摘要
目的观察TTF1影响肝癌HepG-2细胞增殖的情况及其对肝癌HepG-2细胞凋亡的影响,并进一步探讨其可能的凋亡作用机制。
方法采用MTT比色法观察TTF1影响肝癌HepG-2细胞增殖的情况,通过流式细胞仪检测TTF1对HepG-2细胞周期分布的影响,运用免疫细胞化学方法检测凋亡相关基因bcl-2、bax及p53蛋白表达,并用Western blot技术检测Bcl-2、Bax、P53蛋白与Caspase-3、Caspase-8及Caspase-9蛋白表达的情况。
结果TTF1可以抑制肝癌HepG-2细胞的增殖,随着时间的延长和药物浓度的增加,其抑制作用越明显,可见TTF1对肝癌HepG-2细胞的抑制作用具有时间和浓度依赖性,当药物浓度为10μmol/L作用时间为48 h时,对HepG-2细胞的抑制率为47.1%,接近半数抑制浓度(IC50)；TTF1可改变细胞周期分布,10μmol/L TTF1分别作用于HepG-2细胞24 h、48 h、72 h后,G0/G1期比率分别为52.0%、59.6%、64.5%,与对照组比较,有显著性差异(P<0.05),表明多数细胞阻滞在Go/G1期；10μmol/L TTF1分别作用于HepG-2细胞24 h、48 h、72 h后,流式细胞仪检测DNA含量,可见明显的细胞凋亡峰,凋亡率分别为6.3%、7.7%、23.8%,与对照组细胞的凋亡率比较有显著性差异(P<0.01)；用药后与用药前相比,凋亡相关基因bcl-2、bax及p53蛋白表达均有显著性改变(P<0.01)；其中Bcl-2、P53蛋白表达显著下降,Bax蛋白表达显著升高；Caspase-3及Caspase-9随着时间的延长和药物剂量的增加,酶原蛋白条带逐渐变粗,裂解产物逐渐增多,表达逐渐升高,而Caspase-8在时间不同时,随时间变化酶原蛋白条带变粗,裂解产物多,表达升高,在剂量不同时其变化不明显。
结论TTF1可以抑制肝癌HepG-2细胞的增殖,且具有时-效、量-效关系,同时可以改变细胞周期分布,并可引起肝癌HepG-2细胞的凋亡,其作用机制可能与其上调Bax蛋白表达,下调Bcl-2、P53蛋白表达,及激活Caspase-9通过内源性细胞凋亡途径引起Caspase-3级联反应诱导细胞凋亡有关。
Objective:Effects observed TTF1 HepG-2 hepatoma cells and its effect on hepatocellular carcinoma HepG-2 cell apoptosis, and further explore its possible mechanism of apoptosis.
Methods:MTT colorimetric observation of TTF1 affect the proliferation of hepatoma cells HepG-2 situation, by flow cytometry TTF1 on HepG-2 cell cycle distribution, using immunocytochemistry detection of apoptosis-related gene bc 1-2, bax and p53 protein expression detected by Western blot with Bc 1-2, Bax, P53 protein and Caspase-3, Caspase-8 and Caspase-9 protein expression in the case.
Results:TTF1 can inhibit the proliferation of HepG-2 cells, with the time and the concentration of drug increased, the inhibition became more obvious, visible TTF1 on the liver cell line HepG-2 with time and concentration dependent, when concentration was 10μmol/L when the reaction time was 48 h, the inhibition of HepG-2 cells was 47.1%, nearly half of the inhibitory concentration (IC50); TTF1 can change the cell cycle,10μmol/L TTF1 were on the HepG-2 cells for 24 h,48 h,72 h later, G0/G1 phase ratio were 52.0%,59.6%,64.5%, compared with the control group, there were significant differences (P<0.05), indicating that most cells were arrested in G0/G1 phase; 10μmol/ L TTF1 were on the HepG-2 cells for 24 h,48 h,72 h, the DNA content by flow cytometry showed obvious apoptosis peak, the apoptosis rates were 6.3% 7.7%,23.8%, and the apoptosis rate of the control group there was significant difference (P<0.01); compared with the baseline after treatment, apoptosis-related gene bcl-2, bax and p53 protein were significantly change (P<0.01); which Bcl-2, P53 protein was significantly decreased, Bax protein was significantly increased; Caspase-3 and Caspase-9 with the time and the dose increased, zymogen protein band gradually changed thick, cracking products increased gradually, the expression increased gradually, but not Caspase-8 at the same time, over time zymogen protein band became thicker, more pyrolysis products, increased expression, while the change in dose is not obvious.
Conclusion:TTF1 can inhibit the proliferation of HepG-2 cells, and possessed some cases-effective, dose-effect relationship and can change cell cycle distribution, and may cause liver HepG-2 cell apoptosis, its mechanism may be related to increases Bax protein expression, reduced Bcl-2, P53 protein expression, and activation of Caspase-9 through the intrinsic apoptotic pathway induced cascade of Caspase-3 induced apoptosis.
引文
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