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目的研究氯化两面针碱(nitidine chloride,NC)处理后p53通路的分子水平变化,以说明p53途径在NC抑制人肝癌细胞SMMC-7721增殖中的作用。方法应用MTT比色法测定NC对SMMC-7721细胞存活率的影响,细胞克隆集落形成实验测定NC对细胞增殖的抑制作用;实时荧光定量PCR检测不同剂量NC对p53、p21mRNA表达的影响;酶联免疫吸附法测定p53、p21蛋白含量的变化。结果 NC(0.15,0.30,0.60,1.20,2.40,4.80 mg.L-1)浓度依赖性地降低SMMC-7721的存活率,作用48 h时IC50值为(1.03±0.18)mg.L-1;NC可明显抑制细胞集落形成,剂量为0.15,0.30 mg.L-1时,抑制率分别是74.65%、88.48%,剂量为0.60 mg.L-1时,抑制率达到了100%;NC可明显提高癌细胞中p53、p21 mRNA和蛋白的表达,并呈浓度依赖性。结论 p53通路可能在NC诱导的SMMC-7721细胞增殖抑制中起重要作用。
Objective To study the molecular changes of p53 pathway after nitidine chloride (NC) treatment in order to demonstrate the role of p53 pathway in inhibiting the proliferation of human hepatoma SMMC-7721 cells. Methods MTT assay was used to determine the effect of NC on the survival rate of SMMC-7721 cells. The colony formation assay was used to determine the effect of NC on cell proliferation. Real-time fluorescence quantitative PCR was used to detect the effect of different doses of NC on the expression of p53 and p21 mRNA. Determination of p53, p21 protein by immunoadsorption assay. Results NC (0.15,0.30,0.60,1.20,2.40,4.80 mg.L-1) could decrease the survival rate of SMMC-7721 in a concentration-dependent manner. The IC50 value at 48 h was (1.03 ± 0.18) mg.L-1. NC inhibited cell colony formation significantly. When the dosage was 0.15,0.30 mg.L-1, the inhibitory rates were 74.65% and 88.48%, respectively, and the inhibition rate reached 100% when the dosage was 0.60 mg.L-1; Improve the expression of p53, p21 mRNA and protein in cancer cells in a concentration-dependent manner. Conclusion p53 pathway may play an important role in NC-induced inhibition of SMMC-7721 cell proliferation.