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题名 二-(6-羟基-4-氧代-4H-苯并吡喃-3-基)-乙二亚胺在Hela细胞中上调p53的凋亡作用
姓名 蔡体宽
院系 生命科学学院
专业 生物物理
学位名称 理学硕士
外文题名 The antitmor activity of bis(6-hydroxy-4-oxo-4H-chromene-3-yl)- ethylenediimine at Hela cells by upregulated p53 protein
第一导师姓名 王勤
关键词 HeLa;人乳头瘤状病毒;原癌基因;p53;MDM-2;Western blot
外文关键词 HeLa;human papillomavirus(HPV);oncogene;p53;MDM-2;Western blot
学科 理学
摘要 背景:人乳头瘤状病毒(Human Papillomavirus,HPV)感染是宫颈癌的主要病因。HPV编码的E6蛋白与p53蛋白和细胞内的E6相关蛋白(E6 associated protein, E6-AP)结合,经过泛素途径使p53降解,使正常细胞发生癌变,形成恶性肿瘤。p53的细胞内活性作用是维持遗传的稳定性,这主要体现在对细胞周期的阻滞、促进细胞凋亡和DNA修复。MDM2蛋白是细胞内调节p53 浓度及活性的重要蛋白质,两者构成似电流环路中的负反馈,彼此相互进行精细的调节。MDM2 以介导p53 穿过核膜进入胞浆降解和对p53 转录活性的直接抑制作用两种形式实现对p53 功能的调节。黄酮类化合物(flavonoids compounds)是植物次生代谢产物,广泛地存在于自然植物中,结构类型复杂多样,表现出多种多样的药理活性,能防治心脑血管系统的疾病和呼吸系统的疾病,具有抗炎抑菌,降血糖,抗氧化,抗辐射,抗癌,抗肿瘤以及增强免疫能力等药理作用。黄酮类化合物已成为目前新药开发的热点。 方法: 用新合成的黄酮希夫碱二-(6-羟基-4-氧代-4H-苯并吡喃-3-基)-乙二亚胺(bis (6-hydroxy-4-oxo-4H-chromene-3-yl)-ethylenediimine, B6)在不同浓度下对Hela细胞处理不同时间。采用SRB、台盼蓝染色、检测化合物对Hela细胞的抑制作用;在相差显微镜下观察化合物的作用所引起的细胞形态的改变;用AO/EB双染,观察细胞的凋亡形态;琼脂糖凝胶电泳,化合物作用的细胞DNA形成条带;透射电镜观察细胞形成的凋亡小体;流式细胞术检测Hela细胞的凋亡以及细胞周期的阻滞;Western Blot 检测p53、MDM2蛋白表达的变化。 结果: SRB法检测到24h对Hela细胞的IC50为43.51μmol/L,;台盼蓝染色检测到化合物24h对Hela细胞有明显的抑制作用;相差显微镜和AO/EB双染紫外激发荧光显微镜下均观察到细胞质膜分离,细胞产生调亡小体;琼脂糖凝胶电泳紫外激发下可以观察到DNA在180~200bp间形成梯状条带;透射电镜下看到细胞染色质固缩、分离并沿核膜分布,细胞质亦发生固缩,细胞凋亡前体;流式细胞仪检测细胞被阻滞在G1期,细胞发生凋亡,调亡率在0μmol/L、40μmol/L、80μmol/L浓度时分别为0.05% ,6.1%,8.6%;Western Blot检测p53蛋白的量随化合物浓度增加而增加,MDM2蛋白的量随p53蛋白的量增加而增加。以上结果说明该化合物能够诱导Hela细胞发生凋亡,促进p53表达,促使细胞凋亡,是一种具有抗肿瘤活性的化合物。
外文摘要 Background: Substantial epidemiologic and molecular biological data have proved that HPV infection is chief cause of cervical cancer and precancerosis. HPV encodes E6 protein,which can bind p53 protein and E6-AP protein.It can regulate p53 activity via ubiquitin-mediated proteolysis. This can change cell cycle. The p53 gene plays a crucial role for cell cycle control, induction of apoptosis, DNA repair, and genomic stability,and inhibit the formation of tumor tissue vessels, all of which are central to the prevention of human carcinogenesis. Functional p53 must be tightly regulated and the mdm2 (murine double minute 2) gene plays an important role in this regulation. The transcription of the mdm2 oncogene is induced by the p53 gene after DNA damage. The MDM2 protein then binds to p53 and downregulates it in an autoregulatory feedback loop. MDM2 inhibits p53 transcriptional activity, promotes nuclear export to the cytoplasm and rapid degradation of the p53 protein. It, therefore, plays a crucial role as a regulator of p53 in early embryonic development and for recovery of cellular proliferation after repair of DNA damage. Flavonoids compounds are widely present in plants'' metabolism substances. They have quantities of different structures and have important pharmaco-activities .They can prevent cardia and cerebrovascular disease, respiratory system disease , and anti-inflammatory , bacteriostasis, low serum glucose,anti- oxidation, resist radiation, anticancer and antitumor, and build up immune competence. Flavonoids compounds are a topic of general interest of developing new antitumor drug. Methods: The Hela cells are treated with bis (6-hydroxy -4-oxo-4H- chromene- 3-yl)-ethylenediimine at different time and various concentration. Analysis the inhibit of the complex effect to Hela cells via SRB stain and trypan stain. Find the changes of cells treated with complex for 24h by phase contrast microscope and fluorescence microscope under UV provocation with the cells stained by AO/EB. Analysis the DNA of the treated cells by agarose gel electrophoresis. Observe the apoptotic body by transmission electron microscope . Analysis Hela cells'' apoptosis by flow cytometry. Analysis the contents of p53 protein and MDM2 protein by Western blot. Result: The IC50 of the complex is 43.51μmol/L tested through SRB stain and trypan stain. Cytoplasm separated with cell membrane and the formated apoptotic body, we can see through phase contrast microscope and fluore...
研究领域 免疫化学
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