摘要
目的探讨黄芪配伍莪术在体外对小鼠结肠癌细胞CT26黏附和迁移能力的影响及作用机制。方法体外培养CT26细胞,设对照组,分别以0.00625、0.0125、0.025、0.05、0.1、0.2g/ml的黄芪、莪术药物浓度处理24h和48h后,采用CCK-8法检测黄芪配伍莪术对CT26细胞存活率的影响以选择合适的给药浓度。采用划痕实验检测黄芪配伍莪术对CT26细胞迁移能力的影响,采用细胞黏附实验检测黄芪配伍莪术对CT26细胞与细胞及与细胞外基质间黏附能力的影响。药物处理24h后采用Western blot法和RT-PCR法检测CT26细胞中黏附相关因子E-钙黏蛋白(E-cadherin)、抗癌1号蛋白(KAI1)、抑癌基因张力蛋白同源10号染色体缺失的磷酸酶基因(PTEN)、基质蛋白酶诱导因子(CD147)、缺氧诱导因子-1α(HIF-1α)、基质金属蛋白酶9(MMP-9)的蛋白和mRNA表达。结果选择0.0125、0.025、0.05g/ml浓度进行后续实验。与对照组比较,24h时黄芪配伍莪术组0.025、0.05g/ml浓度及48h时0.0125、0.025、0.05g/ml浓度CT26细胞的迁移率均明显降低;与黄芪配伍莪术组0.0125g/ml浓度比较,24、48h 0.025、0.05g/ml浓度CT26细胞迁移率均明显降低;与黄芪配伍莪术组0.025g/ml浓度比较,24、48h 0.05g/ml浓度CT26细胞迁移率均明显降低;除黄芪配伍莪术组0.05g/ml浓度外,各组48h CT26细胞迁移率较本组24h均明显增加(P<0.05或P<0.01)。与对照组比较,黄芪配伍莪术组0.0125、0.025、0.05g/ml浓度与细胞外基质黏附率均明显下降;与黄芪配伍莪术组0.0125g/ml浓度比较,0.025、0.05g/ml浓度与细胞黏附率明显升高,0.05g/ml浓度与细胞外基质黏附率明显下降;与黄芪配伍莪术组0.025g/ml浓度比较,0.05g/ml浓度与细胞黏附率明显升高(P<0.05或P<0.01)。与对照组比较,黄芪配伍莪术组0.025、0.05g/ml浓度CT26细胞的E-cadherin、KAI1、PTEN蛋白及mRNA表达水平显著升高,CD147蛋白表达显著降低;与黄芪配伍莪术组0.0125g/ml浓度比较,0.025g/ml浓度E-cadherin蛋白及mRNA表达明显升高,0.05g/ml浓度E-cadherin、KAI1、PTEN蛋白及mRNA表达显著升高,CD147蛋白及mRNA表达下降;与黄芪配伍莪术组0.025g/ml浓度比较,0.05g/ml浓度的KAI1、PTEN蛋白及mRNA表达明显升高,CD147蛋白及mRNA表达下降(P<0.05或P<0.01)。结论黄芪配伍莪术可以增强CT26细胞间黏附,减弱与细胞外基质黏附能力,并抑制其迁移能力,其作用机制可能与促进同源性黏附相关因子E-cadherin、PTEN、KAI1表达,抑制异源性黏附相关因子CD147、MMP-9、HIF-1α表达有关。
Objective To explore the effects and mechanism of Radix Astragali seu Hedysa and Rhizoma Curcumae synergy on the adhesion and migration ability of mouse colon cancer cells CT26 in vitro. Methods CT26 cells were cultured in vitro, and a blank control group was set up. After treatment with the concentrations of 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2 g/ml of Radix Astragali seu Hedysa and Rhizoma Curcumae respectively for 24 hours and 48 hours, the CCK-8 method was used to detect the effects of Radix Astragali seu Hedysa and Rhizoma Curcumae on the survival rate of CT26 cells in order to select a suitable concentration for administration. The scratch test was used to detect the effects of the compatibility of Radix Astragali seu Hedysa and Rhizoma Curcumae synergy on CT26 cell migration, and the cell adhesion test was used to detect the effects of the compatibility of Radix Astragali seu Hedysa and Rhizoma Curcumae synergy on the adhesion between CT26 cells and cells and extracellular matrix. After 24 hours of drug treatment, Western blot and RT-PCR were used to detect the adhesion-related factors E-cadherin, anti-cancer protein NO.1(KAI1), tumor suppressor gene tension protein homologous NO.10 chromosome-deleted phosphatase gene(PTEN), matrix proteinase-inducible factor(CD147), hypoxia-inducible factor-1α(HIF-1α), matrix metalloproteinase 9(MMP-9) protein and mRNA expression in CT26 cells. Results The concentrations of 0.0125, 0.025 and 0.05 g/ml were selected for subsequent experiments. Compared with the