【作者】 屈展；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 外科学， 2012， 博士

【摘要】 目的：研究表明广泛应用于2型糖尿病临床治疗的药物二甲双胍具有潜在的抗肿瘤效应。本研究旨在研究二甲双胍对肝癌细胞增殖及凋亡的影响,探讨二甲双胍在肝癌细胞中可能的分子靶点及其相关信号通路。方法：1.体外培养人肝癌细胞株HepG2、Huh7、SMMC7721、Be17402和人正常肝细胞株L02。检测不同浓度二甲双胍对细胞增殖、细胞周期及细胞凋亡情况的影响。2.采用Western blot技术检测细胞内蛋白表达水平。采用Real time PCR技术检测细胞内mRNA水平。3.应用阳离子脂质体为载体将Livin siRNA、AMPKα1/2siRNA载入细胞,分析Livin蛋白及AMPK/mTOR通路在二甲双胍抑制肝癌细胞生长现象中的作用。4.建立人肝癌裸鼠移植瘤模型,观察二甲双胍治疗对移植瘤生长的影响。5.采用免疫组化方法,检测移植瘤和正常肝脏组织中细胞周期及细胞凋亡相关蛋白表达的影响。采用ELISA法检测荷瘤鼠血清胰岛素和IGF-1水平。结果：1.在体外细胞实验中,二甲双胍(1mM,5mM,10mM,15mM)作用72小时后,人肝癌细胞的增殖被显著抑制,且抑制程度与药物浓度呈正相关(P<0.05)。二甲双胍(5mM,10mM,15mM)作用72小时后,人肝癌细胞的活力显著低于人正常肝细胞的活力(P<0.05)。2.二甲双胍(5mM,10mM)作用48小时后,人肝癌细胞出现G0/G1期细胞周期阻滞并且细胞凋亡增加(P<0.05),但人正常肝细胞的细胞周期循环及细胞凋亡未明显受到影响。3. Livin siRNA转染48小时后,人肝癌细胞的增殖被显著抑制,出现G0/G1期细胞周期阻滞并且细胞凋亡增加(P<0.05),但人正常肝细胞的细胞周期循环及细胞凋亡未明显受到影响。二甲双胍(5mM,101nM)作用48小时后,人肝癌细胞内Livin蛋白的表达水平下降,且下降程度与药物浓度呈正相关,而人正常肝细胞内Livin蛋白的表达水平无明显变化。Real-time PCR检测发现二甲双胍(5mM,10mM)作用48小时对人肝癌细胞及人正常肝细胞Livin mRNA的表达水平无明显影响。4. AMPKa siRNA转染72小时后,HepG2细胞的生长未受明显影响,但二甲双胍对HepG2细胞生长的抑制作用被显著抑制(P<0.05)。二甲双胍(5mM,10mM)作用72小时能提高HepG2细胞内AMPK蛋白磷酸化水平,降低]nTOR通路下游蛋白p70S6K蛋白的磷酸化水平,并且这种作用呈能浓度依赖性。当HepG2转染AMPKa siRNA后,二甲双胍降低细胞内p70S6K蛋白磷酸化水平及降低Livin蛋白表达水平的作用减弱。5.在裸鼠HepG2细胞及Huh7细胞异位移植瘤模型中,二甲双胍腹腔注射治疗30天能显著缩小移植瘤体积(P<0.05),且抑瘤率与给药剂量呈正相关。6.在裸鼠HepG2细胞原位移植瘤模型中,二甲双胍腹腔注射治疗30天能显著缩小移植瘤体积(P<0.05),低剂量组(125mg/Kg/d)及高剂量组(250mg/Kg/d)的抑瘤率分别为47.37%及86.07%。二甲双胍治疗后移植瘤组织中的Ki67指数显著降低(P<0.05),Cleaved caspase3指数显著升高(P<0.05),且指数的变化与药物剂量呈正相关。在正常肝组织中,二甲双胍治疗对Ki67指数及Cleaved caspase3指数无明显影响。二甲双胍治疗还使得移植瘤组织中AMPK磷酸化水平升高,Livin蛋白表达水平降低。组织HE染色示二甲双胍治疗未导致荷瘤裸鼠主要脏器出现明显病变。各组荷瘤裸鼠的体重、血糖以及血清中胰岛素、IGF-1的水平无明显差别。结论：1.在体内及体外,二甲双胍治疗均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。同时,二甲双胍对正常肝细胞的生长无明显影响。2. Livin蛋白是二甲双胍在细胞内的靶蛋白之一。二甲双胍可能通过降低细胞内Livin蛋白的表达水平,特异性的抑制肿瘤细胞的生长。3.二甲双胍通过激活肝癌细胞内AMPK,抑制mTOR通路,在翻译水平下调Livin蛋白的表达。更多还原

【Abstract】 Aims:Metformin is a biguanide that has been widely used to treat type2diabetes. Several studies have shown that metformin is also effective in treating cancer, including hepatocellular carcinoma (HCC). The objective of this study was to evaluate the antitumor effects of metformin in HCC, as well as to investigate the potential molecular target(s) of metformin-mediated antitumor activity.Methods:HCC cell lines HepG2, Huh7, SMMC7721, Be17402and human normal liver cell line L02were cultured. Cells were treated with different concentrations of metformin and then the cell proliferation, cell cycle and cell apoptosis were measured.Target protein levels were measured by Western blot analysis. Target gene mRNA levels were measured by Real-time PCR.To investigate whether Livin protein and the AMPK/mTOR pathway were involved in the antitumor effects of metformin in HCC, Livin siRNA and AMPKal/2siRNA carried by Lipofectamine2000were transfected into cells.HCC xenograft tumors were established in athymic nude mice, and tumor growth was monitored during metformin treatment.The expression of cell proliferation-related and cell apoptosis- related proteins in tumor tissues and normal