摘要
目的探讨麦冬皂苷B(OPB)对人结肠癌SW620细胞增殖、凋亡及转化生长因子-β1(TGF-β1)/Smad信号通路的影响。方法采用含100 kU·L^-1青霉素、100 mg·L^-1链霉素、体积分数10%胎牛血清的RMPI-1640培养液体外培养结肠癌SW620细胞,取对数生长期细胞接种于96孔板中,细胞培养24 h后分为空白对照组、OPB低剂量组(5μmol·L^-1)、OPB中剂量组(10μmol·L^-1)和OPB高剂量组(20μmol·L^-1),然后将各组细胞培养板移入培养箱中继续培养。取各组培养24、48、72 h的SW620细胞,采用四甲基偶氮唑盐(MTT)法检测SW620的细胞抑制率。取各组培养48 h的SW620细胞,采用流式细胞术检测SW620细胞周期及凋亡情况,酶联免疫吸附试验法检测SW620细胞中TGF-β1蛋白的表达,Western blot法检测SW620细胞中磷酸化Smad3(p-Smad3)、Smad4及细胞周期蛋白D1(Cyclin D1)的表达。结果OPB低、中、高剂量组SW620细胞抑制率显著高于空白对照组(P<0.05),OPB中、高剂量组SW620细胞抑制率显著高于OPB低剂量组(P<0.05),OPB高剂量组SW620细胞抑制率显著高于OPB中剂量组(P<0.05)。与空白对照组比较,OPB低、中、高剂量组G 2/M期SW620细胞比例下降,G 0/G 1和S期SW620细胞比例升高(P<0.05)。与OPB低剂量组比较,OPB中、高剂量组G 2/M期SW620细胞比例降低,G 0/G 1和S期SW620细胞比例升高(P<0.05);与OPB中剂量组比较,OPB高剂量组G 2/M期SW620细胞比例降低,G 0/G 1和S期SW620细胞比例升高(P<0.05)。OPB低、中、高剂量组SW620细胞凋亡率显著高于空白对照组(P<0.05),OPB中、高剂量组SW620细胞凋亡率显著高于OPB低剂量组(P<0.05),OPB高剂量组SW620细胞凋亡率显著高于OPB中剂量组(P<0.05)。OPB低、中、高剂量组细胞培养基中TGF-β1水平低于空白对照组(P<0.05),OPB中、高剂量组细胞培养基中TGF-β1水平低于OPB低剂量组(P<0.05);OPB高剂量组细胞培养基中TGF-β1水平低于OPB中剂量组(P<0.05)。与空白对照组比较,OPB低、中、高剂量组SW620细胞中p-Smad3、Cyclin D1蛋白相对表达量显著下降,Smad4蛋白相对表达量显著升高(P<0.05);与OPB低剂量组比较,OPB中、高剂量组SW620细胞中p-Smad3、Cyclin D1蛋白相对表达量显著下降,Smad4蛋白相对表达量显著升高(P<0.05);与OPB中剂量组比较,OPB高剂量组SW620细胞中p-Smad3、Cyclin D1蛋白相对表达量显著下降,Smad4蛋白相对表达量显著升高(P<0.05)。结论OPB可明显抑制人结肠癌SW620细胞增殖,其机制可能与OPB阻滞TGF-β1/Smad信号通路有关。
Objective To investigate the effect of ophiopogonin B(OPB)on the proliferation,apoptosis and transforming growth factorβ1(TGF-β1)/Smad signal pathway of human colon cancer SW620 cells.Methods The human colorectal cancer SW620 cells were cultured in vitro with RMPI-1640 containing 100 kU·L^-1 penicillin,100 mg·L^-1 streptomycin and volume fraction 10%calf serum.The logarithmic growth phase cells were inoculated into 96 pore plates,and the cells were divided into blank control group,low dose OPB group(5μmol·L^-1),medium dose OPB group(10μmol·L^-1)and high dose OPB group(20μmol·L^-1)after 24 hours of culture.Then the cell culture plates in each group were transferred into the incubator for further culture.The SW620 cells in each group were collected after culturing for 24,48 and 72 hours,the cell inhibition rate of SW620 cells was detected by methyl thiazolyl tetrazolium(MTT)method.The SW620 cells in each group were collected after culturing for 48 hours,the cell cycle and apoptosis of SW620 cell were detected by flow cytometry,the expression of TGF-β1 was detected by enzyme linked immunosorbent assay,and the expressions of phosphorylated Smad3(p-smad3),Smad4 and Cyclin D1 were detected by Western blot.Results The inhibition rate of SW620 cells in the low,medium and high dose OPB groups was significantly higher than that in the blank control group(P<0.05);the inhibition rate of SW620 cells in the medium and high dose OPB group was significantly higher than that in the low dose OPB group(P<0.05);and the inhibition rate of SW620 cells in the high dose OPB group was significantly higher than that in the medium dose OPB group(P<0.05).Compared with the blank control group,the proportion of SW620 cells in G 2/M phase decreased,while that in G 0/G 1 and S phase increased in the low,medium and high dose OPB groups(P<0.05).Compared with the low dose OPB group,the proportion of SW620 cells in G 2/M phase decreased,while that in G 0/G 1 and S phase increased in the medium and high dose OPB groups(P<0.05).Compared with the medium dose OPB group,the proportion of SW620 cells in G 2/M phase decreased,while that in G 0/G 1 and S phase increased in the high dose OPB group(P<0.05).The apoptosis rate of SW620 cells in the low,medium and high dose OPB group was significantly higher than that in the blank control group(P<0.05);the apoptosis rate of SW620 cells in the medium and high dose OPB group was significantly higher than that in the low dose OPB group(P<0.05);and the apoptosis rate of SW620 cells in the high dose OPB group was significantly higher than that in the medium dose OPB group(P<0.05).The level of TGF-β1 in cell culture medium of low,medium and high dose OPB group was lower than that of blank control group(P<0.05),the level of TGF-β1 in cell culture medium in medium and high dose OPB group was lower than that in low dose OPB group(P<0.05),and the level of TGF-β1 in cell culture medium in high dose OPB group was lower than that in medium dose OPB group(P<0.05).Compared with the blank control group,the relative expression of p-Smad3 and Cyclin D1 protein in SW620 cells in the low,medium and high dose OPB group decreased significantly,and the relative expression of Smad4 protein increased significantly(P<0.05).Compared with the low dose OPB group,the relative expression of p-Smad3 and Cyclin D1 protein in SW620 cells in the medium and high dose OPB group decreased significantly,and the relative expression of Smad4 protein increased significantly(P<0.05).Compared with the medium dose OPB group,the relative expression of p-Smad3 and Cyclin D1 protein in SW620 cells in the high dose OPB group decreased significantly,and the relative expression of Smad4 protein increased significantly(P<0.05).Conclusion OPB can inhibit the proliferation of SW620 cells,which may be related to the blocking of TGF-β1/Smad signaling pathway by OPB.
作者
孟鑫
李振想
姜孝奎
MENG Xin;LI Zhen-xiang;JIANG Xiao-kui(Department of General Surgery,the Third Affiliated Hospital of Xinxiang Medical University,Xinxiang 453003,Henan Province,China)
出处
《新乡医学院学报》
CAS
2019年第12期1110-1114,共5页
Journal of Xinxiang Medical University
基金
河南省科技发展计划项目(编号:152102310356)
