FICD及与Sorafenib联合抗肝癌的作用和分子机制
发布时间：2018-08-01 12:08
【摘要】：目的:研究FICD单药以及与Sorafenib联合用药对体外培养的人肝癌细胞HepG2和小鼠荷H22肝癌实体瘤生长的抑制作用,并探索其分子机制。方法:采用四氮唑盐(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliu mromide,MTT)还原法测定FICD单药及与Sorafenib联合用药对体外培养的肿瘤细胞生长的抑制作用。采用荷H22小鼠肝癌实体瘤模型,以肿瘤的重量、抑瘤率为指标,研究FICD单药及与Sorafenib联合对体内肿瘤生长的抑制作用。采用流式细胞仪检测FICD单药及与Sorafenib联合对HepG2细胞凋亡和细胞周期的影响。采用激光共聚焦显微镜法观察细胞凋亡形态。Western blotting检测FICD单药及与Sorafenib联合用药对人肝癌HepG2细胞中β-Catenin、C-Myc、Vimentin、CyclinD1和Ki-67蛋白表达的影响。结果:MTT实验结果表明:FICD浓度依赖性地抑制人肝癌HepG2和SMMC-7721、胶质瘤U251、宫颈癌HeLa、胃癌MGC-231、乳腺癌MAD-MB-231细胞生长,作用48 h IC50分别为10.1μg/m L、14.2μg/m L、16.8μg/m L、17.3μg/m L、18.6μg/m L和15.6μg/m L。根据IC50可知FICD对肝癌HepG2和SMMC-7721细胞作用最强。进一步研究证实FICD能时间、浓度依赖性地抑制人肝癌HepG2细胞生长,作用24 h、48 h和72 h的IC50分别为16.8μg/mL、9.8μg/mL和6.7μg/mL。流式细胞仪和激光共聚焦显微镜法表明FICD能明显诱导HepG2细胞凋亡,并阻止细胞周期于G2/M。Western Blotting实验结果表明FICD使β-Catenin,C-Myc,CyclinD1和Ki-67蛋白下调,而使Vimentin蛋白表达增加。进一步体内实验证实FICD灌胃和腹腔注射给药对H22肝癌细胞生长有相同的抑制作用,50、100、150 mg/kg腹腔注射抑制率分别为40.3%、46.3%和53.0%,灌胃给药的抑制率分别为40.1%、48.2%和53.4%。FICD与Sorafenib联合用药体外对HepG2细胞生长有明显的协同抑制作用,体内对H22肝癌细胞生长也有明显的协同抑制作用。FICD与Sorafenib联合用药还能促进HepG2细胞凋亡。FICD 10μg/mL使HepG2细胞G0/G1期和S期细胞减少,增加G2/M期细胞;Sorafenib 4μg/m L增加G0/G1期细胞而减少S期和G2/M期细胞;两者合用后和Sorafenib 4μg/mL比,G0/G1期细胞减少而S期和G2/M期细胞增加。FICD 10μg/m L和Sorafenib4μg/mL单用或合用均明显降低β-Catenin,C-Myc,CyclinD1和Ki-67蛋白表达水平;两药单用时均明显增加Vimentin蛋白表达量,而联合用药时Vimentin蛋白表达明显减少。结论:FICD体内外均有较好的抗肝癌作用,与Sorafenib联合用药有协同作用,其机制可能与影响Wnt/β-Catenin信号通路而诱导细胞凋亡有关。
[Abstract]:Aim: to study the inhibitory effects of FICD alone or in combination with Sorafenib on the growth of human hepatoma cells HepG2 and mice bearing H22 hepatoma in vitro and to explore its molecular mechanism. Methods: the inhibitory effects of FICD alone or combined with Sorafenib on the growth of tumor cells in vitro were determined by using 3- (4- (4-) -dimethylthiahiazo (-z-y1) -3-di-phenytetrazoliu mromide) reduction assay. The tumor growth inhibition effect of FICD alone or in combination with Sorafenib on tumor growth in vivo was studied by using the tumor weight and tumor inhibition rate of tumor in mice bearing H22 as a solid tumor model. Flow cytometry was used to detect the effect of FICD on apoptosis and cell cycle of HepG2 cells. Apoptosis morphology was observed by laser confocal microscopy. Western blotting was used to detect the expression of 尾 -Cateninine C-Mycentin CyclinD1 and Ki-67 protein in HepG2 cells of hepatocellular carcinoma (HCC). Results the HepG2 and SMMC-7721, U251, HeLa, MGC-231 and MAD-MB-231 cells were inhibited in a dose-dependent manner. The IC50 was 10.1 渭 g / m 路L ~ (-1), 14.2 渭 g / m ~ (-1) 渭 g / m ~ (-1), 17.3 渭 g / m ~ (-1) 渭 g 路m ~ (-1) 路L ~ (-1) and 15.6 渭 g 路m ~ (-1) 路L ~ (-1) at 48 h, respectively. According to IC50, FICD had the strongest effect on HepG2 and SMMC-7721 cells. Further studies confirmed that FICD inhibited the growth of human hepatoma HepG2 cells in a time-and concentration-dependent manner. The IC50 at 24 h for 48 h and 72 h were 16.8 渭 g / mL and 6.7 渭 g / mL, respectively. Flow cytometry and laser confocal microscopy showed that FICD could induce the apoptosis of HepG2 cells, and inhibit the cell cycle. The results of G2/M.Western Blotting experiment showed that FICD could down-regulate the expression of 尾 -Cateninine C-Mycn cyclin D1 and Ki-67 protein, but increase the expression of Vimentin protein. Further in vivo experiments confirmed that FICD had the same inhibitory effect on the growth of H22 hepatoma cells by intragastric administration and intraperitoneal injection. The inhibitory rates of 50100150 mg/kg intraperitoneal injection were 46.3% and 53.0%, respectively. The inhibition rates of intragastric administration were 40.1% and 48.2%, respectively, and the inhibitory rates of 53.4%.FICD combined with Sorafenib were 40.3% and 53.0%, respectively. The growth of HepG2 cells was inhibited in vitro. The combination of FICD and Sorafenib could also promote the apoptosis of HepG2 cells. FICD 10 渭 g/mL reduced the number of HepG2 cells in G0/G1 phase and S phase. The cells in S phase and G 2 / M phase increased by 4 渭 g / mL in G _ 2 / M phase and decreased in S phase and G _ 2 / M phase by increasing Sorafenib _ 4 渭 g 路mL ~ (L), and the protein expression levels of 尾 -Cateninine C-Myccyclin D1 and Ki-67 were significantly decreased in combination with Sorafenib _ 4 渭 g/mL than those in G _ 0 / G _ 1 phase and S phase and G _ 2 / M phase cells in S phase and G _ 2 / M phase, respectively, and the expression levels of 尾 -Cateninine C-Myccyclin D1 and Ki-67 were significantly decreased when combined with or without Sorafenib4 渭 g/mL. The expression of Vimentin protein was significantly increased when the two drugs were used alone, but the expression of Vimentin protein decreased significantly when combined with the drug. ConclusionFICD has good anti-hepatoma effect in vivo and in vitro, and has synergistic effect with Sorafenib. The mechanism may be related to the apoptosis induced by affecting Wnt/ 尾 -Catenin signaling pathway.