【摘要】：目的：本實(shí)驗(yàn)擬通過(guò)MTT法、吖啶橙（AO）/溴乙啶(EB）熒光染色法、流式細(xì)胞術(shù)、Western印跡等方法，觀察尼美舒利在體外對(duì)膠質(zhì)瘤U87細(xì)胞增殖和凋亡的影響，并對(duì)其作用機(jī)制進(jìn)行初步探討。 方法：取膠質(zhì)瘤U87細(xì)胞培養(yǎng)18小時(shí)，根據(jù)尼美舒利作用的不同濃度分組：0.05、0.1、0.2、0.4、0.8mmol/L，不同的作用時(shí)間分組：12、24、48h；然后采用四甲基耦氮唑藍(lán)（MTT）法計(jì)算膠質(zhì)瘤U87細(xì)胞抑制率；吖啶橙（AO）/溴乙啶（EB）熒光染色法和AnnexinV-FITC/PI雙染法流式細(xì)胞術(shù)檢測(cè)U87細(xì)胞凋亡率的變化；最后使用Westem印跡法觀察U87細(xì)胞中Cox-2、Bcl-2和Bax蛋白表達(dá)的變化。 結(jié)果：（1）MTT法分析，發(fā)現(xiàn)用含不同濃度的尼美舒利溶液分別培養(yǎng)U87細(xì)胞12、24、48小時(shí)后，細(xì)胞生長(zhǎng)被明顯抑制，且與藥物作用時(shí)間-濃度正相關(guān)。（2）吖啶橙（AO）/溴乙啶(EB）熒光染色和透射式熒光顯微鏡觀察顯示尼美舒利使膠質(zhì)瘤U87細(xì)胞發(fā)生顯著的凋亡形態(tài)改變；進(jìn)一步應(yīng)用AnnexinV-FITC/PI雙染法流式細(xì)胞儀分析結(jié)果表明，尼美舒利主要使U87細(xì)胞發(fā)生在晚期凋亡，且隨尼美舒利濃度的增加細(xì)胞凋亡率明顯上升。（3）Western印跡凝膠電泳法發(fā)現(xiàn)用0.2、0.4、0.8mmol/L的尼美舒利溶液作用于U87細(xì)胞24小時(shí)后，尼美舒利不僅能劑量性地降低U87細(xì)胞中Bcl-2、Cox-2蛋白的表達(dá)，同時(shí)也可劑量依賴性地升高Bax蛋白的表達(dá)。 結(jié)論：（1）尼美舒利在體外能有效地抑制膠質(zhì)瘤U87細(xì)胞的生長(zhǎng)，且細(xì)胞抑制率呈現(xiàn)時(shí)間-劑量依賴性；（2）尼美舒利能促進(jìn)膠質(zhì)瘤U87細(xì)胞凋亡，，多發(fā)生在晚期，且細(xì)胞凋亡率呈劑量依賴性；（3）尼美舒利在體外能顯著抑制膠質(zhì)瘤U87細(xì)胞的增殖并誘導(dǎo)其凋亡，其作用機(jī)制可能是通過(guò)抑制Cox-2蛋白和下調(diào)Bcl-2蛋白的表達(dá)，升高Bax蛋白的表達(dá)來(lái)實(shí)現(xiàn)其抗腫瘤作用。
[Abstract]:Aim: to observe the effect of nimesulide on the proliferation and apoptosis of U87 cells in vitro by MTT, acridine orange (AO) / ethidium (EB) fluorescence staining, flow cytometry and Western blot, and to explore its mechanism. Methods: U87 glioma cells were cultured for 18 hours. According to the different concentrations of nimesulide, the cells were divided into 0. 05 mmol / L, 0. 2, 0. 4 and 0. 8 mmol / L, respectively. The inhibitory rate of U87 cells was calculated by tetramethyl azolium blue (MTT) assay. The apoptosis rate of U87 cells was detected by flow cytometry with acridine orange (AO) / ethidium bromide (EB) fluorescent staining and AnnexinV-FITC/PI double staining, and the expression of Cox-2,Bcl-2 and Bax in U87 cells was observed by Westem blot. Results: (1) MTT assay showed that the growth of U87 cells cultured with different concentrations of nimesulide for 48 hours was significantly inhibited. (2) fluorescence staining of acridine orange (AO) / bromoethidime (EB) and transmission fluorescence microscopy showed that nimesulide induced apoptosis in U87 cells. The results of AnnexinV-FITC/PI double staining flow cytometry showed that nimesulide mainly caused U87 cell apoptosis in the late stage. The apoptotic rate of U87 cells increased with the increase of nimesulide concentration. (3) Western blot gel electrophoresis showed that nimesulide not only decreased the expression of Bcl-2,Cox-2 protein in U87 cells, but also decreased the expression of Bcl-2,Cox-2 protein in U87 cells after 24 hours treated with 0.2nil 0.4nmol / L nimesulide solution. At the same time, the expression of Bax protein was increased in a dose-dependent manner. Conclusion: (1) nimesulide can effectively inhibit the growth of U87 glioma cells in vitro, and the inhibition rate is time-dose dependent, (2) nimesulide can promote the apoptosis of U87 glioma cells, most of them occur in the late stage. (3) nimesulide could significantly inhibit the proliferation and induce the apoptosis of U87 glioma cells in vitro by inhibiting the expression of Cox-2 protein and down-regulating the expression of Bcl-2 protein. Increase the expression of Bax protein to achieve its anti-tumor effect.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41

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相關(guān)期刊論文 前3條

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本文編號(hào)：2273150