liver tissues were detected by immunohistochemical analysis. The serum insulin and IGF-1levels in tumor-bearing mice were detected by ELISA.Results:Cultured cells were treated with metformin (1mM,5mM,10mM,15mM) for72hours. The proliferation of HCC cells was significantly inhibited by metformin treatment in a dose-dependent manner(P<0.05). Meanwhile, the viabilities of the HCC cells were significantly lower than that of the normal human liver cells after metformin treatment (P <0.05).Metformin treatment induced G0/G1phase cell cycle arrest and cell apoptosis in HCC cells (P<0.05), however, failed to affect the cell cycle distribution and cell apoptosis in normal liver cells.Livin siRNA transfection caused growth inhibition of HCC cells, induced G0/G1phase cell cycle arrest and cell apoptosis (P<0.05). However, it failed to affect the cell cycle distribution and cell apoptosis in normal liver cells. Metformin treatment decreased the expression level of Livin protein in HCC cells in a dose-dependent manner. However, it failed to affect the expression level of Livin protein in normal liver cells. Livin mRNA expression levels of HCC cells and normal liver cells were not affected by metformin treatment.After AMPKa siRNA transfection for72hours, the growth of HepG2cells was not significantly affected, but the inhibitory effect of metformin on the growth of HepG2cells was significantly inhibited. In HepG2cells, Metformin treatment increased AMPK phosphorylation levels and reduced p70S6K protein phosphorylation levels in a dose-dependent manner. Meformin treatment failed to decrease Livin protein levels and p70S6K phosphorylation levels after cells were transfected with AMPKa siRNA.In nude mice ectopic xenograft model, metformin treatment significantly reduced tumor growth in a dosage-dependent manner (P <0.05).In nude mice orthotopic xenograft model, metformin treatment significantly reduced tumor growth in a dosage-dependent manner (P-0.05). In the xenograft tumor tissues, metformin treatment significantly decreased Ki67index and increased Cleaved caspase3index (P<0.05). However, metformin treatment failed to affect the Ki67index and Cleaved caspase3index in normal liver tissues. Metformin treatment increased AMPK phosphorylation levels and decreased Livin protein expression levels in the xenograft tumor tissues in a dosage-dependent manner. Metformin treatment did not cause any pathological changes in organs of nude mice according to HE staining analysis. Metformin treatment didn’t affect body weight, blood glucose and serum insulin, IGF-1levels in tumor-bearing mice. Conclusion:In vitro and in vivo, Metformin inhibits the growth of HCC cells through inducing G0/G1phase cell cycle arrest and cell apoptosis, however, the growth of normal liver cells was not affected.Metformin decreases Livin protein level in HCC cells which causes tumor specific growth inhibition.Metformin activates AMPK/mTOR pathway and subsequently inhibits Livin protein expression at the mRNA translation level.更多还原