定向蛋白质组学对乳腺癌中p53-MDM2相互作用的定量研究

发布时间：2018-03-03 07:29

本文选题：p53蛋白　切入点：MDM2蛋白　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：在乳腺癌的发展进程中,p53蛋白的抑癌功能可能会受与其相互作用蛋白的影响而减弱。在这些相互作用的蛋白中,MDM2作为一个重要的负性调节蛋白能够抑制p53的转录激活。因此,准确定量并监测在疾病状态下p53-MDM2相互作用的改变情况,能够进一步理解蛋白质与蛋白质相互作用(protein-protein interaction,PPI),同时也能为深入研究癌症的发生发展、开发更先进的治疗手段提供参考依据。然而,近年来仅有少部分研究者从事了包括P53-MDM2相互作用在内的关于PPI的定量研究。另外,目前的PPI的研究方法中常常很难同时具有高灵敏度与高特异性的优点。因此,迫切需要发展一种同时具备高灵敏度(低假阴性率)和高特异性(低假阳性率)的PPI定量检测方法。近年来,以液相色谱-串联质谱(LC-MS/MS)为基础的定向蛋白质组学技术凭借其高灵敏度、高选择性以及较宽的动态范围等优点,被广泛应用于分子生物的研究领域。本研究中利用以LC-MS/MS为基础的定向蛋白质组学方法与免疫共沉淀(Co-IP)技术相结合,发展了一种全新的用于P53-MDM2相互作用的定量检测方法。实验结果表明,一种抗原表位在156-214位残基的p53抗体免疫沉淀效果最好。接下来,分别选择了 p53蛋白第321-333位氨基酸序列的多肽KPLDGEYFTLQIR以及MDM2蛋白第327-334位氨基酸序列的多肽ENWLPEDK,作为p53和MDM2在定量分析中的特征性肽段,以稳定同位素标记的合成肽段作为内标,实验得到的定量限为2ng/mL。另外,对乳腺癌细胞样品(MCF-7,MCF-10A)及组织样品(乳腺癌组织,乳腺癌旁组织)中p53蛋白、MDM2蛋白以及p53-MDM2蛋白复合物进行了定量检测,并与蛋白免疫印迹(western blotting)技术得到的结果进行比较。最后,选用了nutlin-3作为PPI的抑制剂,定量检测了在给予nutlin-3抑制剂后,不同时间下MCF-7细胞中p53-MDM2相互作用的变化情况。
[Abstract]:In the development of breast cancer, the suppressor function of p53 protein may be weakened by interacting proteins. MDM2, as an important negative regulatory protein, can inhibit the transcriptional activation of p53. Accurately quantifying and monitoring the changes of p53-MDM2 interaction in disease state can further understand protein-protein interaction in protein-protein interaction, and can also be used to further study the occurrence and development of cancer. However, in recent years, only a small number of researchers have done quantitative research on PPI, including the P53-MDM2 interaction. The current research methods of PPI often have the advantages of high sensitivity and high specificity. It is urgent to develop a high sensitivity (low false negative rate) and high specificity (low false positive rate) quantitative detection method for PPI. The directional proteomics technology based on LC-MS / MS has the advantages of high sensitivity, high selectivity and wide dynamic range. It is widely used in the field of molecular biology. In this study, we combined the LC-MS/MS based directional proteomics with the co-immunoprecipitation co-IP technique. A new method for quantitative detection of P53-MDM2 interaction has been developed. The results show that the immunoprecipitation of p53 antibody with an antigen epitope at the 156-214 residues is the best. The peptide KPLDGEYFTLQIR of the 321-333 amino acid sequence of p53 protein and ENWLPEDK of the 327-334 amino acid sequence of MDM2 protein were selected as the characteristic peptides of p53 and MDM2 in quantitative analysis, and the stable isotope labeled synthetic peptide was used as the internal standard. The quantitative limit was 2ng / mL. In addition, p53 protein / MDM2 protein and p53-MDM2 protein complex in breast cancer cell samples (MCF-7 / MCF-10A) and tissue samples (breast cancer tissues and adjacent breast cancer tissues) were quantitatively detected. The results obtained by Western blotting were compared with those obtained by Western blotting. Finally, nutlin-3 was selected as the inhibitor of PPI to quantitatively detect the changes of p53-MDM2 interaction in MCF-7 cells at different time after nutlin-3 inhibitor was given.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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