【作者】 刘欢；

【导师】 李坚；

【作者基本信息】 中南大学 ， 生物医学工程， 2013， 硕士

【摘要】 目的：肝癌是最常见的恶性肿瘤之一,在我国属于高发病,mTOR信号通路在许多肿瘤的发展中至关重要。AZD8055是新型mTOR激酶抑制剂,对mTOR活性具有很强的抑制作用。本文旨在研究AZD8055对人肝癌细胞系HepG2增殖和凋亡的作用,并初步探讨其作用机制。方法：1、体外培养人肝癌细胞株HepG2,采用MTT、流式细胞术及TUNEL染色检测药物作用不同时间对细胞增殖、细胞周期及细胞凋亡情况的影响,光学显微镜直接观察药物处理24h后细胞形态学变化。2、采用免疫印迹实验(WB)检测细胞内PI3K/Akt/mTOR通路中S6K和Akt的磷酸化水平,采用Real-time PCR技术检测细胞内侵袭相关的重要因子MMP2、MMP9的表达情况。结果：1、在体外细胞实验中,AZD8055(10nM)作用不同时间后,人肝癌细胞的增殖被显著抑制,且抑制程度与作用时间呈正相关(P<0.05)。药物处理24h后HepG2细胞密度明显降低,可见明显的挛缩、凋亡。G0/G1期细胞周期阻滞并且细胞凋亡明显增加。2、药物作用不同时间后,MMP2和MMP9的表达逐渐下调,PI3K/Akt/mTOR通路中S6K和Akt的磷酸化水平降低,并且这种作用呈时间依赖性。结论：1、在体外,AZD8055可抑制人肝癌细胞的生长,阻滞细胞周期于G0/G1期并诱导细胞凋亡。2、MMP2, MMP9与肿瘤侵袭转移相关,AZD8055可能通过降低MMP2和MMP9的表达,抑制肿瘤细胞的侵袭和转移。3、AZD8055通过降低AKT和S6K的磷酸化水平,抑制mTOR通路。更多还原

【Abstract】 Aims:Liver cancer is one of the most common malignant tumors, the incidence of which is high in our country. mTOR signaling pathway is crucial in the development of various tumors. AZD8055is a latest developed oral ATP competitive mTOR kinase inhibitor, having a very strong inhibition of mTOR activity. This study aims to investigate the effect of AZD8055on the proliferation and apoptosis in human hepatoma cell line HepG2,as well as to explore the molecular mechanism.Methods:HCC cell line HepG2were cultured, cells were treated with AZD8055at different times and then the cell proliferation, cell cycle and cell apoptosis were measured. In the situations of per-and post-treatment of AZD8055, variation of cell morphology was observed by microscope.The phosphorylation levels of S6K and Akt in PI3K/Akt/mTOR pathway were detected by immunohistochemical analysis.The mRNA expressions of invasion-related MMP2and MMP9detected by RT-PCR.Results:Cultured cells were treated with10nM AZD8055for different times.24h after treatment of AZD8055, the HepG2cell density was depressed and morphology of cell became smaller androunder. The proliferation of HCC cells was significantly inhibited by AZD8055in a time-dependent manner(P<0.05). Meanwhile, AZD8055treatment induced G0/G1phase cell cycle arrest and cell apoptosis in HCC cells.During the prolonged treatment with AZD8055, the express of MMP2and MMP9were decreased. The phosphorylation levels of S6K and Akt in PI3K/Akt/mTOR pathway were also reduced in a time dependent manner.Conclusion:In virto, AZD8055inhibits the growth of HCC cells through inducing G0/G1phase cell arrest and cell apoptosis.MMP2and MMP9are important factors in tumor invasion and metastasis, the mRNA expressions of invasion-related MMP2and MMP9were gradually decreased with the prolonged duration of the treatment of AZD8055, indicating that AZD8055might inhibit invasion and metastasis of human hepatic carcinoma cells.The phosphorylation of the AKt and S6K were deregulated, suggesting that the activity of mTOR was downregulated.更多还原