姜黄挥发油对皮肤鳞癌A431细胞增殖及凋亡的影响

发布时间：2018-01-26 09:35

本文关键词： 姜黄挥发油皮肤鳞癌A431 凋亡增殖　出处：《贵州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究姜黄挥发油对人皮肤鳞癌(CSCC)A431(以下简称A431)细胞增殖与凋亡的影响,并探究其凋亡机制。方法:应用不同浓度的姜黄挥发油分别作用于皮肤鳞癌A431细胞24h、36h、48h,用Cell Counting Kit-8(CCK-8)检测增殖抑制率;倒置显微镜观察细胞形态学变化;吖啶橙(AO)/溴化乙锭(EB)双染色荧光显微镜观察A431细胞凋亡情况;流式细胞仪检测细胞凋亡;Western blot法检测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)表达量。结果:本实验通过CCK-8检测不同浓度(0、5、10、20、40、80μmol/L)姜黄挥发油作用人皮肤鳞癌A431细胞24h、36h、48h后,表现出不同大小的抑制作用,药物浓度从低浓度至高浓度,抑制率呈增高趋势,且其作用方式呈现剂量依赖性.作用24h后,当药物浓度达到10 mg/L,倒置显微镜下观察到当加入药物后,表现为细胞形态不规则,细胞增殖速度明显变慢,AO/EB染色后可见部分早期凋亡细胞,核染色质着黄绿色或黄色;当药物浓度达到20 mg/L,细胞核固缩,部分肿瘤细胞膜开始破裂,可见部分细胞内容物释放,AO/EB染色后可见部分黄色荧光的早期凋亡细胞和呈橘红色荧光的晚期凋亡细胞;当药物浓度达到40 mg/L,肿瘤细胞明显减少,部分细胞裂解成细胞碎片,并有大量漂浮细胞,AO/EB染色后可见大量细胞核染色质呈橘红色,并可见橘红色坏死细胞及碎片;流式细胞仪检测表明姜黄挥发油对皮肤鳞癌A431细胞具有促进凋亡作用,且随着药物浓度的增加,呈药物浓度-凋亡率正相关性;Western blot法检测结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)表达量随着药物浓度增加而增加,呈一定范围范围内浓度依赖性,且同一药物浓度作用下,Caspase-3蛋白表达量均多于Caspase-9。结论:姜黄挥发油对皮肤鳞癌A431细胞具有增殖抑制及促进其凋亡的作用,其凋亡的机制可能与上调Caspase-3、Caspase-9的表达有关。
[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.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.5

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