小鼠胚胎成纤维细胞重编过程中p53、MDM2、Aurka基因相关性的实验研究

发布时间：2018-04-30 21:11

  本文选题：诱导多能干细胞iPSC + p53基因　； 参考：《西南医科大学》2017年硕士论文

【摘要】：目的:研究携带有转录因子(Oct4、Sox2、Klf4、c-Myc)的腺病毒感染小鼠胚胎成纤维细胞(MEFc),通过诱导出诱导多能干细胞(iPSC),并分析在重编程过程中p53基因、MDM2基因、Aurka基因的变化规律,为提高诱导多能干细胞诱导效率提供思路。方法:1.对前期冻存的MEFc进行复苏,待MEFc生长良好后,加入带有4个转录因子(OSCK)的腺病毒,反复感染两次后,观察细胞形态结构变化;2.对诱导出的细胞进行多能性鉴定:通过对细胞形态学观察、成瘤性、碱性磷酸酶(偶氮偶联法)染色,及干细胞标志基因检测;3.将转染的细胞分别按照诱导前、诱导后24h、诱导后1w、诱导后2w四个时间段,利用Real-Time PCR技术,多次测量p53基因、MDM2基因、Aurka基因的转录情况。实验数据应用SPSS17.0软件进行统计学分析,各个基因内各个时间的数据采用单因素方差分析,统计学检验标准为P0.05。结果:1.对诱导的细胞进行多能性鉴定:a、形态学变化:可观察到细胞边界轮廓清楚,胞体较小鼠成纤维细胞变大,变圆,长度变短,少数呈多变性,仍有明显突起,但突起长度变短。b、成瘤性观察:约7-8周后可见到大鼠根部明显2-3个畸胎瘤,用手触及包块质中等硬度,可滑动,与周围组织无粘连,表面光滑。取出包块组织,可见到包块淡红色块椭圆形柱状物,大小为约1cm×0.5cm×0.3cm。将取下的组织行切片、HE染色处理,可见细胞核成蓝色,胞质成粉红色,在中可见到大量杯状细胞组成的腺体组织、血管、大量的脂肪组织。c、碱性磷酸酶鉴定:可见大量碱性磷酸酶阳性颗粒。d、干细胞标志基因检测:Oct4、Sox2及Nanog在诱导的细胞中均有表达。2.在诱导过程中p53转录变化规律:1、转染前与转染后24h、转染后1w、转染后2w比较,相对表达量差异显著(P0.05)。2、转染后24h与转染后1w及转染后两周比较,差异显著,具有统计学意义(P0.05)。3.转染后1w与转染后两周比较,差异显著,具有统计学意义(P0.05)。p53的转录在四因子腺病毒转染后24h既出现升高,在转染1w后较高,而在转染2w后出现下降。在诱导过程中MDM2转录变化规律:1、转染前与转染后24h、转染后1w、转染后2w比较,RQ差异显著,具有统计学意义(P0.05)。2、转染后24h与转染后1w及转染后两周比较,差异显著,具有统计学意义(P0.05)。3、转染后1w与转染后两周比较,差异显著,具有统计学意义(P0.05)。MDM2的转录在四因子腺病毒转染后24h出现下降,1w时下降明显,而在转染2w后却出现升高。与p53基因的变化规律成反比。在诱导过程中AURKA转录变化规律:1、转染前与转染后24h、转染后1w,RQ差异显著,具有统计学意义(P0.05),与转染后2w比较,RQ差异不显著(P0.05)。2、转染后24h与转染后1w及转染后2w比较,差异显著,具有统计学意义(P0.05)。3、转染后1w与转染后2w比较,差异显著,具有统计学意义(P0.05)。可认为Aurka的转录在四因子腺病毒转染后出现明显升高,1w时较转染后仍高,而在转染2w呈现与正常水平,且Aurka的转录升高水平明显高于p53基因。通过利用MDM2与Aurka的抑制剂发现细胞p53蛋白表达均明显升高。结论1.细胞的重编程过程能够激活p53信号通路,p53转录在转染2w后出现下降,考虑重编程已经完成,可考虑干预重编程过程的时间在2w内。2.细胞重编程过程中MDM2水平下降,通过利用MDM2拮抗剂在诱导多能干细胞中发现p53蛋白表达明显增加,可以通过对MDM2-p53信号通路的作用,影响诱导效率。3.细胞重编程中Aurka的表达水平上调,通过利用VX-680抑制Aurka能够使p53蛋白表达明显增加,同时也能抑制p53传导通路,减少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.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

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相关期刊论文 前2条

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

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