCs相对MPPs高表达。2、成功构建Tcfec过表达载体。3、移植16周后,Tcfec组中阳性细胞比例占有优势,血细胞各谱系分化完全正常。4、过表达Tcfec的HSCs及MPPs细胞库总体数量增加,但对CMP、GMP、MEP祖细胞库无影响。5、Tcfec对于HSPC的细胞周期没有影响。6、Tcfec组体外形成的混合细胞型克隆(GEMM)较对照组增多。7、Tcfec发挥功能是依赖HSCs自身。结论:1、本实验初步揭示Tcfec促进HScs植入,同时不影响HSCs的正常生理功能。2、二次移植实验证明过表达Tcfec的HSCs保持自我更新及多向分化能力。3、本研究鉴定了Tcfec是调控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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R457.7
本文编号：2377782
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R457.7
【参考文献】
相关期刊论文 前1条
1 闫洪敏;刘静;薛梅;王志东;朱玲;丁丽;王恒湘;;造血干细胞移植与非移植治疗重型再生障碍性贫血的比较[J];中国组织工程研究与临床康复;2011年01期
，本文编号：2377782
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