二氢青蒿素抑制BGC-823胃癌细胞增殖及转移的研究

发布时间：2018-08-20 08:36

【摘要】：第一部分二氢青蒿素抑制胃癌细胞株BGC-823增殖及其机制的影响目的：在体外细胞水平及活体动物水平探讨DHA对人胃癌细胞株BGC-823增殖、细胞周期、凋亡等生物学表型的影响,并初步探讨其分子机制。方法：采用MTT法检测DHA、5氟尿嘧啶(5-FU)对BGC-823细胞的杀伤作用；流式细胞术检测DHA对BGC-823细胞周期与凋亡的影响；Western blot检测细胞凋亡相关蛋白Bax、Bcl-2、Caspase-3蛋白的表达；分光光度方法检测Caspase-3活性。建立裸鼠胃癌动物模型评估DHA在活体水平对胃癌的治疗效果。结果：与对照组相比,不同浓度DHA(10、20、40μmol/L)均能显著抑制BGC-823胃癌细胞的体外增殖。小剂量DHA (10μmol/L)可与5-FU协同杀伤胃癌细胞,促进5-FU的细胞毒效应;与5-FU单药组相比,联合用药组的细胞存活率显著下降,5-FU杀伤BGC-823的半数抑制率浓度(ICso)=3.04μg/mL,而联合用药组的IC50下降为1.42 μg/mL。细胞周期和凋亡分析显示,DHA可以使BGC-823细胞在G2/M期发生阻滞,并可诱导BGC-823凋亡。DHA处理BGC-823细胞后,抗凋亡蛋白Bcl-2表达显著下调,促凋亡蛋白Bax和Caspase-3表达显著上调。DHA能够显著抑制裸鼠皮下胃癌移植瘤的生长,并诱导其细胞凋亡。结论：DHA能够显著抑制胃癌细胞的增殖,阻滞细胞周期进程,促进细胞凋亡,并能增加BGC-823细胞对5-FU的敏感性,其机理可能与下调Bcl-2表达,上调Bax表达有关。第二部分二氢青蒿素对BGC-823胃癌细胞侵袭及转移能力的影响及其机制的研究目的：二氢青蒿素是青蒿素的一种衍生物,具有较为广泛的抗肿瘤活性。本课题研究DHA对人胃癌细胞株BGC-823体外迁移、粘附、侵袭能力及活体肿瘤转移能力的影响并初步探讨其分子机制。方法：采用Transwell小室技术检测DHA对胃癌细胞BGC-823细胞迁移和侵袭能力的影响,黏附实验检测DHA作用后细胞黏附能力的变化。实时定量RT-PCR方法和酶联免疫分析法分别检测VEGF mRNA和蛋白的表达水平。采用尾静脉注射方式建立裸鼠胃癌肺转移模型,采用DHA灌胃方式研究DHA对裸鼠胃癌转移的影响。结果：与对照组相比,DHA (20、40μmol/L)处理组BGC-823胃癌细胞的体外迁移、粘附、侵袭能力均显著降低(P0.05)。DHA处理胃癌细胞VEGF mRNA表达水平显著下降,细胞上清中VEGF水平也比对照组显著减少(P0.05)。DHA处理可以显著减少裸鼠肺部胃癌转移灶的数量(P0.01)。结论：DHA能够显著抑制胃癌肿瘤转移能力,其机理可能与下调VEGF表达有关。
[Abstract]:Part I effects of dihydroartemisinin on the proliferation of gastric cancer cell line BGC-823 and its mechanism objective: to investigate the effects of DHA on the proliferation, cell cycle, apoptosis and other biological phenotypes of human gastric cancer cell line BGC-823 in vitro and in vivo. The molecular mechanism was also discussed. Methods: MTT assay was used to detect the cytotoxicity of DHA5 fluorouracil (5-FU) to BGC-823 cells, flow cytometry was used to detect the effect of DHA on the cell cycle and apoptosis of BGC-823 cells. Western blot was used to detect the expression of apoptosis-related protein Baxia Bcl-2Caspase-3, and the activity of Caspase-3 was detected by spectrophotometry. Animal models of gastric cancer in nude mice were established to evaluate the therapeutic effect of DHA on gastric cancer in vivo. Results: compared with the control group, the proliferation of BGC-823 gastric cancer cells was significantly inhibited by different concentrations of DHA (10 ~ 2040 渭 mol/L). Low dose DHA (10 渭 mol/L) could synergistically kill gastric cancer cells with 5-FU and promote the cytotoxic effect of 5-FU. Compared with 5-FU single drug group, the cell survival rate of the combined treatment group was significantly lower than that of the 5-FU group (ICso) 3.04 渭 g / mL), while the IC50 of the combined treatment group was 1.42 渭 g / mL. Cell cycle and apoptosis analysis showed that DHA could block BGC-823 cells in G _ 2 / M phase and induce BGC-823 apoptosis. DHA could significantly down-regulate the expression of anti-apoptotic protein Bcl-2 after BGC-823 cells were treated with DHA. The expression of pro-apoptotic protein Bax and Caspase-3 increased significantly. DHA could significantly inhibit the growth and induce apoptosis of xenografts of subcutaneous gastric cancer in nude mice. ConclusionDHA can significantly inhibit the proliferation of gastric cancer cells, block cell cycle progression, promote cell apoptosis, and increase the sensitivity of BGC-823 cells to 5-FU. The mechanism may be related to down-regulation of Bcl-2 expression and up-regulation of Bax expression. Effect of dihydroartemisinin on invasion and metastasis of BGC-823 gastric cancer cells and its mechanism objective: dihydroartemisinin is a derivative of artemisinin and has extensive antitumor activity. The aim of this study was to investigate the effects of DHA on the migration, adhesion, invasion and metastasis of human gastric cancer cell line BGC-823 in vitro and to explore its molecular mechanism. Methods: Transwell chamber technique was used to detect the effect of DHA on the migration and invasion of gastric cancer BGC-823 cells. Adhesion assay was used to detect the changes of cell adhesion ability after DHA treatment. Real-time quantitative RT-PCR and enzyme-linked immunosorbent assay (Elisa) were used to detect the expression of VEGF mRNA and protein respectively. The lung metastasis model of gastric cancer in nude mice was established by tail vein injection, and the effect of DHA on gastric cancer metastasis in nude mice was studied by DHA. Results: compared with the control group, the migration, adhesion and invasiveness of BGC-823 gastric cancer cells treated with DHA (20 渭 mol/L) were significantly decreased (P0.05) .DHA treated with DHA significantly decreased the expression of VEGF mRNA in gastric cancer cells. The level of VEGF in the supernatant was significantly lower than that in the control group (P0.05). DHA treatment could significantly reduce the number of metastatic foci of lung gastric cancer in nude mice (P0.01). ConclusionDHA can significantly inhibit the ability of gastric cancer to metastasize, and its mechanism may be related to the down-regulation of VEGF expression.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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