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目的:研究蟾毒灵诱导人胰腺癌细胞凋亡以及凋亡相关基因表达的JNK信号转导通路,揭示其抗胰腺癌的部分机制.方法:MTT法观察蟾毒灵对人胰腺癌BxPC-3细胞的生长抑制作用;0.16、0.32、0.64mg/L蟾毒灵分别作用人胰腺癌BxPC-3细胞48h后,流式细胞仪(flow cytometry,FCM)检测细胞周期和细胞凋亡;Western印迹法检测蟾毒灵作用BxPC-3细胞后SAPK/JNK信号通路的激活情况,荧光定量PCR检测Survivin基因mRNA的表达水平;并比较阻断JNK信号通路后丹参酮ⅡA对胰腺癌细胞凋亡Survivin基因mRNA的表达.结果:MTT法测得蟾毒灵对人胰腺癌BxPC-3细胞具有显著的抑制作用,其作用效果与剂量和作用时间成正相关;0.16、0.32、0.64mg/L浓度蟾毒灵作用人胰腺癌细胞后的细胞凋亡率分别为19.36%±0.39%、40.69%±0.44%、59.63%±1.14%,与对照组2.24%±0.37%比较均有显著性差异(P<0.01);蟾毒灵作用人胰腺癌细胞1h后JNK信号通路被激活,2h达峰值;阻断JNK信号通路后,凋亡率明显降低(P<0.01);0.32mg/L蟾毒灵作用人胰腺癌细胞48h后Survivin mRNA的表达明显下降;阻断JNK信号通路后,蟾毒灵作用人胰腺癌细胞的Survivin mRNA的表达明显上升.结论:蟾毒灵通过JNK信号转导通路下调人胰腺癌BxPC-3细胞Survivin mRNA的表达,可能是其诱导胰腺癌细胞凋亡的机制.
OBJECTIVE: To study the JNK signal transduction pathway induced by bufalin in human pancreatic cancer cell apoptosis and related gene expression, and to reveal its mechanism of anti-pancreatic cancer.Methods: MTT assay was used to detect the expression of Buprelin in human pancreatic cancer BxPC-3 The inhibitory effects of Bufalin on human pancreatic cancer BxPC-3 cells were evaluated by flow cytometry (FCM) at different concentrations of 0.16, 0.32 and 0.64 mg / L bufalin for 48 h. Western blotting To detect the activation of SAPK / JNK signaling pathway after buprenorphine treatment of BxPC-3 cells and to detect the expression of Survivin mRNA by fluorescence quantitative PCR. Compare the inhibitory effects of tanshinone ⅡA on the expression of Survivin mRNA Expression.Results: MTT assay showed that Bufalin had a significant inhibitory effect on human pancreatic cancer BxPC-3 cells, and its effect was positively correlated with dose and duration of action; and buprenorphine at concentrations of 0.16, 0.32 and 0.64 mg / L The apoptosis rates of pancreatic cancer cells were 19.36% ± 0.39%, 40.69% ± 0.44% and 59.63% ± 1.14%, respectively, which were significantly different from those of control group (2.24% ± 0.37%, P <0.01) Poison effect of human pancreatic cancer cells after 1h JNK signaling pathway is activated, (P <0.01). After treated with 0.32mg / L bufalin for 48h, the expression of Survivin mRNA was significantly decreased. After blocking the JNK signaling pathway, Bufalin significantly increased the expression of Survivin mRNA in human pancreatic cancer cells.Conclusion: Bufalin down-regulated the expression of Survivin mRNA in human pancreatic cancer BxPC-3 cells via JNK signal transduction pathway, which may be the result of apoptosis of pancreatic cancer cells mechanism.