姜黄素联合L-FP抗胃癌MGC-803细胞的作用及其机制的研究

发布时间：2018-03-25 11:07

本文选题：姜黄素　切入点：L-FP(氟尿嘧啶　出处：《湖北中医药大学》2016年硕士论文

【摘要】：目的:姜黄素(curcumin,CUR)是中药姜黄的主要活性成分,是一种脂溶性酚类色素,具有抗肿瘤、抗炎症、抑制突变等多种作用。小剂量顺铂(DDP)与5-氟尿嘧啶(5-FU)联合用药(L-FP)是临床上治疗胃癌的常用化疗方案。本文旨在探讨姜黄素联合L-FP对体外培养的胃癌MGC-803细胞的生长、增殖、迁移和凋亡的影响及其机制,为临床上联合应用姜黄素和L-FP治疗胃癌提供理论基础和实验依据。方法:采用细胞生物学和分子生物学的研究技术与方法,观察姜黄素和/或不同剂量的L-FP对体外培养的胃癌MGC-803细胞的生长、克隆形成、迁移、细胞周期、细胞凋亡的作用以及对相关调控蛋白Caspase-3、Caspase-8、Bax和Bcl-2的影响。主要观察指标及方法如下:1.MTT实验:用姜黄素和/或不同剂量的L-FP分别处理96孔板培养的胃癌MGC-803细胞24h、48h和72h后,使用酶标仪检测每孔细胞的OD值,并计算细胞的生长抑制率。2.平板克隆形成实验:用姜黄素和/或不同剂量的L-FP处理6孔板培养的MGC-803细胞,24h后换不含药物的培养液继续培养10天,吉姆萨染色后计数并计算克隆形成率。3.Transwell迁移实验:用含有姜黄素和/或不同剂量的L-FP的培养液培养Transwell小室中的胃癌MGC-803细胞,10h后,MTT方法检测并计算迁移率。4.AO/EB双染法:用姜黄素和/或不同剂量的L-FP处理6孔板培养的MGC-803细胞24h后,AO/EB双染在荧光显微镜下拍照,观察细胞形态、计数凋亡细胞数并计算凋亡率。5.流式细胞术:姜黄素和/或不同剂量的L-FP处理6孔板培养的胃癌MGC-803细胞24h后,用PI单染方法检测细胞周期;用Annexin V-FITC/PI双染方法检测细胞凋亡。6.酶标仪比色法:用姜黄素和/或不同剂量的L-FP处理6孔板培养的 MGC-803细胞24h后,用酶标仪比色法检测Caspase-3和Caspase-8的活性,并计算其相对活性。7.Western blot方法:用姜黄素和/或不同剂量的L-FP处理6孔板培养的MGC-803细胞24h后,提取总蛋白,用Western blot法检测Bax和Bcl-2的表达。结果:1.姜黄素联合不同剂量的L-FP对体外培养的胃癌MGC-803细胞生长的影响:用姜黄素和/或不同剂量的L-FP处理胃癌MGC-803细胞24h、48h和72h后,与对照组相比,各实验组均表现出了显著的抑制生长效应(*P0.05或**P0.01或***P0.001);其中药物处理24h和72h的抑制率,姜黄素联合低剂量L-FP组与中剂量L-FP组相比和姜黄素联合中剂量L-FP组与高剂量L-FP组相比,无显著性差异(P0.05);而48h的抑制率,姜黄素联合L-FP组显著高于姜黄素或L-FP单用组(*P0.05)。2.姜黄素联合不同剂量的L-FP对体外培养的胃癌MGC-803细胞克隆形成的影响:与对照组相比,各实验组细胞的克隆形成能力均显著下降(*P0.05);并且姜黄素联合L-FP组抑制率显著高于姜黄素或L-FP单用组(*P0.05)。3.姜黄素联合不同剂量的L-FP对体外培养的胃癌MGC-803细胞迁移的影响:与对照组相比,各实验组通过Transwell小室的细胞量明显降低(*P0.05或**P0.01);其中姜黄素联合L-FP组迁移率与姜黄素或L-FP单用组无显著差异(P0.05)。4.姜黄素联合不同剂量的L-FP对体外培养的胃癌MGC-803细胞周期的影响:与对照组相比,各实验组主要阻滞细胞于S期(*P0.05),而且姜黄素联合L-FP组细胞在S期所占比例显著高于姜黄素或L-FP单用组(*P0.05)。5.姜黄素联合不同剂量的L-FP对体外培养的胃癌MGC-803细胞凋亡的影响:与对照组相比,各实验组均能显著诱导MGC-803细胞发生凋亡(*P0.05),其中姜黄素联合L-FP组的凋亡率显著高于姜黄素或L-FP单用组(*P0.05),并且Annexin-V/PI双染流式细胞仪检测的各组细胞凋亡率与AO/EB双染荧光显微镜检测的各组细胞凋亡率没有显著差异(P0.05)。6.姜黄素联合不同剂量的L-FP对体外培养的胃癌MGC-803细胞相关调控蛋白的影响:与对照组相比,各实验组细胞显著增强了Bax的表达,降低了Bcl-2的表达,提高了Caspase-3和Caspase-8的活性(*P0.05或**P0.01);除了姜黄素联合中剂量L-FP组的Bax相对表达量与高剂量L-FP单用组无显著差异(P0.05),其余姜黄素联合L-FP组的Bax相对表达量显著高于姜黄素或L-FP单用组,Bcl-2相对表达量显著低于姜黄素或L-FP单用组(*P0.05或**P0.01);姜黄素联合低剂量L-FP组的Caspase-3和Caspase-8相对活性与中剂量L-FP单用组无显著差异(P0.05),而姜黄素联合中剂量L-FP组的Caspase-3和Caspase-8相对活性显著高于高剂量L-FP单用组(*P0.05或**P0.01)。结论:1.姜黄素能增强L-FP对体外培养的胃癌MGC-803细胞的生长抑制作用。2.姜黄素能增强L-FP对体外培养的胃癌MGC-803细胞克隆形成和迁移能力的抑制作用。3.姜黄素能增强L-FP对体外培养的胃癌MGC-803细胞周期中S期的阻滞作用。4.姜黄素能增强L-FP对体外培养的胃癌MGC-803细胞凋亡的诱导作用,其机制与抑制Bcl-2表达、促进Bax表达和提高Caspase-3和Caspase-8活性有关。对于细胞凋亡的检测而言,Annexin-V/PI双染流式细胞仪检测方法与AO/EB双染荧光显微镜检测方法的结果无显著性差异。
[Abstract]:Objective: curcumin (curcumin, CUR) is the main active ingredient of turmeric, is a fat soluble phenolic pigment, anti-tumor, anti-inflammatory, inhibiting mutation and so on. The low dose of cisplatin (DDP) and 5- fluorouracil (5-FU) combination therapy (L-FP) is a commonly used chemotherapy treatment of gastric cancer. This paper aims to investigate the effect of curcumin and L-FP on cultured human gastric cancer MGC-803 cell growth, proliferation, migration and apoptosis effect and its mechanism, to provide theoretical and experimental basis for clinical application of curcumin combined with L-FP in the treatment of gastric cancer. Methods: the techniques and methods of using cell biology and molecular biology, to