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
本文编号：2157528
[Abstract]:Aim: to study the inhibitory effects of FICD alone or in combination with Sorafenib on the growth of human hepatoma cells HepG2 and mice bearing H22 hepatoma in vitro and to explore its molecular mechanism. Methods: the inhibitory effects of FICD alone or combined with Sorafenib on the growth of tumor cells in vitro were determined by using 3- (4- (4-) -dimethylthiahiazo (-z-y1) -3-di-phenytetrazoliu mromide) reduction assay. The tumor growth inhibition effect of FICD alone or in combination with Sorafenib on tumor growth in vivo was studied by using the tumor weight and tumor inhibition rate of tumor in mice bearing H22 as a solid tumor model. Flow cytometry was used to detect the effect of FICD on apoptosis and cell cycle of HepG2 cells. Apoptosis morphology was observed by laser confocal microscopy. Western blotting was used to detect the expression of 尾 -Cateninine C-Mycentin CyclinD1 and Ki-67 protein in HepG2 cells of hepatocellular carcinoma (HCC). Results the HepG2 and SMMC-7721, U251, HeLa, MGC-231 and MAD-MB-231 cells were inhibited in a dose-dependent manner. The IC50 was 10.1 渭 g / m 路L ~ (-1), 14.2 渭 g / m ~ (-1) 渭 g / m ~ (-1), 17.3 渭 g / m ~ (-1) 渭 g 路m ~ (-1) 路L ~ (-1) and 15.6 渭 g 路m ~ (-1) 路L ~ (-1) at 48 h, respectively. According to IC50, FICD had the strongest effect on HepG2 and SMMC-7721 cells. Further studies confirmed that FICD inhibited the growth of human hepatoma HepG2 cells in a time-and concentration-dependent manner. The IC50 at 24 h for 48 h and 72 h were 16.8 渭 g / mL and 6.7 渭 g / mL, respectively. Flow cytometry and laser confocal microscopy showed that FICD could induce the apoptosis of HepG2 cells, and inhibit the cell cycle. The results of G2/M.Western Blotting experiment showed that FICD could down-regulate the expression of 尾 -Cateninine C-Mycn cyclin D1 and Ki-67 protein, but increase the expression of Vimentin protein. Further in vivo experiments confirmed that FICD had the same inhibitory effect on the growth of H22 hepatoma cells by intragastric administration and intraperitoneal injection. The inhibitory rates of 50100150 mg/kg intraperitoneal injection were 46.3% and 53.0%, respectively. The inhibition rates of intragastric administration were 40.1% and 48.2%, respectively, and the inhibitory rates of 53.4%.FICD combined with Sorafenib were 40.3% and 53.0%, respectively. The growth of HepG2 cells was inhibited in vitro. The combination of FICD and Sorafenib could also promote the apoptosis of HepG2 cells. FICD 10 渭 g/mL reduced the number of HepG2 cells in G0/G1 phase and S phase. The cells in S phase and G 2 / M phase increased by 4 渭 g / mL in G _ 2 / M phase and decreased in S phase and G _ 2 / M phase by increasing Sorafenib _ 4 渭 g 路mL ~ (L), and the protein expression levels of 尾 -Cateninine C-Myccyclin D1 and Ki-67 were significantly decreased in combination with Sorafenib _ 4 渭 g/mL than those in G _ 0 / G _ 1 phase and S phase and G _ 2 / M phase cells in S phase and G _ 2 / M phase, respectively, and the expression levels of 尾 -Cateninine C-Myccyclin D1 and Ki-67 were significantly decreased when combined with or without Sorafenib4 渭 g/mL. The expression of Vimentin protein was significantly increased when the two drugs were used alone, but the expression of Vimentin protein decreased significantly when combined with the drug. ConclusionFICD has good anti-hepatoma effect in vivo and in vitro, and has synergistic effect with Sorafenib. The mechanism may be related to the apoptosis induced by affecting Wnt/ 尾 -Catenin signaling pathway.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
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