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目的:体内外观察葫芦素B对人喉癌细胞系Hep-2的抑制增殖和诱导凋亡作用并探讨其作用机制,为临床应用提供实验依据。方法:用0.1、1.0、10.0和100.0μmol/L的葫芦素B作用于Hep-2细胞24、48和72h,MTT法检测细胞增殖。用0.1、1.0和10.0μmol/L的葫芦素B作用于Hep-2细胞24h或10μmol/L葫芦素B作用8、12和24h,流式细胞仪检测细胞周期分布和细胞凋亡率,荧光显微镜观察细胞凋亡。Westernblot检测p-STAT3、cyclinB1和Bcl-2的蛋白表达。建立喉癌裸鼠移植瘤模型,观察葫芦素B的体内抑瘤作用。结果:MTT结果显示葫芦素B对Hep-2细胞的增殖抑制作用具有明显的剂量和时间依赖性;流式细胞仪检测发现随着葫芦素B浓度增高或作用时间延长,处于G2/M期细胞的比例逐渐升高,同时伴有G0/G1期细胞减少,细胞凋亡率逐渐升高,经统计学分析,各实验组之间及其与对照组之间的均差异有统计学意义(P<0.05或P<0.01);荧光显微镜观察可见典型细胞凋亡;Westernblot检测显示葫芦素B可剂量依赖性抑制p-STAT3、cyclinB1和Bcl-2的蛋白表达。体内实验发现低、中、高剂量组的抑瘤率分别是32.43%、43.24%和70.27%。结论:葫芦素B通过抑制STAT3活化,抑制cyclinB1和Bcl-2的蛋白表达,引起喉癌细胞G2/M期阻滞、增殖抑制和凋亡,发挥对喉癌的抗癌效应。
OBJECTIVE: To observe the inhibitory effect of cucurbitacin B on Hep-2 human laryngeal carcinoma cell line Hep-2 in vitro and in vivo and its mechanism of action to provide experimental evidence for clinical application. Methods: Hep-2 cells were treated with 0.1, 1.0, 10.0 and 100.0 μmol / L of cucurbitacin B for 24,48 and 72 h, respectively. The cell proliferation was detected by MTT assay. The cells were treated with 0.1, 1.0 and 10.0 μmol / L of cucurbitacin B for 24h or 10 μmol / L cucurbitacin B for 8, 12 and 24 h respectively. The cell cycle distribution and apoptosis rate were detected by flow cytometry. Fluorescence microscopy Observation of apoptosis. Western blot was used to detect the protein expression of p-STAT3, cyclinB1 and Bcl-2. Establishment of laryngeal carcinoma xenograft model in nude mice to observe the cucurbitacin B in vivo antitumor effect. Results: MTT results showed that the cucurbitacin B inhibited the proliferation of Hep-2 cells in a dose-and time-dependent manner. Flow cytometry showed that with the increasing concentration of cucurbitacin B or the prolonged action time, (P <0.05) .At the same time, the percentage of cells in G0 / G1 phase decreased and the rate of apoptosis increased gradually.According to statistical analysis, there was significant difference between each experimental group and control group (P <0.05 or P <0.01). Apoptosis of typical cells was observed by fluorescence microscopy. Western blot analysis showed that cucurbitacin B could inhibit the protein expression of p-STAT3, cyclinB1 and Bcl-2 in a dose-dependent manner. In vivo experiments found that low, medium and high dose groups of tumor inhibition rates were 32.43%, 43.24% and 70.27%. CONCLUSION: Cucurbitacin B can inhibit G2 / M arrest, proliferation inhibition and apoptosis of laryngeal carcinoma cells by inhibiting the activation of STAT3, inhibiting the expression of cyclinB1 and Bcl-2, and exert anti-cancer effects on laryngeal carcinoma.