FICD及與Sorafenib聯(lián)合抗肝癌的作用和分子機制
發(fā)布時間：2018-08-01 12:08
【摘要】：目的:研究FICD單藥以及與Sorafenib聯(lián)合用藥對體外培養(yǎng)的人肝癌細胞HepG2和小鼠荷H22肝癌實體瘤生長的抑制作用,并探索其分子機制。方法:采用四氮唑鹽(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliu mromide,MTT)還原法測定FICD單藥及與Sorafenib聯(lián)合用藥對體外培養(yǎng)的腫瘤細胞生長的抑制作用。采用荷H22小鼠肝癌實體瘤模型,以腫瘤的重量、抑瘤率為指標,研究FICD單藥及與Sorafenib聯(lián)合對體內(nèi)腫瘤生長的抑制作用。采用流式細胞儀檢測FICD單藥及與Sorafenib聯(lián)合對HepG2細胞凋亡和細胞周期的影響。采用激光共聚焦顯微鏡法觀察細胞凋亡形態(tài)。Western blotting檢測FICD單藥及與Sorafenib聯(lián)合用藥對人肝癌HepG2細胞中β-Catenin、C-Myc、Vimentin、CyclinD1和Ki-67蛋白表達的影響。結(jié)果:MTT實驗結(jié)果表明:FICD濃度依賴性地抑制人肝癌HepG2和SMMC-7721、膠質(zhì)瘤U251、宮頸癌HeLa、胃癌MGC-231、乳腺癌MAD-MB-231細胞生長,作用48 h IC50分別為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。根據(jù)IC50可知FICD對肝癌HepG2和SMMC-7721細胞作用最強。進一步研究證實FICD能時間、濃度依賴性地抑制人肝癌HepG2細胞生長,作用24 h、48 h和72 h的IC50分別為16.8μg/mL、9.8μg/mL和6.7μg/mL。流式細胞儀和激光共聚焦顯微鏡法表明FICD能明顯誘導(dǎo)HepG2細胞凋亡,并阻止細胞周期于G2/M。Western Blotting實驗結(jié)果表明FICD使β-Catenin,C-Myc,CyclinD1和Ki-67蛋白下調(diào),而使Vimentin蛋白表達增加。進一步體內(nèi)實驗證實FICD灌胃和腹腔注射給藥對H22肝癌細胞生長有相同的抑制作用,50、100、150 mg/kg腹腔注射抑制率分別為40.3%、46.3%和53.0%,灌胃給藥的抑制率分別為40.1%、48.2%和53.4%。FICD與Sorafenib聯(lián)合用藥體外對HepG2細胞生長有明顯的協(xié)同抑制作用,體內(nèi)對H22肝癌細胞生長也有明顯的協(xié)同抑制作用。FICD與Sorafenib聯(lián)合用藥還能促進HepG2細胞凋亡。FICD 10μg/mL使HepG2細胞G0/G1期和S期細胞減少,增加G2/M期細胞;Sorafenib 4μg/m L增加G0/G1期細胞而減少S期和G2/M期細胞;兩者合用后和Sorafenib 4μg/mL比,G0/G1期細胞減少而S期和G2/M期細胞增加。FICD 10μg/m L和Sorafenib4μg/mL單用或合用均明顯降低β-Catenin,C-Myc,CyclinD1和Ki-67蛋白表達水平;兩藥單用時均明顯增加Vimentin蛋白表達量,而聯(lián)合用藥時Vimentin蛋白表達明顯減少。結(jié)論:FICD體內(nèi)外均有較好的抗肝癌作用,與Sorafenib聯(lián)合用藥有協(xié)同作用,其機制可能與影響Wnt/β-Catenin信號通路而誘導(dǎo)細胞凋亡有關(guān)。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7
本文編號：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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7
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