壬基酚对人结肠癌SW480细胞活性及跨膜G蛋白偶联雌激素膜受体30表达的影响

Influence of nonylphenol on cytoactive and the expression of G protein-coupled estrogen receptor in human colon cancer SW480 cells

目的分析壬基酚对人结肠癌SW480细胞活性及跨膜G蛋白偶联雌激素膜受体30(GPR30)表达的影响。方法采用实验研究方法。通过体外培养人结肠癌SW480细胞,采用细胞增殖、周期及凋亡检测以及基因和蛋白检测实验,分析壬基酚影响人结肠癌SW480细胞增殖、细胞周期、凋亡以及GPR30表达的情况。细胞分组:SW480细胞加入培养基设为对照组,加入培养基+1×10^(‒8) mol/L雌二醇设为雌二醇组,加入培养基+1×10^(-8) mol/L壬基酚设为壬基酚组,加入培养基+1×10^(-8) mol/L壬基酚+1×10^(-7) mol/L GPR30特异性拮抗剂G15设为壬基酚+G15组。观察指标:(1)4组人结肠癌SW480细胞增殖指数。(2)4组人结肠癌SW480细胞的周期占比。(3)4组人结肠癌SW480细胞的凋亡指数。(4)4组人结肠癌SW480细胞GPR30信使RNA(mRNA)表达情况。(5)4组人结肠癌SW480细胞GPR30蛋白表达情况。正态分布的计量资料以x±s表示,组间比较采用单因素方差分析,两两比较采用最小显著差异法检验。结果(1)4组人结肠癌SW480细胞增殖指数。细胞增殖实验结果显示:对照组、雌二醇组、壬基酚组、壬基酚+G15组人结肠癌SW480细胞的增殖指数分别为100.00±0.00、89.19±4.86、148.96±6.04、120.40±3.39,4组比较,差异有统计学意义(F=21.45,P<0.05);壬基酚组与对照组比较,差异有统计学意义(P<0.05);雌二醇组、壬基酚+G15组分别与对照组比较,差异均无统计学意义(P>0.05)。(2)4组人结肠癌SW480细胞的周期占比。细胞周期实验结果显示:对照组、雌二醇组、壬基酚组、壬基酚+G15组人结肠癌SW480细胞的S期细胞占比分别为39.96%±2.02%、36.67%±0.62%、43.85%±1.02%、38.29%±1.42%,4组比较,差异有统计学意义(F=10.08,P<0.05);雌二醇组、壬基酚组分别与对照组比较,差异均有统计学意义(P<0.05);壬基酚+G15组与对照组比较,差异无统计学意义(P>0.05)。(3)4组人结肠癌SW480细胞的凋亡指数。细胞凋亡实验结果显示:对照组、雌二醇组、壬基酚组、壬基酚+G15组人结肠癌SW480细胞的凋亡指数分别为1.67±0.18、4.80±0.31、0.75±0.11、2.20±0.19,4组比较,差异有统计学意义(F=136.79,P<0.05);雌二醇组、壬基酚组分别与对照组比较,差异均有统计学意义(P<0.05);壬基酚+G15组与对照组比较,差异无统计学意义(P>0.05)。(4)4组人结肠癌SW480细胞GPR30 mRNA表达情况。实时荧光定量聚合酶链式反应检测结果显示:对照组、雌二醇组、壬基酚组、壬基酚+G15组人结肠癌SW480细胞GPR30 mRNA相对表达率分别为1.00±0.00、0.86±0.05、1.89±0.27、0.64±0.12,4组比较,差异有统计学意义(F=26.61,P<0.05);壬基酚组、壬基酚+G15组分别与对照组比较,差异均有统计学意义(P<0.05);雌二醇组与对照组比较,差异无统计学意义(P>0.05)。(5)4组人结肠癌SW480细胞GPR30蛋白表达情况。Western blot检测结果显示:对照组、雌二醇组、壬基酚组、壬基酚+G15组人结肠癌SW480细胞GPR30蛋白相对表达率分别为1.83±0.16、1.68±0.15、3.10±0.30、1.26±0.11,4组比较,差异有统计学意义(F=34.05,P<0.05);壬基酚组、壬基酚+G15组分别与对照组比较,差异均有统计学意义(P<0.05);雌二醇组与对照组比较,差异无统计学意义(P>0.05)。结论低剂量壬基酚可增加人结肠癌SW480细胞增殖指数与S期细胞占比,降低细胞凋亡指数,并促进GPR30 mRNA和蛋白表达。

