速生桉叶水提物对肝癌细胞株HepG2生长的抑制和凋亡作用

Growth Inhibition and Apoptosis of Heptocellular Carcinoma HepG2 Cells by the Eucalyptus robusta Leaf Water Extract

关键词:  速生桉叶水提物  HepG2细胞  细胞生长  细胞凋亡

Key words:  ELWE  HepG2 cells  cell proliferation  cell apoptosis

摘要:

[目的] 探讨速生桉叶水提物（Eucalyptus robusta leaf water extract，ELWE）在体外对HepG2细胞的抗肿瘤作用及其机制。[方法] 用荧光显微镜观察ELWE对HepG2细胞核形态学的改变，用甲基噻唑基四唑（Methyl thiazolyl tetrazolium，MTT）比色法检测ELWE对HepG2细胞生长的影响；用免疫印迹法（Western blotting，WB）检测ELWE对激活型半胱氨酸蛋白酶3（caspase-3）蛋白的表达影响；用反转录实时荧光定量聚合酶链式反应技术（Real-time reverse transcription polymerase chain reaction，RT-RT-PCR）检测ELWE对Wnt/β-catenin信号通路相关因子β-catenin、Tcf-4、CD44v6表达水平的影响。[结果] ELWE处理HepG2细胞48 h、72 h，稀释5倍和10倍ELWE的处理组细胞均出现凋亡的形态学改变；MTT法检测稀释5倍和10倍的ELWE处理组，在处理48 h细胞生长抑制率分别为（44.13±10.93）%和（25.93±8.37）%，与未处理组相比，两组值差异显著且具有统计学意义（P<0.05）；WB法检测稀释10倍的ELWE处理组，同样在48 h进行凋亡相关蛋白因子caspase-3表达情况的检测，处理组caspase-3蛋白表达水平上调，为对照组的1.73倍，差异显著并具有统计学意义（P<0.01）；同样处理组的β-catenin、Tcf-4、CD44v6 3个基因的mRNA表达水平均下降，与对照组相比差异均显著并均具有统计学意义（P<0.01）。[结论] ELWE在10倍稀释处理48 h条件下对HepG2细胞生长有抑制作用和促进细胞凋亡的作用。该作用与caspase-3介导的凋亡程序和Wnt/β-catenin信号通路有关。

Abstract:

[Objective] To investigate the anti-tumor effect and the mechanism of Eucalyptus robusta leaf water extract (ELWE) on HepG2 cells in vitro.[Methods] The antitumor activity of ELWE were studied by observing morphological changes and nuclear damage of HepG2 cells by fluorescence microscope. The effect of ELWE on the growth of HepG2 cells was detected by methyl thiazolyl tetrazolium (MTT) colorimetry. Western blotting (WB) was used to detect the effect of ELWE on the expression of activated caspase-3 protein. The effect of ELWE on the expression levels of β-catenin, Tcf-4 and CD44v6, which were signal pathway correlation factors of Wnt/β-catenin, was detected by real-time reverse transcription polymerase chain reaction (RT-RT-PCR).[Results] The HepG2 cells were treated by ELWE for 48 h and 72 h, the morphological changes of apoptosis were observed in the treatment group of ELWE which was diluted 5 times and 10 times. Using MTT method to detect ELWE treatment group which was diluted 5 times and 10 times,HepG2 cells growth inhibition ratio were (44.13±10.93)% and (25.93±8.37)% respectively after 48 h.Compared with the untreated group, the difference between the two groups was significant and had statistical significance (P<0.05). The ELWE treatment group was diluted 10 times by WB method. The expression of apoptosis-related protein factor caspase-3 was also detected at 48 h. The expression level of caspase-3 protein was up-regulated in the treatment group, which was 1.73 times that of the control group. The difference was significant and had statistical significance (P<0.01).Moreover,the mRNA expression levels of β-catenin, Tcf-4 and CD44v6 in the same treatment group decreased,and the difference was significant and had statistical significance compared with the control group (P<0.01).[Conclusion] ELWE inhibited the growth of HepG2 cells and promoted apoptosis in a 10-fold dilution treatment for 48 h. This effect was related to the caspase-3 mediated apoptosis program and the Wnt/β-catenin signal pathway.