blank control group, the migration rates of CT26 cells at 0.025, 0.05 g/ml concentration of Radix Astragali seu Hedysa and Rhizoma Curcumae group at 24 h and 0.0125, 0.025, 0.05 g/ml concentration at 48 hour were significantly reduced. The migration rate of CT26 cells at 0.025 and 0.05 g/ml concentrations at 24 hours and 48 hours was significantly reduced;the migration rate of CT26 cells at 0.05 g/ml concentration at 24 hours and 48 hours was significantly reduced compared to the concentration at 0.025 g/ml. Except for the 0.05 g/ml concentration, the migration rate of CT26 cells in all groups at 48 hour was significantly higher than that at 24 hour within group(P<0.05 or P<0.01). Compared with the blank control group, at the concentration of 0.0125, 0.025, 0.05 g/ml, the adhesion rate of CT26 cell with extracellular matrix in the Radix Astragali seu Hedysa and Rhizoma Curcumae groups were significantly decreased;compared with the concentration of 0.0125 g/ml, the concentration of 0.025, 0.05 g/ml, the adhesion rate of CT26 cell with cells increased significantly, and the 0.05 g/ml concentration the extracellular matrix adhesion rate decreased significantly;compared with the 0.025 g/ml concentration, the 0.05 g/ml concentration cell adhesion rate increased significantly(P<0.05 or P<0.01). Compared with the blank control group, the expression levels of E-cadherin, KAI1, PTEN protein and mRNA in CT26 cells of 0.025 and 0.05 g/ml concentration in Radix Astragali seu Hedysa and Rhizoma Curcumae group were significantly increased, and the expression of CD147 protein was significantly reduced;compared with the concentration of 0.0125 g/ml, under 0.025 g/ml concentration E-cadherin protein and mRNA expression increased significantly, under 0.05 g/ml concentration E-cadherin, KAI1, PTEN protein and mRNA expression increased significantly, CD147 protein and mRNA expression decreased;compared with 0.025 g/ml concentration, under 0.05 g/ml concentration KAI1, PTEN protein and mRNA expression increased significantly, CD147 protein and mRNA expression decreased(P<0.05 or P<0.01). Conclusion The synergy of Radix Astragali seu Hedysa and Rhizoma Curcumae can enhance the adhesion between CT26 cells, weaken the adhesion ability with extracellular matrix, and inhibit its migration ability. The mechanism may be related to the promotion of the expression of homologous adhesion-related factors E-cadherin, PTEN, KAI1, and inhibition of heterologous adhesion-related factors CD147, MMP-9, and HIF-1α.
作者
吴幸冬
唐德才
WU Xingdong;TANG Decai(Nanjing University of Chinese Medicine,Nanjing,210000)
出处
《中医杂志》
CSCD
北大核心
2020年第13期1176-1183,共8页
Journal of Traditional Chinese Medicine
基金
国家自然科学基金(81873021)。
关键词
结肠癌
黄芪
莪术
CT26细胞
细胞黏附
细胞迁移
colon cancer
Radix Astragali seu Hedysa
Rhizoma Curcumae
CT26 cells
adhesion
migration
