本文選題：誘導(dǎo)多能干細(xì)胞iPSC + p53基因��； 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:研究攜帶有轉(zhuǎn)錄因子(Oct4、Sox2、Klf4、c-Myc)的腺病毒感染小鼠胚胎成纖維細(xì)胞(MEFc),通過(guò)誘導(dǎo)出誘導(dǎo)多能干細(xì)胞(iPSC),并分析在重編程過(guò)程中p53基因、MDM2基因、Aurka基因的變化規(guī)律,為提高誘導(dǎo)多能干細(xì)胞誘導(dǎo)效率提供思路。方法:1.對(duì)前期凍存的MEFc進(jìn)行復(fù)蘇,待MEFc生長(zhǎng)良好后,加入帶有4個(gè)轉(zhuǎn)錄因子(OSCK)的腺病毒,反復(fù)感染兩次后,觀察細(xì)胞形態(tài)結(jié)構(gòu)變化;2.對(duì)誘導(dǎo)出的細(xì)胞進(jìn)行多能性鑒定:通過(guò)對(duì)細(xì)胞形態(tài)學(xué)觀察、成瘤性、堿性磷酸酶(偶氮偶聯(lián)法)染色,及干細(xì)胞標(biāo)志基因檢測(cè);3.將轉(zhuǎn)染的細(xì)胞分別按照誘導(dǎo)前、誘導(dǎo)后24h、誘導(dǎo)后1w、誘導(dǎo)后2w四個(gè)時(shí)間段,利用Real-Time PCR技術(shù),多次測(cè)量p53基因、MDM2基因、Aurka基因的轉(zhuǎn)錄情況。實(shí)驗(yàn)數(shù)據(jù)應(yīng)用SPSS17.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,各個(gè)基因內(nèi)各個(gè)時(shí)間的數(shù)據(jù)采用單因素方差分析,統(tǒng)計(jì)學(xué)檢驗(yàn)標(biāo)準(zhǔn)為P0.05。結(jié)果:1.對(duì)誘導(dǎo)的細(xì)胞進(jìn)行多能性鑒定:a、形態(tài)學(xué)變化:可觀察到細(xì)胞邊界輪廓清楚,胞體較小鼠成纖維細(xì)胞變大,變圓,長(zhǎng)度變短,少數(shù)呈多變性,仍有明顯突起,但突起長(zhǎng)度變短。b、成瘤性觀察:約7-8周后可見(jiàn)到大鼠根部明顯2-3個(gè)畸胎瘤,用手觸及包塊質(zhì)中等硬度,可滑動(dòng),與周?chē)M織無(wú)粘連,表面光滑。取出包塊組織,可見(jiàn)到包塊淡紅色塊橢圓形柱狀物,大小為約1cm×0.5cm×0.3cm。將取下的組織行切片、HE染色處理,可見(jiàn)細(xì)胞核成藍(lán)色,胞質(zhì)成粉紅色,在中可見(jiàn)到大量杯狀細(xì)胞組成的腺體組織、血管、大量的脂肪組織。c、堿性磷酸酶鑒定:可見(jiàn)大量堿性磷酸酶陽(yáng)性顆粒。d、干細(xì)胞標(biāo)志基因檢測(cè):Oct4、Sox2及Nanog在誘導(dǎo)的細(xì)胞中均有表達(dá)。2.在誘導(dǎo)過(guò)程中p53轉(zhuǎn)錄變化規(guī)律:1、轉(zhuǎn)染前與轉(zhuǎn)染后24h、轉(zhuǎn)染后1w、轉(zhuǎn)染后2w比較,相對(duì)表達(dá)量差異顯著(P0.05)。2、轉(zhuǎn)染后24h與轉(zhuǎn)染后1w及轉(zhuǎn)染后兩周比較,差異顯著,具有統(tǒng)計(jì)學(xué)意義(P0.05)。3.轉(zhuǎn)染后1w與轉(zhuǎn)染后兩周比較,差異顯著,具有統(tǒng)計(jì)學(xué)意義(P0.05)。p53的轉(zhuǎn)錄在四因子腺病毒轉(zhuǎn)染后24h既出現(xiàn)升高,在轉(zhuǎn)染1w后較高,而在轉(zhuǎn)染2w后出現(xiàn)下降。在誘導(dǎo)過(guò)程中MDM2轉(zhuǎn)錄變化規(guī)律:1、轉(zhuǎn)染前與轉(zhuǎn)染后24h、轉(zhuǎn)染后1w、轉(zhuǎn)染后2w比較,RQ差異顯著,具有統(tǒng)計(jì)學(xué)意義(P0.05)。2、轉(zhuǎn)染后24h與轉(zhuǎn)染后1w及轉(zhuǎn)染后兩周比較,差異顯著,具有統(tǒng)計(jì)學(xué)意義(P0.05)。3、轉(zhuǎn)染后1w與轉(zhuǎn)染后兩周比較,差異顯著,具有統(tǒng)計(jì)學(xué)意義(P0.05)。MDM2的轉(zhuǎn)錄在四因子腺病毒轉(zhuǎn)染后24h出現(xiàn)下降,1w時(shí)下降明顯,而在轉(zhuǎn)染2w后卻出現(xiàn)升高。與p53基因的變化規(guī)律成反比。在誘導(dǎo)過(guò)程中AURKA轉(zhuǎn)錄變化規(guī)律:1、轉(zhuǎn)染前與轉(zhuǎn)染后24h、轉(zhuǎn)染后1w,RQ差異顯著,具有統(tǒng)計(jì)學(xué)意義(P0.05),與轉(zhuǎn)染后2w比較,RQ差異不顯著(P0.05)。2、轉(zhuǎn)染后24h與轉(zhuǎn)染后1w及轉(zhuǎn)染后2w比較,差異顯著,具有統(tǒng)計(jì)學(xué)意義(P0.05)。3、轉(zhuǎn)染后1w與轉(zhuǎn)染后2w比較,差異顯著,具有統(tǒng)計(jì)學(xué)意義(P0.05)。可認(rèn)為Aurka的轉(zhuǎn)錄在四因子腺病毒轉(zhuǎn)染后出現(xiàn)明顯升高,1w時(shí)較轉(zhuǎn)染后仍高,而在轉(zhuǎn)染2w呈現(xiàn)與正常水平,且Aurka的轉(zhuǎn)錄升高水平明顯高于p53基因。通過(guò)利用MDM2與Aurka的抑制劑發(fā)現(xiàn)細(xì)胞p53蛋白表達(dá)均明顯升高。結(jié)論1.細(xì)胞的重編程過(guò)程能夠激活p53信號(hào)通路,p53轉(zhuǎn)錄在轉(zhuǎn)染2w后出現(xiàn)下降,考慮重編程已經(jīng)完成,可考慮干預(yù)重編程過(guò)程的時(shí)間在2w內(nèi)。2.細(xì)胞重編程過(guò)程中MDM2水平下降,通過(guò)利用MDM2拮抗劑在誘導(dǎo)多能干細(xì)胞中發(fā)現(xiàn)p53蛋白表達(dá)明顯增加,可以通過(guò)對(duì)MDM2-p53信號(hào)通路的作用,影響誘導(dǎo)效率。3.細(xì)胞重編程中Aurka的表達(dá)水平上調(diào),通過(guò)利用VX-680抑制Aurka能夠使p53蛋白表達(dá)明顯增加,同時(shí)也能抑制p53傳導(dǎo)通路,減少P53引起的抑制作用。
