本文關(guān)鍵詞： 塞來(lái)昔布 茶多酚 細(xì)胞凋亡 Bcl-2Bax caspase-3caspase-9　出處：《天津醫(yī)科大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的： 研究塞來(lái)昔布與茶多酚合用對(duì)乳腺癌MDA-MB-231細(xì)胞增殖及凋亡的影響,并初步探討兩藥合用誘導(dǎo)細(xì)胞凋亡的作用機(jī)制。 方法： MTT比色法檢測(cè)塞來(lái)昔布與茶多酚單用及合用對(duì)細(xì)胞的增殖抑制作用。倒置相差顯微鏡觀察對(duì)細(xì)胞形態(tài)學(xué)的影響。Hoechst33258熒光染色觀察細(xì)胞凋亡形態(tài)學(xué)改變。流式細(xì)胞術(shù)Annexin V-FITC/PI雙染檢測(cè)細(xì)胞凋亡率。Real-timePCR檢測(cè)Bcl-2和Bax的mRNA的表達(dá)。Western Blot檢測(cè)凋亡相關(guān)蛋白Bcl-2、Bax、caspase-3、caspase-9的表達(dá)。 結(jié)果： 1.MTT結(jié)果顯示,塞來(lái)昔布與茶多酚單用均能顯著抑制乳腺癌MDA-MB-231細(xì)胞的增殖(P0.05)。最小有效量的塞來(lái)昔布(10μmol/L)與不同濃度的茶多酚(6.25、12.5、25、50、100μg/mL)合用對(duì)細(xì)胞的增殖抑制率高于各單藥組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 2.倒置相差顯微鏡下觀察,塞來(lái)昔布(10μmol/L)與茶多酚(50.100μg/mL)單藥組及合用組均出現(xiàn)了細(xì)胞皺縮,折光性變差,細(xì)胞碎片增多的現(xiàn)象,且合用組與各單藥組比較,正常細(xì)胞數(shù)明顯減少,形態(tài)學(xué)改變更加明顯。 3. Hoechst33258熒光染色后,塞來(lái)昔布(10μmol/L)與茶多酚(50、100μg/mL)單藥組及合用組均出現(xiàn)了細(xì)胞核皺縮,染色質(zhì)凝聚的典型凋亡形態(tài)學(xué)特征,且合用組與各單藥組比較,凋亡形態(tài)學(xué)變化更加顯著。 4.流式細(xì)胞術(shù)結(jié)果顯示,塞來(lái)昔布(10μmol/L)與茶多酚(50、100μg/mL)單用及合用均能誘導(dǎo)細(xì)胞凋亡,且兩藥合用的凋亡率顯著高于各單藥組。 5. Real-time PCR結(jié)果顯示塞來(lái)昔布(10μmol/L)和茶多酚(100μg/mL)能使抑凋亡基因Bcl-2的mRNA的表達(dá)降低,促凋亡基因Bax的mRNA的表達(dá)升高,且兩藥合用能起到協(xié)同作用。 6. Western Blot結(jié)果表明塞來(lái)昔布(10μmol/L)和茶多酚(100μg/mL)單用及合用均能顯著降低抑凋亡蛋白Bcl-2的表達(dá),提高促凋亡蛋白Bax的表達(dá),并使凋亡相關(guān)蛋白caspase-3、caspase-9的活化形式cleaved caspase-3、cleaved caspase-9表達(dá)增高,且聯(lián)合用藥組與各單藥組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論： 1.塞來(lái)昔布與茶多酚單用及合用均能抑制乳腺癌MDA-MB-231細(xì)胞的增殖,且聯(lián)合用藥組的增殖抑制率明顯高于各單藥組。表明兩藥合用能夠增強(qiáng)抑制細(xì)胞增殖的作用。 2.塞來(lái)昔布與茶多酚單用及合用均能誘導(dǎo)細(xì)胞凋亡,兩藥合用具有協(xié)同誘導(dǎo)細(xì)胞凋亡的作用。 3.塞來(lái)昔布與茶多酚合用誘導(dǎo)細(xì)胞凋亡的機(jī)制可能為兩種藥物作用于靶點(diǎn)Bcl-2和Bax,通過(guò)下調(diào)抑凋亡基因Bcl-2并上調(diào)促凋亡基因Bax,經(jīng)線粒體信號(hào)途徑,活化caspase-9,從而激活caspase-3,引發(fā)下游級(jí)聯(lián)反應(yīng),誘導(dǎo)細(xì)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R96

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