本文選題：誘導多能干細胞iPSC + p53基因�。� 參考：《西南醫(yī)科大學》2017年碩士論文

【摘要】：目的:研究攜帶有轉錄因子(Oct4、Sox2、Klf4、c-Myc)的腺病毒感染小鼠胚胎成纖維細胞(MEFc),通過誘導出誘導多能干細胞(iPSC),并分析在重編程過程中p53基因、MDM2基因、Aurka基因的變化規(guī)律,為提高誘導多能干細胞誘導效率提供思路。方法:1.對前期凍存的MEFc進行復蘇,待MEFc生長良好后,加入帶有4個轉錄因子(OSCK)的腺病毒,反復感染兩次后,觀察細胞形態(tài)結構變化;2.對誘導出的細胞進行多能性鑒定:通過對細胞形態(tài)學觀察、成瘤性、堿性磷酸酶(偶氮偶聯(lián)法)染色,及干細胞標志基因檢測;3.將轉染的細胞分別按照誘導前、誘導后24h、誘導后1w、誘導后2w四個時間段,利用Real-Time PCR技術,多次測量p53基因、MDM2基因、Aurka基因的轉錄情況。實驗數據應用SPSS17.0軟件進行統(tǒng)計學分析,各個基因內各個時間的數據采用單因素方差分析,統(tǒng)計學檢驗標準為P0.05。結果:1.對誘導的細胞進行多能性鑒定:a、形態(tài)學變化:可觀察到細胞邊界輪廓清楚,胞體較小鼠成纖維細胞變大,變圓,長度變短,少數呈多變性,仍有明顯突起,但突起長度變短。b、成瘤性觀察:約7-8周后可見到大鼠根部明顯2-3個畸胎瘤,用手觸及包塊質中等硬度,可滑動,與周圍組織無粘連,表面光滑。取出包塊組織,可見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,可見細胞核成藍色,胞質成粉紅色,在中可見到大量杯狀細胞組成的腺體組織、血管、大量的脂肪組織。c、堿性磷酸酶鑒定:可見大量堿性磷酸酶陽性顆粒。d、干細胞標志基因檢測:Oct4、Sox2及Nanog在誘導的細胞中均有表達。2.在誘導過程中p53轉錄變化規(guī)律:1、轉染前與轉染后24h、轉染后1w、轉染后2w比較,相對表達量差異顯著(P0.05)。2、轉染后24h與轉染后1w及轉染后兩周比較,差異顯著,具有統(tǒng)計學意義(P0.05)。3.轉染后1w與轉染后兩周比較,差異顯著,具有統(tǒng)計學意義(P0.05)。p53的轉錄在四因子腺病毒轉染后24h既出現升高,在轉染1w后較高,而在轉染2w后出現下降。在誘導過程中MDM2轉錄變化規(guī)律:1、轉染前與轉染后24h、轉染后1w、轉染后2w比較,RQ差異顯著,具有統(tǒng)計學意義(P0.05)。2、轉染后24h與轉染后1w及轉染后兩周比較,差異顯著,具有統(tǒng)計學意義(P0.05)。3、轉染后1w與轉染后兩周比較,差異顯著,具有統(tǒng)計學意義(P0.05)。MDM2的轉錄在四因子腺病毒轉染后24h出現下降,1w時下降明顯,而在轉染2w后卻出現升高。與p53基因的變化規(guī)律成反比。在誘導過程中AURKA轉錄變化規(guī)律:1、轉染前與轉染后24h、轉染后1w,RQ差異顯著,具有統(tǒng)計學意義(P0.05),與轉染后2w比較,RQ差異不顯著(P0.05)。2、轉染后24h與轉染后1w及轉染后2w比較,差異顯著,具有統(tǒng)計學意義(P0.05)。3、轉染后1w與轉染后2w比較,差異顯著,具有統(tǒng)計學意義(P0.05)�？烧J為Aurka的轉錄在四因子腺病毒轉染后出現明顯升高,1w時較轉染后仍高,而在轉染2w呈現與正常水平,且Aurka的轉錄升高水平明顯高于p53基因。通過利用MDM2與Aurka的抑制劑發(fā)現細胞p53蛋白表達均明顯升高。結論1.細胞的重編程過程能夠激活p53信號通路,p53轉錄在轉染2w后出現下降,考慮重編程已經完成,可考慮干預重編程過程的時間在2w內。2.細胞重編程過程中MDM2水平下降,通過利用MDM2拮抗劑在誘導多能干細胞中發(fā)現p53蛋白表達明顯增加,可以通過對MDM2-p53信號通路的作用,影響誘導效率。3.細胞重編程中Aurka的表達水平上調,通過利用VX-680抑制Aurka能夠使p53蛋白表達明顯增加,同時也能抑制p53傳導通路,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.

【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R329.2

【參考文獻】

相關期刊論文 前2條

1 Wen-biao CHEN;Jian-rong HUANG;Xiang-qi YU;Xiao-cong LIN;Yong DAI;;運用高通量測序研究Alport綜合征多能干細胞的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,林晨,黃志強,馬慶久,付明,張雪艷,梁蕭,要秀,吳e，

本文編號：1826232