论文总字数：23136字

摘 要

根据2015 年中国癌症报告显示：中国新发癌症病例约占全球的22%，中国癌症死亡比例约占全球的27%。因此，抗肿瘤药物的作用机制研究和新的作用靶点的研究十分重要。在真核细胞中，泛素蛋白酶体系统（UPS）负责蛋白质的降解，并在维持细胞内蛋白质动态平衡中担当重要角色。UPS的底物蛋白参与诸如细胞周期调节、细胞生长及增殖、信号传导、DNA修复及细胞凋亡信号传导过程。实验发现，在某些肿瘤细胞中UPS信号通路被上调，而抑制UPS后导致该细胞凋亡。

本课题重点研究先导化合物M22的抗肿瘤作用机制。将化合物M22和肿瘤细胞A549共温孵，48h后裂解细胞；提取蛋白；用试剂盒进行蛋白定量；采用western blot技术，与一抗（anti-NEDD8抗体，anti-Ubc12抗体，anti-Ubc10等抗体)温孵，孵二抗并涂显色液后读取灰度值。通过条带的变化研究先导化合物对肿瘤细胞内NEDD8通路的影响。研究证明M22 为ATP 竞争性NAE 抑制剂，能选择性抑制细胞内NAE 活性，下调neddylation，抑制CRLs 介导的底物蛋白降解，抑制肿瘤细胞增殖，并诱导细胞凋亡。M22 与硼替佐米联合用药对抑制肿瘤细胞增值具有协同作用。

关键词：M22 抗肿瘤作用机制 NAE抑制剂 泛素蛋白酶系统

Study on the mechanism of antitumor drugs

targeting the ubiquitin pathway

Abstract

According to the 2015 China cancer report: China's new cancer cases accounted for about 22% of the world, the proportion of cancer deaths in China accounted for about of the world's 27%.Over the years, the mortality of all diseases caused by malignant tumors accounted for the first one.Therefore, it is very important to study the action mechanism of anti tumor drugs and the new targets.In eukaryotic cells, the ubiquitin proteasome system (UPS) is responsible for the degradation of proteins and plays an important role in maintaining the homeostasis of intracellular proteins.It was found that the UPS signaling pathway was up-regulated in some tumor cells, and the apoptosis was induced by the inhibition of UPS.

This topic focuses on the anti-tumor mechanism of the lead compound M22.M22 and tumor cells were incubated with A549 co incubation, 48h after cleavage cell; extracted protein; the protein was quantified by kit;Using blot Western technology, and an anti (anti-NEDD8 antibody, anti-Ubc12 antibody, anti-Ubc10 and other antibodies) incubation, incubation two anti and coated with the color of the color after reading the gray value.The effects of the lead compounds on the NEDD8 pathway of tumor cells were studied by the changes of bands.Studies have shown that M22 is a competitive NAE inhibitor of ATP, can selectively inhibit the intracellular NAE activity, down regulate neddylation, inhibit the degradation of CRLs mediated substrate protein, inhibit tumor cell proliferation, and induce cell apoptosis.M22 and bortezomib combined with drugs has a synergistic effect on the proliferation of tumor cells.

Key words:M22;anti tumor action mechanism;NAE inhibitor;ubiquitin proteasome system

目 录

摘要 II

ABSTRACT II

第一章 研究背景 1

1.1、发现 1

1.2、泛素—蛋白酶体通路 1

1.2.1、泛素-蛋白酶体途径 1

1.2.2、泛素-蛋白酶体途径的功能 3

1.3、NEDD8 通路 3

1.4、Western Blot 7

第二章 M22 对离体NAE的抑制活性研究 8

2.1、实验材料 8

2.1.1、化合物 8

2.1.2、主要试剂 8

2.1.3、主要抗体 8

2.1.4、主要仪器 9

2.1.5、主要试剂配置 9

2.1.6、药物溶液配置 10

2.2、实验步骤 10

2.2.1、M22 对离体NAE 的抑制活性（Cell-free）.....................................10

2.2.2、M22 的ATP竞争性NAE抑制实验（Cell-free） 10

2.3、实验结果与讨论 11

第三章 M22细胞内作用机制研究 13

3.1、实验材料 13

3.1.1、细胞系 13

3.1.2、主要试剂 13

3.1.3、主要抗体 14

3.1.4、主要仪器 14

3.1.5、主要试剂配置 15

3.2、实验步骤 15

3.2.1、蛋白提取及蛋白样品制备 15

3.2.2、western 检测细胞内蛋白含量 16

3.3、实验结论 17

第四章 总结与展望 20

4.1、总结 20

4.2、展望 21

参考文献 22

致谢 25

第一章 研究背景

1.1、发现

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