[Abstract]:Objective: To study the adenovirus infected mouse embryonic fibroblasts (MEFc) carrying Oct4 (Sox2, Klf4, c-Myc), and to induce induced pluripotent stem cells (iPSC) by inducing the induced pluripotent stem cells (iPSC), and to analyze the variation rules of the p53, MDM2, and Aurka genes in the reprogramming process, and provide a way to improve the induction of induction of pluripotent stem cells. Method: 1. pairs MEFc was resuscitated during the period of frozen storage. After good growth of MEFc, adenovirus with 4 transcription factors (OSCK) was added to the cell. After repeated infection for two times, the morphological changes of cells were observed. 2. of the induced cells were identified: by morphological observation, tumorigenicity, alkaline phosphatase (azo coupling) staining, and stem cell markers Due to detection, 3. the transfected cells were induced, after induction, after induction, after induction of 24h, after induction of 1W, and after induction of 2W four time periods, using Real-Time PCR technology to measure the p53 gene, MDM2 gene and Aurka gene multiple times. The experimental data were statistically analyzed with SPSS17.0 software, and the data of each time in each gene were single factor. Difference analysis, the statistical test standard was P0.05. results: 1. the pluripotent identification of induced cells: A, morphological changes: the cell boundary contour is clear, the cell body is larger than the mouse fibroblast, the circle, the length becomes shorter, the few are variable, but the protuberance length becomes shorter.B, the tumor observation: about 7-8 weeks later visible. There were 2-3 teratoma at the root of the rat. It could be slid with medium hardness of the mass, slipping, without adhesion to the surrounding tissue, and smooth surface. Taking out the tissue of the package, we could see the oval shaped columnar with a light red block. The size of the tissue was about 1cm * 0.5cm x 0.3cm., the tissues were sliced and HE stained, so the nucleus was blue and the cytoplasm became pink. Color, we can see a large number of glandular tissue, blood vessels, a large number of adipose tissue.C, alkaline phosphatase identification: a large number of alkaline phosphatase.D, stem cell marker gene detection: Oct4, Sox2 and Nanog expression of.2. in the induction of p53 transcriptional changes in the induction process: 1, before transfection and 2 after transfection 4h, 1W after transfection, the relative expression amount was significantly different (P0.05).2 after transfection. The difference was significant between 24h and 1W after transfection and two weeks after transfection. The difference was statistically significant (P0.05) after.3. transfection, and the difference was significant between 1W and two weeks after transfection. The difference was statistically significant (P0.05) the