Ddx5和Ddx17对干细胞及重编程的作用机制研究

发布时间：2018-07-05 11:53

  本文选题：ES + 重编程　； 参考：《大连医科大学》2017年硕士论文

【摘要】：实验目的:1.研究Ddx5和Ddx17在胚胎干细胞(Embryonic stem cell,ES cell)、诱导多能干细胞(induced pluripotent stem cell,i PS cell)中的作用。2.探究Ddx5和Ddx17在干细胞及重编程中对下游基因的选择性剪接作用。实验方法:为了研究Ddx5和Ddx17在ES及i PS中的作用,首先检测了二者在ES、MEF(mouse embryonic fibroblasts,小鼠胚胎成纤维细胞)中的表达,及构建了带MKO红色荧光的真核表达载体。用此载体建立受Dox(强力霉素)控制的Tet-on调控系统的过表达ES稳转细胞株,并利用PCR、q PCR技术在基因组水平和RNA水平进行检测,同时构建了敲低的载体及用此载体建立了敲低的ES稳转细胞株。利用q PCR方法检测在ES中过表达和敲低Ddx5以及过表达Ddx17对多能性基因表达的影响。另外,用q PCR检测在无LIF(Leukemia Inhibitory Factor,白血病抑制因子)培养的ES分化过程中,Ddx5和Ddx17的基因表达变化。及在过表达、敲低Ddx5和过表达Ddx17,在无LIF培养条件下进行ES分化,检测对Nanog、Oct4等多能性基因及与胚胎发育相关的三个胚层的基因Fgf5、T、Gata6的表达变化。利用构建成功的二次重编程系统小鼠制备MEF。利用MEF细胞在含有Dox的ES培养基中诱导重编程,并用q PCR技术检测在重编程过程中Ddx5和Ddx17的基因表达变化及用流式细胞术和碱性磷酸酶染色检测它们对重编程效率的影响。由于Ddx5、Ddx17的这些不同作用,之后研究了Ddx5和Ddx17对下游基因在干细胞和重编程中的选择性剪接作用。在已发表的文献数据库中查找了大量相关基因,设计引物,收取2天的样品,提RNA进行RT-PCR检测,分析二者对候选基因的选择性剪接变化。对有明显选择性变化的基因进行下一步研究。针对有选择性剪接变化的基因各自的亚型,分别构建真核表达载体,检测其在ES、MEF中的表达及重编程过程中的基因表达变化。研究不同亚型在重编程过程中有什么作用。实验结果:1、真核表达载体及ES稳转细胞株构建成功。1)Ddx5过表达、敲低的真核表达载体及相应的ES稳转细胞株构建成功。2)Ddx17过表达的真核表达载体及ES稳转细胞株构建成功。2、Ddx5和Ddx17在ES中发挥着重要作用。1)与MEF细胞相比,Ddx5在ES中表达高,Ddx17在ES中表达较低。2)过表达Ddx5后,多能性基因表达上调;敲低则相反。过表达Ddx17后,多能性基因表达上调。3)在无LIF培养的ES分化过程中,Ddx5和Ddx17的表达下降,呈递减模式。3、Ddx5和Ddx17在重编程中发挥着重要作用。1)在重编程过程中Ddx5、Ddx17的表达明显上升,呈递增模式。2)过表达Ddx5后可促进重编程效率,敲低则明显降低。3)过表达Ddx17后可降低重编程效率。4、在ES中,过表达Ddx5后对Hras、CD44等有选择性剪接作用;在重编程中对H-ras等有选择性剪接作用。5、在ES、重编程中Ddx5和Ddx17对Hras的选择性剪接作用不同。1)在ES及重编程中发现,过表达Ddx5对Hras有选择性剪接作用,可使亚型2的表达上调,亚型1的表达下调。而Ddx17对Hras无选择性剪接作用。2)在普通MEF(129-rt TA-MEF)中,Ddx5和Ddx17对Hras的选择性剪接变化不明显。6、Hras亚型1的真核表达载体构建成功;Hras总体RNA水平在ES、MEF中的表达变化差异不大;在重编程过程中亚型1的表达逐渐明显上升,呈递增模式。并且Hras总体RNA水平在重编程过程中表达上调。实验结论:Ddx5和Ddx17在ES和重编程过程中都发挥着作用,但是有所不同,它们对下游基因Hras的选择性剪接作用不同。
[Abstract]:Objective: 1. to study the role of Ddx5 and Ddx17 in embryonic stem cells (Embryonic stem cell, ES cell) and the role of inducible pluripotent stem cells (induced pluripotent stem cell, I). The expression of two people in ES, MEF (mouse embryonic fibroblasts, mouse embryonic fibroblasts) and the construction of a eukaryotic expression vector with MKO red fluorescence were first detected, and a ES stabilized cell line expressed by the Tet-on regulatory system controlled by Dox (doxycycline) was established by using this vector, and PCR, Q PCR technology was used at the genome level and in the genome level. At the same time, the level of RNA was detected, and a low knockout carrier was constructed and a low ES stable cell line was set up with this carrier. The effects of overexpression and knockout Ddx5 and over expressed Ddx17 on the expression of pluripotent genes in ES were detected by Q PCR. In addition, Q PCR detection was used in the culture of non LIF (Leukemia Inhibitory), leukemia inhibitory factor. In the process of ES differentiation, the gene expression changes of Ddx5 and Ddx17, and overexpression, low Ddx5 and overexpression of Ddx17, ES differentiation under no LIF culture conditions, detection of Nanog, Oct4 and other genes and gene Fgf5, T, Gata6, three genes related to embryonic development. Using the constructed two reprogramming system mouse system MEF. used MEF cells