塞来昔布与茶多酚合用对乳腺癌细胞增殖及凋亡的影响和机制

发布时间：2018-01-20 15:47

本文关键词： 塞来昔布茶多酚细胞凋亡 Bcl-2Bax caspase-3caspase-9　出处：《天津医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：研究塞来昔布与茶多酚合用对乳腺癌MDA-MB-231细胞增殖及凋亡的影响,并初步探讨两药合用诱导细胞凋亡的作用机制。方法： MTT比色法检测塞来昔布与茶多酚单用及合用对细胞的增殖抑制作用。倒置相差显微镜观察对细胞形态学的影响。Hoechst33258荧光染色观察细胞凋亡形态学改变。流式细胞术Annexin V-FITC/PI双染检测细胞凋亡率。Real-timePCR检测Bcl-2和Bax的mRNA的表达。Western Blot检测凋亡相关蛋白Bcl-2、Bax、caspase-3、caspase-9的表达。结果： 1.MTT结果显示,塞来昔布与茶多酚单用均能显著抑制乳腺癌MDA-MB-231细胞的增殖(P0.05)。最小有效量的塞来昔布(10μmol/L)与不同浓度的茶多酚(6.25、12.5、25、50、100μg/mL)合用对细胞的增殖抑制率高于各单药组,差异具有统计学意义(P0.05)。 2.倒置相差显微镜下观察,塞来昔布(10μmol/L)与茶多酚(50.100μg/mL)单药组及合用组均出现了细胞皱缩,折光性变差,细胞碎片增多的现象,且合用组与各单药组比较,正常细胞数明显减少,形态学改变更加明显。 3. Hoechst33258荧光染色后,塞来昔布(10μmol/L)与茶多酚(50、100μg/mL)单药组及合用组均出现了细胞核皱缩,染色质凝聚的典型凋亡形态学特征,且合用组与各单药组比较,凋亡形态学变化更加显著。 4.流式细胞术结果显示,塞来昔布(10μmol/L)与茶多酚(50、100μg/mL)单用及合用均能诱导细胞凋亡,且两药合用的凋亡率显著高于各单药组。 5. Real-time PCR结果显示塞来昔布(10μmol/L)和茶多酚(100μg/mL)能使抑凋亡基因Bcl-2的mRNA的表达降低,促凋亡基因Bax的mRNA的表达升高,且两药合用能起到协同作用。 6. Western Blot结果表明塞来昔布(10μmol/L)和茶多酚(100μg/mL)单用及合用均能显著降低抑凋亡蛋白Bcl-2的表达,提高促凋亡蛋白Bax的表达,并使凋亡相关蛋白caspase-3、caspase-9的活化形式cleaved caspase-3、cleaved caspase-9表达增高,且联合用药组与各单药组比较差异具有统计学意义(P0.05)。结论： 1.塞来昔布与茶多酚单用及合用均能抑制乳腺癌MDA-MB-231细胞的增殖,且联合用药组的增殖抑制率明显高于各单药组。表明两药合用能够增强抑制细胞增殖的作用。 2.塞来昔布与茶多酚单用及合用均能诱导细胞凋亡,两药合用具有协同诱导细胞凋亡的作用。 3.塞来昔布与茶多酚合用诱导细胞凋亡的机制可能为两种药物作用于靶点Bcl-2和Bax,通过下调抑凋亡基因Bcl-2并上调促凋亡基因Bax,经线粒体信号途径,活化caspase-9,从而激活caspase-3,引发下游级联反应,诱导细胞凋亡。
[Abstract]:Objective: To study the effect of celecoxib combined with tea polyphenol on the proliferation and apoptosis of breast cancer MDA-MB-231 cells, and to explore the mechanism of apoptosis induced by the combination of two drugs. Methods: MTT colorimetric assay was used to detect the inhibitory effect of celecoxib and tea polyphenols on cell proliferation. The effect of inverted phase contrast microscope on cell morphology. Hoechst33258 fluorescence staining. Morphological changes of apoptosis. Flow cytometry Annexin. Detection of apoptosis rate by V-FITC / Pi double staining. Real-time PCR to detect mRNA expression of Bcl-2 and Bax. Western. The apoptosis-related protein Bcl-2 was detected by Blot. Expression of caspase-3 and caspase-9 in Baxia caspase-3. Results: 1. MTT results showed that. Both celecoxib and tea polyphenols could significantly inhibit the proliferation of breast cancer MDA-MB-231 cells (P 0.05). The minimum effective dose of celecoxib was 10 渭 mol / L). With different concentrations of tea polyphenols 6.25. The inhibition rate of cell proliferation was significantly higher than that of each single drug group (P 0.05). 2. Under inverted phase contrast microscope, cell shrinkage was observed in the single drug group (celecoxibul 10 渭 mol / L) and tea polyphenol group (50.100 渭 g / mL) and in the combination group. The number of normal cells was significantly decreased and the morphological changes were more obvious in the combined group than in the single drug group. 3. After Hoechst33258 fluorescence staining, nuclear shrinkage was observed in the single drug group (10 渭 mol / L) of celecoxib (10 渭 mol / L) and 50 渭 g / mL tea polyphenols (50 渭 g / mL), as well as in the combination group. The typical apoptotic morphological features of chromatin condensation and the morphological changes of apoptosis in the combined group were more significant than those in the single drug group. 4. Flow cytometry showed that celecoxib (10 渭 mol / L) and tea polyphenol (50 渭 g / mL) alone could induce apoptosis. The apoptotic rate of two drugs combined was significantly higher than that of each single drug group. 5. Real-time PCR results showed that celecoxib (10 渭 mol / L) and tea polyphenols (100 渭 g / mL). It can reduce the expression of mRNA of Bcl-2. The expression of mRNA in apoptotic gene Bax was increased, and the combination of the two drugs could play a synergistic effect. 6. Western Blot results showed that celecoxib (10 渭 mol / L) and tea polyphenols (100 渭 g / mL). Both monotherapy and combination could significantly reduce the expression of Bcl-2. The expression of pro-apoptotic protein Bax and the activation of caspase-3 caspase-9 in the form of cleaved caspase-3 were increased. The expression of cleaved caspase-9 was increased, and there was significant difference between the combined treatment group and the single drug group (P 0.05). Conclusion: 1. Celecoxib combined with tea polyphenols could inhibit the proliferation of breast cancer MDA-MB-231 cells. The inhibition rate of proliferation in combination group was significantly higher than that in single drug group, which indicated that the combination of two drugs could enhance the inhibitory effect of cell proliferation. 2. Celecoxib combined with tea polyphenols could induce apoptosis. 3. The mechanism of apoptosis induced by celecoxib combined with tea polyphenols may be that two drugs act on target Bcl-2 and Bax, down-regulate Bcl-2 and up-regulate the apoptosis-promoting gene Bax. Through mitochondrial signaling pathway, caspase-9 was activated, caspase-3 was activated, downstream cascade reaction was induced and apoptosis was induced.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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