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  本文關(guān)鍵詞： 姜黃揮發(fā)油 皮膚鱗癌A431 凋亡 增殖　出處：《貴州醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:研究姜黃揮發(fā)油對人皮膚鱗癌(CSCC)A431(以下簡稱A431)細(xì)胞增殖與凋亡的影響,并探究其凋亡機(jī)制。方法:應(yīng)用不同濃度的姜黃揮發(fā)油分別作用于皮膚鱗癌A431細(xì)胞24h、36h、48h,用Cell Counting Kit-8(CCK-8)檢測增殖抑制率;倒置顯微鏡觀察細(xì)胞形態(tài)學(xué)變化;吖啶橙(AO)/溴化乙錠(EB)雙染色熒光顯微鏡觀察A431細(xì)胞凋亡情況;流式細(xì)胞儀檢測細(xì)胞凋亡;Western blot法檢測半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)表達(dá)量。結(jié)果:本實驗通過CCK-8檢測不同濃度(0、5、10、20、40、80μmol/L)姜黃揮發(fā)油作用人皮膚鱗癌A431細(xì)胞24h、36h、48h后,表現(xiàn)出不同大小的抑制作用,藥物濃度從低濃度至高濃度,抑制率呈增高趨勢,且其作用方式呈現(xiàn)劑量依賴性.作用24h后,當(dāng)藥物濃度達(dá)到10 mg/L,倒置顯微鏡下觀察到當(dāng)加入藥物后,表現(xiàn)為細(xì)胞形態(tài)不規(guī)則,細(xì)胞增殖速度明顯變慢,AO/EB染色后可見部分早期凋亡細(xì)胞,核染色質(zhì)著黃綠色或黃色;當(dāng)藥物濃度達(dá)到20 mg/L,細(xì)胞核固縮,部分腫瘤細(xì)胞膜開始破裂,可見部分細(xì)胞內(nèi)容物釋放,AO/EB染色后可見部分黃色熒光的早期凋亡細(xì)胞和呈橘紅色熒光的晚期凋亡細(xì)胞;當(dāng)藥物濃度達(dá)到40 mg/L,腫瘤細(xì)胞明顯減少,部分細(xì)胞裂解成細(xì)胞碎片,并有大量漂浮細(xì)胞,AO/EB染色后可見大量細(xì)胞核染色質(zhì)呈橘紅色,并可見橘紅色壞死細(xì)胞及碎片;流式細(xì)胞儀檢測表明姜黃揮發(fā)油對皮膚鱗癌A431細(xì)胞具有促進(jìn)凋亡作用,且隨著藥物濃度的增加,呈藥物濃度-凋亡率正相關(guān)性;Western blot法檢測結(jié)果半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)表達(dá)量隨著藥物濃度增加而增加,呈一定范圍范圍內(nèi)濃度依賴性,且同一藥物濃度作用下,Caspase-3蛋白表達(dá)量均多于Caspase-9。結(jié)論:姜黃揮發(fā)油對皮膚鱗癌A431細(xì)胞具有增殖抑制及促進(jìn)其凋亡的作用,其凋亡的機(jī)制可能與上調(diào)Caspase-3、Caspase-9的表達(dá)有關(guān)。
[Abstract]:Objective: to study the effect of volatile oil of turmeric on proliferation and apoptosis of human skin squamous cell carcinoma cell line CSCCnA431 (hereinafter referred to as A431). Methods: different concentrations of volatile turmeric oil were applied to A431 cells of skin squamous cell carcinoma for 24 h and 36 h and 48 h respectively. The inhibitory rate of proliferation was detected by Cell Counting Kit-8 CCK-8. The morphological changes of the cells were observed by inverted microscope. The apoptosis of A431 cells was observed by acridine orange AOA / ethidium bromide (EBB) double staining fluorescence microscope. Apoptosis was detected by flow cytometry. Cysteine aspartate protease 3 (caspase-3) was detected by Western blot. Results: the expression of caspase-9 was detected by CCK-8. 80 渭 mol / L turmeric essential oil treated human skin squamous cell carcinoma A431 cells for 24 h or 36 h for 48 h showed different inhibitory effects, the concentration of drugs ranged from low concentration to high concentration. The inhibition rate was increased in a dose-dependent manner. After 24 hours of treatment, when the drug concentration reached 10 mg / L, it was observed under inverted microscope that when the drug was added. After AO-EB staining, some early apoptotic cells were observed, and the chromatin was yellowish green or yellow. When the drug concentration reaches 20 mg / L, the nucleus shrinks, some tumor cell membranes begin to rupture, and some cell contents are released. After AO/EB staining, some early apoptotic cells with yellow fluorescence and late apoptosis cells with orange fluorescence could be seen. When the drug concentration reached 40 mg / L, the tumor cells decreased significantly, some of the cells cracked into cell fragments, and a large number of floating cells were stained with AO- / EB, and a large number of nuclear chromatin was orange. Orange necrotic cells and fragments can be seen. Flow cytometry analysis showed that turmeric volatile oil could promote apoptosis of skin squamous cell carcinoma A431 cells, and with the increase of drug concentration, there was a positive correlation between drug concentration and apoptosis rate. Cysteine aspartate protease 3 (caspase-3) was detected by Western blot assay. The expression of cysteine aspartate protease-9 caspase-9) increased with the increase of drug concentration in a range of concentration dependent and under the same drug concentration. Conclusion: turmeric volatile oil can inhibit the proliferation and promote the apoptosis of skin squamous cell carcinoma A431 cells. The mechanism of apoptosis may be related to the up-regulation of Caspase-3 and Caspase-9 expression.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.5

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