to induce reprogramming in ES medium containing Dox, and detected the changes in gene expression of Ddx5 and Ddx17 during reprogramming with Q PCR technology and the effect of their effects on reprogramming efficiency by flow cytometry and alkaline phosphatase staining. The difference between Ddx5, Ddx17 and Ddx5 and Ddx17 pairs was then studied. The selective splicing of the downstream genes in the stem cells and reprogramming. In the published literature database, a large number of related genes were found, the primers were designed, the samples were collected for 2 days, the RT-PCR detection was carried out by RNA, and the selective splicing of the candidate genes was analyzed. The next step in the study of genes with obvious selective changes was carried out. The subtypes of genes that have selective splicing changes, construct eukaryotic expression vectors, detect their expression in ES, MEF, and change the gene expression during reprogramming. What is the role of different subtypes in the reprogramming process. Experimental results: 1, eukaryotic expression vector and ES stable.1) Ddx5 overexpression and low knockout Eukaryotic expression vector and corresponding ES stable cell line were constructed successfully.2) Ddx17 overexpressed eukaryotic expression vector and ES stable cell line were constructed successfully.2, Ddx5 and Ddx17 played an important role in ES. Compared with MEF cells, Ddx5 was expressed in ES. On the contrary, after overexpression of Ddx17, the expression of pluripotent gene expression was up regulation of.3) in the process of ES differentiation without LIF culture, the expression of Ddx5 and Ddx17 decreased, Ddx5 and Ddx17 played an important role in reprogramming.3, Ddx5 and Ddx17 played an important role in the reprogramming process. After overexpression of Ddx17, the reprogramming efficiency.4 can be reduced after overexpression of.3). In ES, Hras, CD44, etc. have selective splicing after overexpressing Ddx5; in reprogramming, there is selective splicing on H-ras and so on. In ES, and in reprogramming, the selective shearing action of Ddx5 and Ddx17 is different. The selective splicing of Hras can increase the expression of subtype 2 and reduce the expression of subtype 1. While Ddx17 has no selective splicing of Hras.2) in ordinary MEF (129-rt TA-MEF), the selective splicing changes of Ddx5 and Ddx17 to Hras are not obvious.6, and the eukaryotic expression body of Hras subtype 1 is constructed successfully. There was little difference in the change; the expression of Central Asian type 1 in the reprogramming process was gradually increased and increased. And the overall RNA level of Hras was up-regulated during reprogramming. Experimental conclusions: Ddx5 and Ddx17 played a role in ES and reprogramming, but they were different in the selective splicing of the downstream gene Hras.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

【参考文献】

相关期刊论文 前1条

1 Kenian Chen;Xiaojing Dai;Jiaqian Wu;;Alternative splicing: An important mechanism in stem cell biology[J];World Journal of Stem Cells;2015年01期

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本文编号：2100123