白藜芦醇对人肝癌细胞株Bel-7402作用的实验研究
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摘要
目的：研究白藜芦醇(resverartol, Res)对人肝癌细胞株Bel-7402增殖、凋亡及其沉默信息调节因子1 (silent information regulator 1, SIRT1)表达的影响。探讨Res在人类肝癌防治中的作用及其机制。
方法：四甲基偶氮唑蓝(Methyl thiazolyl tetrazolium, MTT)法检测不同浓度(0,12.5,25,50,100,200μM) Res和不同作用时间(24,48,72h)处理人肝癌细胞株Bel-7402的细胞抑制率；倒置显微镜和荧光显微镜观察细胞形态学改变；流式细胞术检测细胞的凋亡率和细胞周期分布；RT-PCR法检测SIRT1 mRNA的表达；western blotting法检测SIRT1蛋白的表达。
结果：Res在12.5～200μM浓度,作用时间24～72h范围内,以浓度和时间依赖方式抑制Bel-7402细胞增殖(P<0.05)；72小时半数抑制浓度为62.84μM; 200μM Res抑制率达77.62+1.51%(P<0.05)。倒置显微镜和荧光显微镜下分别观察到典型的细胞增殖抑制和凋亡的形态学改变。经0,12.5,25,50,100,200μM Res处理24 h后,流式细胞术结果显示,凋亡细胞的细胞周期主要发生S期阻滞；细胞凋亡率分别为2.56%、16.8%、20.4%、35.8%、38.0%、42.3%(P<0.01)。RT-PCR和western blotting结果显示,经相同处理(0～200μM Res处理24 h)后,Bel-7402细胞SIRT1 mRNA和蛋白表达均明显增加(P<0.01),在Res浓度为25μM时达到峰值,然后逐渐下降。
结论：Res通过增加SIRT1表达以浓度依赖性和时间依赖性方式抑制人肝癌细胞株Bel-7402增殖,诱导其凋亡。
Objectives To investigate the effect of resveratrol on the proliferation, apoptosis, and expression level of silent information regulator 1 (SIRT1) of human hepatocarcinoma cell line Bel-7402.
Methods Human hepatocarcinoma cell line Bel-7402 was cultured and treated with different concentrations(0,12.5,25,50,100, and 200μM) of resveratrol for 24,48 and 72 hours. The growth inhibition rate of Bel-7402 cells was detected by MTT method. The morphological changes of Bel-7402 cells were observed by the inverted microscope and the fluorescent microscope. The flow cytometry was applied to analyze the cell apoptosis rate and cell distribution in the cell cycle. The expression level of SIRT1, mRNA and protein production, was tested with RT-PCR and Western blotting, respectively.
Results Resveratrol inhibited the proliferation of Bel-7402 cells in a time- and concentration- dependent manner ranging from 12.5 to 200μM and from 24 to 72 hours. After 72 hours of treatment, IC5o for resveratrol was 62.84μM; After treatment with 200μM resveratrol, the inhibition rate for Bel-7402 cells was 77.62±1.51%(P<0.05). Both the inverted microscope and the fluorescent microscope showed typical morphological changes in Bel-7402 cells treated with resveratrol. Results of the flow cytometry showed that resveratrol induced Bel-7402 cell cycle is mainly with S-phase arrest. After treatment with the concentration of 0,12.5,25,50,100, and 200μM resveratrol for 24 hours, the apoptosis rate of Bel-7402 cells was 2.56%,16.8%,20.4%,35.8%, 38.0%and 42.3%, respectively, the expression level of SIRT1 mRNA and SIRT1 protein production was significantly increased (P<0.01), and reached the peaked at the concentration of 25μM resveratrol treatment, then declined gradually as resveratrol concentration level increases.
Conclusions Resveratrol can inhibit the proliferation and induce the apoptosis of Bel-7402 cells in a time- and concentration-dependent manner by increasing the expression level of SIRT1.
方法：四甲基偶氮唑蓝(Methyl thiazolyl tetrazolium, MTT)法检测不同浓度(0,12.5,25,50,100,200μM) Res和不同作用时间(24,48,72h)处理人肝癌细胞株Bel-7402的细胞抑制率；倒置显微镜和荧光显微镜观察细胞形态学改变；流式细胞术检测细胞的凋亡率和细胞周期分布；RT-PCR法检测SIRT1 mRNA的表达；western blotting法检测SIRT1蛋白的表达。
结果：Res在12.5～200μM浓度,作用时间24～72h范围内,以浓度和时间依赖方式抑制Bel-7402细胞增殖(P<0.05)；72小时半数抑制浓度为62.84μM; 200μM Res抑制率达77.62+1.51%(P<0.05)。倒置显微镜和荧光显微镜下分别观察到典型的细胞增殖抑制和凋亡的形态学改变。经0,12.5,25,50,100,200μM Res处理24 h后,流式细胞术结果显示,凋亡细胞的细胞周期主要发生S期阻滞；细胞凋亡率分别为2.56%、16.8%、20.4%、35.8%、38.0%、42.3%(P<0.01)。RT-PCR和western blotting结果显示,经相同处理(0～200μM Res处理24 h)后,Bel-7402细胞SIRT1 mRNA和蛋白表达均明显增加(P<0.01),在Res浓度为25μM时达到峰值,然后逐渐下降。
结论：Res通过增加SIRT1表达以浓度依赖性和时间依赖性方式抑制人肝癌细胞株Bel-7402增殖,诱导其凋亡。
Objectives To investigate the effect of resveratrol on the proliferation, apoptosis, and expression level of silent information regulator 1 (SIRT1) of human hepatocarcinoma cell line Bel-7402.
Methods Human hepatocarcinoma cell line Bel-7402 was cultured and treated with different concentrations(0,12.5,25,50,100, and 200μM) of resveratrol for 24,48 and 72 hours. The growth inhibition rate of Bel-7402 cells was detected by MTT method. The morphological changes of Bel-7402 cells were observed by the inverted microscope and the fluorescent microscope. The flow cytometry was applied to analyze the cell apoptosis rate and cell distribution in the cell cycle. The expression level of SIRT1, mRNA and protein production, was tested with RT-PCR and Western blotting, respectively.
Results Resveratrol inhibited the proliferation of Bel-7402 cells in a time- and concentration- dependent manner ranging from 12.5 to 200μM and from 24 to 72 hours. After 72 hours of treatment, IC5o for resveratrol was 62.84μM; After treatment with 200μM resveratrol, the inhibition rate for Bel-7402 cells was 77.62±1.51%(P<0.05). Both the inverted microscope and the fluorescent microscope showed typical morphological changes in Bel-7402 cells treated with resveratrol. Results of the flow cytometry showed that resveratrol induced Bel-7402 cell cycle is mainly with S-phase arrest. After treatment with the concentration of 0,12.5,25,50,100, and 200μM resveratrol for 24 hours, the apoptosis rate of Bel-7402 cells was 2.56%,16.8%,20.4%,35.8%, 38.0%and 42.3%, respectively, the expression level of SIRT1 mRNA and SIRT1 protein production was significantly increased (P<0.01), and reached the peaked at the concentration of 25μM resveratrol treatment, then declined gradually as resveratrol concentration level increases.
Conclusions Resveratrol can inhibit the proliferation and induce the apoptosis of Bel-7402 cells in a time- and concentration-dependent manner by increasing the expression level of SIRT1.
引文
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