斑蝥素酸镁对胃癌体外抗癌活性及其机制的研究

发布时间：2018-03-10 22:06

本文选题：斑蝥素酸镁　切入点：抗癌活性　出处：《遵义医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:比较自主合成新药──斑蝥素酸镁与斑蝥素、斑蝥素酸钠在体外的抗癌活性,筛选出抗癌活性最好的药物,并初步探讨其抗癌作用机制。方法:1.利用CCK-8(Cell Counting Kit-8)法检测不同浓度的斑蝥素酸镁、斑蝥素、斑蝥素酸钠在体外对人胃癌细胞MKN-28、SGC-7901、BGC-823增殖的影响;2.倒置相差显微镜下观察不同浓度的斑蝥素酸镁、斑蝥素、斑蝥素酸钠作用后三种胃癌细胞形态的变化;3.以人正常胃粘膜细胞GES-1为对照,评价斑蝥素酸镁的细胞毒性作用4.利用透射电镜观察斑蝥素酸镁作用后人胃癌细胞BGC-823超微结构的变化;5.流式细胞技术检测斑蝥素酸镁对人胃癌细胞BGC-823的细胞周期和细胞凋亡的影响;6.Western-Blot蛋白印迹法检测斑蝥素酸镁作用后人胃癌细胞BGC-823中周期素依赖性蛋白激酶1(CDK1)、细胞周期蛋白B1(Cyclin B1)蛋白的表达水平。结果:1.斑蝥素酸镁、斑蝥素、斑蝥素酸钠对MKN-28、SGC-7901、BGC-823三株胃癌细胞增殖均有明显抑制作用,呈剂量-效应关系(P0.05);斑蝥素酸镁的抑制作用优于斑蝥素和斑蝥素酸钠;2.倒置相差显微镜观察显示:斑蝥素酸镁、斑蝥素、斑蝥素酸钠作用24h后,胃癌细胞皱缩、变圆,部分细胞破碎;3.斑蝥素酸镁在0.52μmol/L时,已经对胃癌细胞BGC-823表现出明显的抑制作用,而对于人正常胃粘膜细胞GES-1,在1.04μmol/L时抑制率仍然很低。在相同的药物浓度作用下,斑蝥素酸镁对胃癌细胞BGC-823增殖的抑制作用明显强于人正常胃粘膜细胞GES-1;其半数抑制浓度IC50分别为4.868μmol/L、9.645μmol/L。4.透射电镜:半数抑制浓度的斑蝥素酸镁作用于BGC-823细胞24h后,观察到细胞核异形、裂解,内质网及线粒体明显肿胀;5.碘化丙锭(propidiumiodide,PI)单染色检测结果显示:斑蝥素酸镁作用于胃癌细胞BGC-823后,G0/G1期细胞比率减少,S期基本不变,G2/M期细胞增加,表现为G2/M期阻滞(P0.05);膜连蛋白-异硫氰酸荧光素(Annexin V-FITC/PI)双染流式细胞术检测结果:随着斑蝥素酸镁浓度增加,BGC-823细胞的凋亡率也逐渐升高(P0.05);6.Western-Blot结果显示:斑蝥素酸镁作用24h后,BGC-823细胞中CDK1、CyclinB1蛋白表达水平显著降低(**P0.05,##P0.05)。结论:1.斑蝥素酸镁对人胃癌细胞的抑制作用优于斑蝥素和斑蝥素酸钠;2.斑蝥素酸镁能将胃癌细胞BGC-823阻滞在G2/M期,并诱导BGC-823细胞发生凋亡,其机制可能与下调CDK1、Cyclin B1蛋白的表达有关;3.斑蝥素酸镁的抗癌细胞毒性具有一定程度的细胞选择性和敏感性。
[Abstract]:Objective: to compare the anticancer activity of magnesium cantharidate with that of cantharidin, sodium cantharidin in vitro, and to screen out the drugs with the best anticancer activity. CCK-8(Cell Counting Kit-8 method was used to detect different concentrations of magnesium cantharidate and cantharidin. Effect of Cantharidin Sodium on Proliferation of Human gastric Cancer Cell Line MKN-28 SGC-7901 BGC-823 in Vitro. Morphological changes of three gastric cancer cells after treatment with sodium cantharidin 3. GES-1 of normal gastric mucosal cells was used as control. To evaluate the cytotoxicity of magnesium cantharidin 4. To observe the ultrastructural changes of BGC-823 in human gastric cancer cells after treated with magnesium cantharidin by transmission electron microscope. Flow cytometry was used to detect the cell cycle of Cantharidin magnesium on human gastric cancer cell line BGC-823. 6. Western-Blot Western blot was used to detect the expression of cyclin dependent protein kinase (CDK1) and cyclin B1 (cyclin B1) protein in human gastric cancer cell line BGC-823 treated with magnesium cantharidate. Cantharidin and sodium cantharidate inhibited the proliferation of MKN-28 SGC-7901 and BGC-823 gastric cancer cells in a dose-effect manner, and the inhibitory effects of magnesium cantharidate were superior to those of cantharidin and sodium cantharidate. Cantharidin, sodium cantharidin, after 24 hours, the gastric cancer cells shrinked and became round, and some of the cells were broken. Magnesium cantharidin has shown obvious inhibitory effect on the BGC-823 of gastric cancer cells at 0.52 渭 mol/L. The inhibitory rate of GES-1 on normal gastric mucosal cells was still very low at 1.04 渭 mol/L. The inhibitory effect of magnesium cantharidate on BGC-823 proliferation of gastric cancer cells was significantly stronger than that on normal gastric mucosal cells GES-1, and its half inhibitory concentration (IC50) was 4.868 渭 mol / L, 9.645 渭 mol / L 路L. 4. Transmission electron microscope showed that magnesium cantharidate at half inhibitory concentration could inhibit the proliferation of BGC-823 cells for 24 hours. The results of single staining for propidiumiodide iodide (Pi) showed that the ratio of G _ 0 / G _ 1 phase to G _ 0 / G _ 1 phase of gastric cancer cells increased after treated with magnesium cantharidate, and the ratio of G _ 0 / G _ 1 phase to G _ 2 / M phase increased significantly after treatment with Cantharidin. The results of flow cytometry showed that the apoptosis rate of BGC-823 cells increased with the increase of magnesium cantharidate concentration, and the results of Western-Blot showed that: the effect of Cantharidin on magnesium Cantharidin was found in BGC-823 cells with the increase of the concentration of magnesium cantharidin. The results of Western-Blot showed that: the effect of Cantharidin on magnesium Cantharidin was determined by flow cytometry, and the apoptosis rate of BGC-823 cells increased with the increase of concentration of magnesium cantharidate. After 24 hours, the expression of CDK1 and CyclinB1 in BGC-823 cells decreased significantly. Conclusion: 1. The inhibitory effect of magnesium cantharidin on human gastric cancer cells is superior to that of cantharidin and sodium cantharidate. Magnesium cantharidin can block the BGC-823 of gastric cancer cells at G 2 / M stage. The mechanism may be related to the down-regulation of CDK1 Cyclin B1 protein expression. Cantharidin magnesium has a certain degree of cell selectivity and sensitivity to anticancer cytotoxicity.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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