Objective To investigate the influence of nonylphenol(NP)on cytoactive and the expression of G protein-coupled estrogen receptor 30(GPR30)in human colon cancer SW480 cells.Methods The experimental study was conducted.The human colon cancer SW480 cells were cultured in vitro.The influence of NP on proliferation,cell cycle,apoptosis and the expression of GPR30 in human colon cancer SW480 cells were analyzed by cell proliferation,cell cycle detection,cell apoptosis and gene expression and protein expression experiments.Cell grouping:SW480 cells cultured with medium were set as the control group,cultured with medium+1×10^(-8) mol/L estradiol were set as the estradiol group,cultured with medium+1×10^(-8) mol/L NP were set as the NP group,cultured with medium+1×10^(-8) mol/L NP+1×10^(-7) mol/L GPR30 specific antagonist G15 were set as the NP+G15 group,respectively.Observation indicators:(1)proliferation index of human colon cancer SW480 cells in the 4 groups;(2)cycle proportion of human colon cancer SW480 cells in the 4 groups;(3)apoptosis index of human colon cancer SW480 cells in the 4 groups;(4)GPR30 messenger RNA(mRNA)expression of human colon cancer SW480 cells in the 4 groups;(5)GPR30 protein expression of human colon cancer SW480 cells in the 4 groups.Measurement data with normal distribution were represented as Mean±SD and one way ANOVA was used for comparison between groups.The least significant difference method was used to test the pairwise comparison.Results(1)Proliferation index of human colon cancer SW480 cells in the 4 groups.Results of the cell proliferation experiments showed that the proliferation indexes of human colon cancer SW480 cells in the control group,the estradiol group,the NP group and the NP+G15 group were 100.00±0.00,89.19±4.86,148.96±6.04 and 120.40±3.39,respectively,showing a significant difference among the 4 groups(F=21.45,P<0.05).There was a significant difference between the control group and the NP group(P<0.05),and there was no significant difference between the control group and the estradiol group,between the control group and the NP+G15 group(P>0.05).(2)Cycle proportion of human colon cancer SW480 cells in the 4 groups.Results of the cell cycle detection experiments showed that the proportions of human colon cancer SW480 cells in the S phase of the cell cycles in the control group,the estradiol group,the NP group and the NP+G15 group were 39.96%±2.02%,36.67%±0.62%,43.85%±1.02%and 38.29%±1.42%,respectively,showing a significant difference among the 4 groups(F=10.08,P<0.05).There were significant differences between the control group and the estradiol group,between the control group and the NP group(P<0.05),and there was no significant difference between the control group and the NP+G15 group(P>0.05).(3)Apoptosis index of human colon cancer SW480 cells in the 4 groups.Results of the cell apoptosis experiments showed that the apoptosis indexes of human colon cancer SW480 cells in the control group,the estradiol group,the NP group and the NP+G15 group were 1.67±0.18,4.80±0.31,0.75±0.11 and 2.20±0.19,respectively,showing a significant difference among the 4 groups(F=136.79,P<0.05).There were significant differences between the control group and the estradiol group,between the control group and the NP group(P<0.05),and there was no significant difference between the control group and the NP+G15 group(P>0.05).(4)GPR30 mRNA expression of human colon cancer SW480 cells in the 4 groups.Results of quantitative real-time polymerase chain reaction detection showed that the relative expression rates of GPR30 mRNA in human colon cancer SW480 cells of the control group,the estradiol group,the NP group and the NP+G15 group were 1.00±0.00,0.86±0.05,1.89±0.27 and 0.64±0.12,respectively,showing a significant difference among the 4 groups(F=26.61,P<0.05).There were significant differences between the control group and the NP group,between the control group and the NP+G15 group(P<0.05),and there was no significant difference between the control group and the estradiol group(P>0.05).(5)GPR30 protein expression human colon cancer SW480 cells in the 4 groups.Results of Western blot detection showed that the relative expression rates of GPR30 protein in human colon cancer SW480 cells of the control group,the estradiol group,the NP group and the NP+G15 group were 1.83±0.16,1.68±0.15,3.10±0.30 and 1.26±0.11,respectively,showing a significant difference among the 4 groups(F=34.05,P<0.05).There were significant differences between the control group and the NP group,between the control group and the NP+G15 group(P<0.05),and there was no significant difference between the control group and the estradiol group(P>0.05).Conclusion Low dose of NP can increase the proliferation index and the proportion of cells in the S phase of the cell cycles,decrease the apoptosis index,and promote the mRNA and protein expression of GPR30 in human colon cancer SW480 cells.