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目的肿瘤侵袭转移的发生是影响结肠癌治疗的重要原因,结肠癌细胞黏附能力的下降以及迁移能力的增强是促进结肠癌细胞侵袭转移的重要原因。有研究发现,蟾毒灵具有抑制结肠癌细胞侵袭能力的作用,但其作用机制尚不明确。本研究探讨蟾毒灵对人结肠癌HCT116细胞侵袭的影响,并探索其可能的作用机制。方法用蟾毒灵溶液干预结肠癌HCT116细胞,采用细胞划痕实验与细胞黏附实验检测蟾毒灵溶液对结肠癌HCT116细胞侵袭能力的影响;ELISA法检测蟾毒灵溶液干预结肠癌HCT116细胞培养基中细胞外泌TGFβ1蛋白的含量;蛋白质印迹法检测蟾毒灵干预后结肠癌HCT116细胞中P-smad3、smad4和钙黏链蛋白(E-cadherin)表达的变化;免疫荧光实验检测蟾毒灵干预后各组HCT116细胞中P-smad3与smad4蛋白表达的变化。结果细胞划痕实验结果显示,低、中、高剂量蟾毒灵均可降低HCT116细胞迁移能力;细胞黏附实验显示,对照组、低剂量组、中剂量组和高剂量组黏附细胞平均值分别为833.33±7.80、877.00±17.75、1 304.00±15.82和1 406.00±20.53,相对黏附率分别为100%、105.24%、156.48%和168.73%,差异有统计学意义,χ2=297.597,P<0.001。ELISA检测结果显示,对照组、低、中和高剂量细胞培养液中转化生长因子β(transforming growth factor-β,TGFβ1)蛋白含量分别为(1 198.78±38.96)、(1 106.58±35.76)、(1 040.80±47.47)和(987.74±37.52)pg/mL,F=16.357,P=0.005。蛋白质印迹法检测结果显示,与对照组相比,中、高剂量蟾毒灵处理后HCT116细胞中P-smad3表达呈现明显下降;而smad4蛋白表达明显上调,组间差异有统计学意义,F=56.993,P<0.001;低剂量蟾毒灵也可上调smad4蛋白的表达,t=5.342,P=0.006。各用药组HCT116细胞中E-cadherin蛋白的表达水平也随着蟾毒灵剂量的增加而增加,分别为对照组的2.514±0.385(t=4.833,P=0.008)、3.524±0.397(t=9.200,P=0.001)和3.937±0.318(t=10.675,P<0.001)倍;免疫荧光法检测发现,蟾毒灵处理后结肠癌HCT116细胞中P-smad3与smad4蛋白的表达呈现出与蛋白质印迹法检测相似的趋势。结论蟾毒灵可通过抑制TGFβ1/smad信号通路的信号传导抑制结肠癌HCT116细胞的侵袭。
Objective The incidence of tumor invasion and metastasis is an important reason for the treatment of colon cancer. The decrease of adhesion and the enhancement of migration ability of colon cancer cells are the important reasons for the invasion and metastasis of colon cancer cells. Some studies have found that bufalin can inhibit the invasion of colon cancer cells, but its mechanism of action is not yet clear. This study was to investigate the effect of bufalin on the invasion of human colon cancer HCT116 cells and to explore its possible mechanism. Methods The human colon cancer HCT116 cells were treated with bufalin solution. The cell invasion assay was used to detect the invasion of HCT116 colon cancer cells by cell scratch assay and cell adhesion assay. The inhibitory effect of bufalin on colon cancer HCT116 cell culture medium TGFβ1 was detected by Western blotting. The expression of P-smad3, Smad4 and E-cadherin in HCT116 colon cancer cells were detected by Western blotting. The expression of TGFβ1 was detected by immunofluorescence in the presence of bufalin Changes of P-smad3 and smad4 protein expression in HCT116 cells in each group. Results Cell scratch test results showed that low, medium and high doses of bufalin can reduce HCT116 cell migration ability; cell adhesion experiments showed that the control group, low dose group, medium dose group and high dose group, the average adhesion cells were 833.33 ± 7.80, 877.00 ± 17.75, 1 304.00 ± 15.82 and 1 406.00 ± 20.53, respectively. The relative adhesion rates were 100%, 105.24%, 156.48% and 168.73%, respectively. There was significant difference between them (χ2 = 297.597, P <0.001). The results of ELISA showed that the contents of transforming growth factor-β (TGFβ1) in control, low, medium and high dose cell culture medium were (1 198.78 ± 38.96), (1 106.58 ± 35.76) and 1 040.80 ± 47.47) and (987.74 ± 37.52) pg / mL, F = 16.357, P = 0.005. Compared with the control group, the expression of P-smad3 in HCT116 cells treated with middle and high doses of bufalin significantly decreased compared with the control group, while the expression of smad4 protein was significantly increased, the difference between the two groups was statistically significant (F = 56.993, P <0.001; low dose bufalin also up-regulated smad4 protein expression, t = 5.342, P = 0.006. The expression level of E-cadherin protein in HCT116 cells in each drug group also increased with the increase of the dose of bufalin, which were 2.514 ± 0.385 (t = 4.833, P = 0.008), 3.524 ± 0.397 , P = 0.001), and 3.937 ± 0.318 (t = 10.675, P <0.001). Immunofluorescence showed that the expression of P-smad3 and smad4 protein in HCT116 colon cancer cells treated with bufalin showed a positive correlation with the Western blotting Detect similar trends. Conclusion Bufalin can inhibit the invasion of colon cancer HCT116 cells by inhibiting the signal transduction of TGFβ1 / smad signaling pathway.