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目的:观察华蟾素注射液对食管癌细胞ECA109增殖的抑制作用,分析华蟾素联合X射线照射对细胞周期的影响,探讨华蟾素注射液与放疗联合效应的机制。方法:实验分空白组、华蟾素组、放疗组、华蟾素+放疗组,作用食管癌ECA109细胞48 h后,应用噻唑蓝(MTT)比色法检测华蟾素注射液对ECA109细胞增殖的影响;流式细胞术(FCM)检测细胞周期的变化,反转录-PCR(RT-PCR)和免疫印迹法(Western blot)检测各组泛素连接酶(RNF2),p16和周期蛋白依赖性激酶(CDK4)mRNA和蛋白表达。结果:华蟾素可显著抑制食管癌ECA109细胞的增殖,且呈浓度和时间依赖性。与空白组比较,各药物组中DNA合成前期(G0/G1期)细胞比例明显升高(P<0.05),S期细胞比例明显下降(P<0.05)。与空白组比较,华蟾素+放疗组中RNF2 mRNA和蛋白明显降低(P<0.05),且各药物组均能促进p16 mRNA和蛋白表达(P<0.05),抑制CDK4 mRNA和蛋白水平(P<0.05)。结论:华蟾素注射液能有效抑制人食管癌ECA109细胞增殖,并将细胞阻滞于G0/G1期,机制可能与降低RNF2,CDK4水平,上调p16基因水平相关。
OBJECTIVE: To observe the inhibitory effect of cinobufacini injection on the proliferation of esophageal carcinoma cell line ECA109, and to study the effect of cinobufacini combined with X-ray irradiation on the cell cycle and to explore the mechanism of the combined effect of cinobufacini injection and radiotherapy. Methods: The ECA109 cells were divided into blank group, cinobufacini group, radiotherapy group, cinobufotalin + radiotherapy group and ECA109 cells for 48 h. MTT assay was used to detect the proliferation of ECA109 cells . The changes of cell cycle were detected by flow cytometry (FCM). The expressions of ubiquitin ligase (RNF2), p16 and cyclin dependent proteins in each group were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot Sex kinase (CDK4) mRNA and protein expression. Results: Cinobufacini significantly inhibited the proliferation of ECA109 cells in a concentration-and time-dependent manner. Compared with the blank group, the percentage of cells in the pre-G0 / G1 phase (P <0.05) and the proportion of cells in the S phase were significantly decreased in each drug group (P <0.05). Compared with the blank group, RNF2 mRNA and protein were significantly decreased in the cinobufotalin + radiotherapy group (P <0.05), and all the drug groups were able to promote the expression of p16 mRNA and protein (P <0.05), and inhibit the expression of CDK4 mRNA and protein <0.05). CONCLUSION: Cinobufacini injection can effectively inhibit the proliferation of human esophageal carcinoma ECA109 cells and arrest the cells in G0 / G1 phase. The mechanism may be related to decreasing the levels of RNF2 and CDK4 and up-regulating the level of p16 gene.