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目的:研究1,25-二羟维生素D3诱导人喉癌Hep-2细胞凋亡及其对PI3K/AKT/Bcl-2信号通路的影响。方法:用不同剂量(10-8、10-7、10-6 mol/L)1,25-二羟维生素D3处理Hep-2细胞24、48、72 h,四甲基偶氮唑蓝法(MTT)检测Hep-2细胞的增殖情况,计算抑制率。流式细胞术检测Hep-2细胞的凋亡率。Western blot检测用药前后细胞中PI3K、AKT及其磷酸化、Bax和Bcl-2蛋白的表达水平。结果:MTT结果显示,1,25-二羟维生素D3可以抑制Hep-2细胞增殖(P<0.05),1,25-二羟维生素D3浓度为10-6 mol/L抑制率可达30.71%,在上述浓度内随着浓度增加和时间延长,抑制作用逐渐增强,呈时间-剂量依赖性。流式细胞术检测到10-8、10-7、10-6 mol/L 1,25-二羟维生素D3作用48 h后,Hep-2凋亡细胞比例显著增加,凋亡率分别为12.13%、14.05%、16.17%,高于空白对照组的6.82%(P<0.05)。Western blot检测结果显示1,25-二羟维生素D3处理48 h后,Hep-2细胞PI3K、AKT、p-AKT、Bcl-2蛋白表达降低,Bax蛋白表达升高(P<0.05)。结论:在一定浓度范围内,1,25-二羟维生素D3(10-6、10-7、10-8mol/L)能够抑制人喉癌Hep-2细胞增殖,呈时间-剂量依赖性,其抑制作用与诱导细胞凋亡有关;1,25-二羟维生素D3可通过影响PI3K/AKT/Bcl-2,从而诱导Hep-2细胞凋亡。
AIM: To investigate the apoptosis of human laryngeal carcinoma Hep-2 cells induced by 1,25-dihydroxyvitamin D3 and its effect on PI3K / AKT / Bcl-2 signaling pathway. Methods: Hep-2 cells were treated with 1,25-dihydroxyvitamin D3 (10-8,10-7,10-6 mol / L) for 24,48,72 h at different doses (tetramethylthiazole blue method MTT) detection of Hep-2 cell proliferation, calculate the inhibition rate. Flow cytometry was used to detect the apoptosis rate of Hep-2 cells. Western blot was used to detect the expression of PI3K, AKT, phosphorylation of Bax and Bcl-2 in the cells before and after treatment. Results: The results of MTT showed that 1,25-dihydroxyvitamin D3 could inhibit the proliferation of Hep-2 cells (P <0.05), and the inhibitory rate of 1,25-dihydroxyvitamin D3 at 10-6 mol / L was 30.71% With the increase of concentration and prolongation of time, the inhibitory effect gradually increased in a time-and dose-dependent manner. The percentage of apoptotic cells in Hep-2 cells was significantly increased after treated with 10-8,10-7,10-6 mol / L 1,25-dihydroxyvitamin D3 by flow cytometry for 48 h, the apoptotic rates were 12.13% , 14.05% and 16.17% respectively, higher than that of the control group (6.82%, P <0.05). The results of Western blot showed that the expression of PI3K, AKT, p-AKT and Bcl-2 protein and the protein expression of Bax increased in Hep-2 cells treated with 1,25-dihydroxyvitamin D3 for 48 h (P <0.05). CONCLUSION: 1,25-dihydroxyvitamin D3 (10-6,10-7,10-8 mol / L) can inhibit the proliferation of human laryngeal carcinoma Hep-2 cells in a time-and dose-dependent manner 1,25-dihydroxyvitamin D3 can induce apoptosis of Hep-2 cells by affecting PI3K / AKT / Bcl-2.