苦参碱通过ERK信号通路对HepG2肝癌细胞增殖及迁移的影响

发布时间：2019-05-15 03:53

【摘要】：目的:观察苦参碱对肝癌细胞的抑制效果及机制。方法:选取HepG2肝癌细胞系,MTT筛选苦参碱作用于HepG2肝癌细胞的浓度梯度,并观察苦参碱对HepG2肝癌细胞形态学的影响。实验分为空白对照组、实验组、阳性对照组3组,空白对照组加入不含药的细胞培养基,实验组分别加入苦参碱,使其终浓度为1、2、4 mg/mL,阳性对照组加用终浓度为20umol/L的ERK信号通路阻断剂PD98059,培养24小时后,MTT检测细胞增殖、Transwell小室检测细胞迁移数目、逆转录聚合酶链反应(RT-PCR)检测ERK信号通路上的关键蛋白ERK的核酸表达变化,蛋白质免疫印迹法(Western-Blot)检测ERK、p-ERK蛋白质表达变化。结果:1.细胞形态学变化:空白对照组细胞排列紧密,呈梭形,形态饱满,贴壁良好;实验组细胞形态随着浓度升高细胞密度逐渐减低,贴壁不牢,并在高浓度时出现坏死的细胞碎片。2.MTT结果显示,苦参碱对HepG2细胞增殖的抑制作用具有时间和浓度依赖性。并且在24h时间段内1 mg/m L、2 mg/mL、4 mg/mL浓度时苦参碱对HepG2作用最敏感。3.细胞迁移能力变化:实验组与空白对照组相比,1 mg/m L、2 mg/m L、4mg/m L组细胞迁移能力显著减少(P0.01),4 mg/m L与阳性对照组比较无差异(P0.05)。4.ERK核酸表达:经苦参碱作用后ERK mRNA的表达量显著下降,实验组中4 mg/m L、2 mg/m L与空白对照组相比有差异(P0.05);实验组与阳性对照组比较核酸表达量未下降,4mg/m L、2mg/m L有差异(P0.05)。5.ERK和p-ERK蛋白的表达:实验组与空白对照组相比,p-ERK蛋白表达量在2 mg/m L、4 mg/mL浓度时下调显著,有统计学意义(P0.01),ERK蛋白表达量在2 mg/m L、4 mg/m L浓度时下调显著,有统计学差异(P0.05);与阳性对照组相比,1 mg/m L、2 mg/mL苦参碱作用后p-ERK蛋白下调量不及PD98059(P0.05),且1 mg/mL具有显著性差异(P0.01)。结论:1.苦参碱可抑制HepG2肝癌细胞的增殖和迁移,其具体机制可能通过下调ERK信号转导通路中的关键蛋白p-ERK和ERK蛋白的表达。2.苦参碱对HepG2肝癌细胞的抑制作用具有时间和浓度依赖性。
[Abstract]:Objective: to observe the inhibitory effect and mechanism of Matrine on HCC cells. Methods: HepG2 HCC cell line was selected and MTT was selected to screen the concentration gradient of Matrine on HepG2 HCC cells, and the effect of Matrine on the morphology of HepG2 HCC cells was observed. The experiment was divided into three groups: blank control group, experimental group and positive control group. The blank control group was supplemented with non-drug cell culture medium, and the experimental group was added with Matrine to make the final concentration of Matrine 1, 2, 4 mg/mL,. The positive control group was cultured with ERK signal pathway blocker PD98059, with final concentration of 20umol/L for 24 hours. The proliferation of cells was detected by MTT and the number of cell migration was detected by Transwell chamber. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the nucleic acid expression of the key protein ERK in ERK signaling pathway, and Western-Blot was used to detect the expression of ERK,p-ERK protein. Result: 1. Morphological changes of cells in the blank control group: the cells in the blank control group were closely arranged, fusiform, full in shape and good adherent to the wall. The cell morphology of the experimental group decreased gradually with the increase of concentration, the adherent cell density was not firm, and the cell fragments appeared at high concentration. 2. The results showed that the inhibitory effect of Matrine on the proliferation of HepG2 cells was time-and concentration-dependent. At the concentration of 1 mg/mL, 2 mg/mL,4 mg/mL in 24 h, Matrine was the most sensitive to HepG2. The changes of cell migration ability: compared with the blank control group, the cell migration ability of the experimental group was significantly lower than that of the blank control group (1 mg/m / L, 2 mg/m / L, 4 mg 路L) (P 0.01). There was no significant difference in the expression of ERK mRNA between the 4 mg/m / L group and the positive control group (P 0.05). 4. The expression of ERK nucleic acid: the expression of ERK decreased significantly after treatment with Matrine, and the expression of ERK in the experimental group was 4 mg/m / L. 2There was significant difference between mg/m / L and blank control group (P 0.05). Compared with the positive control group, the expression of nucleic acid in the experimental group was not decreased, but the expression of 4mg/m L and 2 mg / L was different (P 0.05). 5. The expression of ERK and p-ERK protein in the experimental group was higher than that in the blank control group. The expression of p-ERK protein decreased significantly at the concentration of 2 mg/mL and 4 mg/mL, which was statistically significant (P 0.01). The expression of), ERK protein decreased significantly at the concentration of 2 mg/mL and 4 mg/mL (P 0.05). Compared with the positive control group, the down regulation of p-ERK protein in 1 mg/mL and 2 mg/mL Matrine was lower than that in PD98059 (P 0.05), and there was significant difference in 1 mg/mL (P 0.01). Conclusion: 1. Matrine can inhibit the proliferation and migration of HepG2 hepatocellular carcinoma cells, and its mechanism may be by down-regulating the expression of p-ERK and ERK proteins in ERK signal transduction pathway. 2. The inhibitory effect of Matrine on HepG2 HCC cells was time-and concentration-dependent.
【学位授予单位】：甘肃中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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