observe the effect of curcumin and / or different doses of L-FP on cultured human gastric cancer MGC-803 cell growth, colony formation, migration, cell cycle, apoptosis and protein related to the regulation of Caspase-3, Caspase-8, Bax and Bcl-2 The following effects. Main outcome measures and methods: 1.MTT experiment: treatment of gastric cancer cell line MGC-803 24h was cultured in 96 well plates respectively with curcumin and / or different doses of L-FP, 48h and 72h, using the ELISA OD was detected in each hole cells, and calculate the cell growth inhibition rate of.2. plate clone formation experiment: Curcumin and / or different doses of L-FP treatment 6 pore plate cultured MGC-803 cells after 24h culture medium not containing drugs to cultured for 10 days, after Giemsa staining counts and colony formation rate of.3.Transwell Migration Experiment: containing curcumin and / or different doses of L-FP cultured Transwell cell in gastric cancer MGC-803 cells, 10h, MTT method to detect and calculate the migration rate of.4.AO/EB double staining with curcumin and / or different doses of L-FP treatment of MGC-803 cells cultured in 24h 6 after AO/EB staining under fluorescence microscope and photographed, observe the cell Form, counting the number of apoptotic cells and apoptotic rate of.5. flow cytometry: Curcumin and / or different doses of L-FP treatment of MGC-803 gastric cancer cells 24h cultured in 6 well plates after stained with PI method to detect cell cycle; colorimetric method with Annexin V-FITC/PI double staining method to detect apoptosis.6. eliasa than by curcumin and / or different doses of L-FP treatment of MGC-803 cells cultured in 24h 6 after Caspase-3 and Caspase-8 detector colorimetric method with enzyme activity, and calculated the relative activity of.7.Western blot method: Curcumin and / or different doses of L-FP treatment of MGC-803 cells cultured in 24h 6 after extraction of total protein the expression of Bax and Bcl-2, with the detection of Western by blot. Results: 1. effects of curcumin combined with different doses of L-FP on the growth of in vitro cultured gastric cancer MGC-803 cells by curcumin and / or different doses of L-FP treatment of MGC-803 gastric cancer cells 24h, 48H And after 72h, compared with the control group, the experimental group showed significant growth inhibition effect (*P0.05 or **P0.01 or ***P0.001); inhibit drug treatment 24h and 72h was compared among them, dose L-FP group and high dose group L-FP curcumin combined with low dose of L-FP group compared with the medium dose L-FP group and curcumin in combination, no significant difference (P0.05); and the 48h inhibition rate of curcumin combined with L-FP group was significantly higher than that of curcumin or L-FP alone group (*P0.05) of.2. L-FP combined with different doses of curcumin on the cloning of gastric cancer MGC-803 cells cultured in vitro formation: compared with the control group, the experimental group cells clone forming ability were significantly decreased (*P0.05); and curcumin combined with inhibition rate of L-FP group was significantly higher than that of curcumin or L-FP alone group (*P0.05).3. L-FP of curcumin combined with different doses of the migration