本文選題：ES + 重編程��； 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：實驗?zāi)康?1.研究Ddx5和Ddx17在胚胎干細胞(Embryonic stem cell,ES cell)、誘導(dǎo)多能干細胞(induced pluripotent stem cell,i PS cell)中的作用。2.探究Ddx5和Ddx17在干細胞及重編程中對下游基因的選擇性剪接作用。實驗方法:為了研究Ddx5和Ddx17在ES及i PS中的作用,首先檢測了二者在ES、MEF(mouse embryonic fibroblasts,小鼠胚胎成纖維細胞)中的表達,及構(gòu)建了帶MKO紅色熒光的真核表達載體。用此載體建立受Dox(強力霉素)控制的Tet-on調(diào)控系統(tǒng)的過表達ES穩(wěn)轉(zhuǎn)細胞株,并利用PCR、q PCR技術(shù)在基因組水平和RNA水平進行檢測,同時構(gòu)建了敲低的載體及用此載體建立了敲低的ES穩(wěn)轉(zhuǎn)細胞株。利用q PCR方法檢測在ES中過表達和敲低Ddx5以及過表達Ddx17對多能性基因表達的影響。另外,用q PCR檢測在無LIF(Leukemia Inhibitory Factor,白血病抑制因子)培養(yǎng)的ES分化過程中,Ddx5和Ddx17的基因表達變化。及在過表達、敲低Ddx5和過表達Ddx17,在無LIF培養(yǎng)條件下進行ES分化,檢測對Nanog、Oct4等多能性基因及與胚胎發(fā)育相關(guān)的三個胚層的基因Fgf5、T、Gata6的表達變化。利用構(gòu)建成功的二次重編程系統(tǒng)小鼠制備MEF。利用MEF細胞在含有Dox的ES培養(yǎng)基中誘導(dǎo)重編程,并用q PCR技術(shù)檢測在重編程過程中Ddx5和Ddx17的基因表達變化及用流式細胞術(shù)和堿性磷酸酶染色檢測它們對重編程效率的影響。由于Ddx5、Ddx17的這些不同作用,之后研究了Ddx5和Ddx17對下游基因在干細胞和重編程中的選擇性剪接作用。在已發(fā)表的文獻數(shù)據(jù)庫中查找了大量相關(guān)基因,設(shè)計引物,收取2天的樣品,提RNA進行RT-PCR檢測,分析二者對候選基因的選擇性剪接變化。對有明顯選擇性變化的基因進行下一步研究。針對有選擇性剪接變化的基因各自的亞型,分別構(gòu)建真核表達載體,檢測其在ES、MEF中的表達及重編程過程中的基因表達變化。研究不同亞型在重編程過程中有什么作用。實驗結(jié)果:1、真核表達載體及ES穩(wěn)轉(zhuǎn)細胞株構(gòu)建成功。1)Ddx5過表達、敲低的真核表達載體及相應(yīng)的ES穩(wěn)轉(zhuǎn)細胞株構(gòu)建成功。2)Ddx17過表達的真核表達載體及ES穩(wěn)轉(zhuǎn)細胞株構(gòu)建成功。2、Ddx5和Ddx17在ES中發(fā)揮著重要作用。1)與MEF細胞相比,Ddx5在ES中表達高,Ddx17在ES中表達較低。2)過表達Ddx5后,多能性基因表達上調(diào);敲低則相反。過表達Ddx17后,多能性基因表達上調(diào)。3)在無LIF培養(yǎng)的ES分化過程中,Ddx5和Ddx17的表達下降,呈遞減模式。3、Ddx5和Ddx17在重編程中發(fā)揮著重要作用。1)在重編程過程中Ddx5、Ddx17的表達明顯上升,呈遞增模式。2)過表達Ddx5后可促進重編程效率,敲低則明顯降低。3)過表達Ddx17后可降低重編程效率。4、在ES中,過表達Ddx5后對Hras、CD44等有選擇性剪接作用;在重編程中對H-ras等有選擇性剪接作用。5、在ES、重編程中Ddx5和Ddx17對Hras的選擇性剪接作用不同。1)在ES及重編程中發(fā)現(xiàn),過表達Ddx5對Hras有選擇性剪接作用,可使亞型2的表達上調(diào),亞型1的表達下調(diào)。而Ddx17對Hras無選擇性剪接作用。2)在普通MEF(129-rt TA-MEF)中,Ddx5和Ddx17對Hras的選擇性剪接變化不明顯。6、Hras亞型1的真核表達載體構(gòu)建成功;Hras總體RNA水平在ES、MEF中的表達變化差異不大;在重編程過程中亞型1的表達逐漸明顯上升,呈遞增模式。并且Hras總體RNA水平在重編程過程中表達上調(diào)。實驗結(jié)論:Ddx5和Ddx17在ES和重編程過程中都發(fā)揮著作用,但是有所不同,它們對下游基因Hras的選擇性剪接作用不同。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78

【參考文獻】

相關(guān)期刊論文 前1條

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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本文編號：2100123