transcription of.P53 was found not only after the transfection of four factor adenovirus. The increase was higher after transfection of 1W, but decreased after transfection of 2W. In the course of induction, the changes of MDM2 transcriptional changes: 1, 24h before transfection and transfection, 1W after transfection and 2W after transfection, RQ difference was significant (P0.05).2, and the difference was significant between 24h and 1W after transfection and two weeks after transfection, with statistical significance (P0.05), 1W after transfection compared with the two weeks after transfection, the difference was significant. The transcription of.MDM2 was significantly decreased after transfection of four factor adenovirus, 24h decreased obviously, but increased after 1W transfection, but increased after transfection of 2W, it was inversely proportional to the change rule of p53 gene. In the course of induction, the regulation of AURKA transcriptional changes: 1, 24h before transfection and transfection. The difference of 1W, RQ was significant (P0.05). Compared with 2W after transfection, the difference of RQ was not significant (P0.05).2. The difference was significant between 24h and 1W after transfection and 2W after transfection. The difference was statistically significant (P0.05). After transfection of the adenovirus, the 1W was higher than that of the transfection, while the transfection was still higher than that of the transfection, while the transfection of 2W presented with the normal level, and the level of Aurka was significantly higher than that of the p53 gene. The expression of p53 protein in the cells was obviously increased by using the inhibitors of MDM2 and Aurka. Conclusion the reprogramming process of 1. cells can activate the p53 signaling pathway and p53 transcript. After transfection of 2W, the reprogramming has been completed, and the time of the intervention reprogramming process is considered to decrease in the MDM2 level during the reprogramming process of.2. cells in 2W. The expression of p53 protein in the induced pluripotent stem cells is obviously increased by using MDM2 antagonist, which can affect the induction efficiency.3. by the role of the MDM2-p53 signaling pathway. In reprogramming of cell reprogramming, the expression level of Aurka is up. By using VX-680 to inhibit Aurka, the expression of p53 protein can be significantly increased, and it can also inhibit the p53 conduction pathway and reduce the inhibitory effect caused by P53.

【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R329.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Wen-biao CHEN;Jian-rong HUANG;Xiang-qi YU;Xiao-cong LIN;Yong DAI;;運(yùn)用高通量測(cè)序研究Alport綜合征多能干細(xì)胞的microRNA及其靶基因(英文)[J];Journal of Zhejiang University-Science B(Biomedicine & Biotechnology);2015年03期

2 魯建國(guó),林晨,黃志強(qiáng),馬慶久,付明,張雪艷,梁蕭,要秀,吳e，

本文編號(hào)：1826232