effect on in vitro cultured gastric cancer cells: MGC-803 and control group. Ratio of each experimental group by Transwell assay cells were significantly decreased (*P0.05 or **P0.01); the migration rate of curcumin combined with L-FP group and curcumin or L-FP alone group had no significant difference (P0.05) effect of.4. L-FP combined with different doses of curcumin in vitro cultured gastric cancer MGC-803 cell cycle: compared with the control group, the experiment the main group of cells were arrested in S phase (*P0.05), and curcumin combined with L-FP group the proportion of cells in S phase was significantly higher than that of curcumin or L-FP alone group (*P0.05) of.5. L-FP combined with different doses of curcumin on apoptosis of cultured human gastric cancer cell line MGC-803: compared with the control group, the experimental group were significantly induced apoptosis of MGC-803 cells (*P0.05), the apoptosis of curcumin combined with L-FP group was significantly higher than that of curcumin or L-FP alone group (*P0.05), and Annexin-V/PI double stained cells were detected by flow cytometry. The apoptosis rate With AO/EB double staining of apoptosis were detected by fluorescence microscopy was no significant difference (P0.05) effect of.6. L-FP combined with different doses of curcumin on cultured gastric cancer cells MGC-803 related regulatory protein: compared with the control group, the experimental group cells significantly enhanced the expression of Bax, reduce the expression of Bcl-2, increased by Caspase-3 and the activity of Caspase-8 (*P0.05 or **P0.01); L-FP group in addition to the dose of curcumin combined with Bax in the relative expression of high dose L-FP group had no significant difference (P0.05), the rest of curcumin combined with L-FP group Bax expression was significantly higher than that of curcumin or L-FP alone group, the relative expression of Bcl-2 was significantly lower than that of curcumin or L-FP the single group (*P0.05 or **P0.01); the relative activity of curcumin combined with low dose of L-FP group Caspase-3 and Caspase-8 and L-FP in single dose group had no significant difference (P0.05), and Jiang Huang combined with medium dose L-FP The relative activity of group Caspase-3 and Caspase-8 were significantly higher than that of the high dose L-FP group (*P0.05 or **P0.01). Conclusion: 1..3. curcumin can enhance the inhibitory effect of curcumin.2. inhibition of curcumin on cultured L-FP human gastric cancer cell line MGC-803 growth could enhance the L-FP on cloning of MGC-803 gastric cancer cells in vitro and migration ability to enhance L-FP can enhance the induction effect of L-FP on apoptosis of cultured human gastric cancer cell line MGC-803 to.4. inhibition in vitro of curcumin in gastric cancer MGC-803 cell cycle in S phase, the mechanism and inhibition of Bcl-2 expression, promote Bax expression and increase Caspase-3 and Caspase-8 activities. For the detection of apoptosis, there was no significant difference in Annexin-V/PI staining AO/EB method and flow cytometry staining with fluorescence microscope detection results.

【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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