目的:氟化钠(NaF)染毒人心肌细胞(AC16),实时荧光定量PCR (qRT-PCR)法检测Wnt/β-catenin信号通路中β-连环蛋白(β-catenin)、细胞周期蛋白(CyclinD1)、myc癌基因(c-myc)、蓬乱蛋白1 (Dvl-1) mRNA的表达情况,为氟中毒所致心肌损伤机制...目的:氟化钠(NaF)染毒人心肌细胞(AC16),实时荧光定量PCR (qRT-PCR)法检测Wnt/β-catenin信号通路中β-连环蛋白(β-catenin)、细胞周期蛋白(CyclinD1)、myc癌基因(c-myc)、蓬乱蛋白1 (Dvl-1) mRNA的表达情况,为氟中毒所致心肌损伤机制提供理论依据。方法:体外培养AC16细胞,设置不同浓度梯度NaF进行染毒,将其分为0 umol/LNaF组(对照组)、680 umol/L NaF组、1360 umol/L NaF组、2720 umol/LNaF组,染毒24 h后置于倒置荧光显微镜下观察其细胞形态,CCK-8法检测细胞存活率;qRT-PCR检测染毒24 h细胞中Wnt/β-catenin信号通路β-catenin、CyclinD1、c-myc、Dvl-1 mRNA的表达水平。结果:AC16细胞成功造模后CCK-8结果显示,不同浓度NaF染毒AC16细胞24小时,对照组细胞存活率为100% ± 0.08%,染毒组细胞存活率分别为83.66% ± 0.06%、42.75% ± 0.07%、31.75% ± 0.02%。经单因素的方差分析得出(F = 168.97, P β-catenin表达量分别为(1.00 ± 0.73, 1.02 ± 0.23, 5.48 ± 0.72, 2.94 ± 0.10)、c-myc (1.00 ± 0.78, 1.96 ± 0.94, 3.34 ± 0.75, 3.83 ± 0.25)、CyclinD1 (1.00 ± 0.95, 0.98 ± 0.24, 12.36 ± 0.97, 1.05 ± 0.13)以及Dvl-1 (1.00 ± 0.64, 1.51 ± 0.24, 37.54 ± 0.33, 18.96 ± 0.26)。对照组、低、中剂量组随染毒浓度升高,Wnt/β-catenin信号通路β-catenin、CyclinD1、c-myc、Dvl-1 mRNA表达升高,高剂量组反而降低;经单因素方差分析可得,β-catenin (F = 8.16, P = 0.008);c-myc (F = 4.34, P = 0.043);CyclinD1 (F = 6.48, P = 0.016);Dvl-1 (F = 41.90, P 0.05),中、高剂量组差异有统计学意义(P β-catenin信号通路导致心肌细胞的损伤,β-catenin、CyclinD1、c-myc、Dvl-1 mRNA表达升高,高剂量氟β-catenin、CyclinD1、c-myc、Dvl-1 mRNA表达反而降低,可能由于Wnt信号通路过度激活反而降低了对心肌细胞的损伤。Objective: Sodium fluoride (NaF) infected human cardiomyocytes (AC16);Real-time quantitative fluorescent PCR (qRT-PCR) was used to detect the expression of β-catenin (β-catenin), CyclinD1, myc oncogene (c-myc) and Dvl-1 mRNA in Wnt/β-Catenin signaling pathway, to provide a theoretical basis for the mechanism of myocardial injury caused by fluorosis. Methods: AC16 cells were cultured in vitro and exposed with different concentration gradients of NaF. They were divided into 0 umol/LNaF group (control group), 680 umol/LNaF group, 1360 umol/LNaF group and 2720 umol/LNaF group. 24 h after exposure, the cell morphology was observed under inverted fluorescence microscope, and the cell survival rate was detected by CCK-8 method. The expression levels of Wnt/β-catenin signaling pathways β-catenin, CyclinD1, c-myc, Dvl-1 mRNA were detected by qRT-PCR in 24 h infected cells. Results: After AC16 cells were successfully modeled, CCK-8 results showed that the survival rate of AC16 cells in the control group was 100% ± 0.08% after 24 hours of exposure to NaF. The survival rates of the infected group were 83.66% ± 0.06%, 42.75% ± 0.07% and 31.75% ± 0.02%, respectively, according to one-way analysis of variance (F = 168.97, P β-catenin in control group, 680 umol/LNaF group, 1360 umol/LNaF group and 2720 umol/LNaF group were (1.00 ± 0.73, 1.02 ± 0.23, 5.48 ± 0.72 and 2.94 ± 0.10, respectively), c-myc (1.00 ± 0.78, 1.96 ± 0.94, 3.34 ± 0.75, 3.83 ± 0.25), CyclinD1 (1.00 ± 0.95, 0.98 ± 0.24, 12.36 ± 0.97, 1.05 ± 0.13), Dvl-1 (1.00 ± 0.64, 1.51 ± 0.24, 37.54 ± 0.33, 18.96 ± 0.26);control group, low dose group and medium dose group increased with the concentration of exposure. The expression of β-catenin, CyclinD1, c-myc, Dvl-1 mRNA in Wnt/β-catenin signaling pathway increased;The high dose group decreased;By one-way ANOVA, β-catenin (F = 8.16, P = 0.008) was obtained. c-myc (F = 4.34, P = 0.043);CyclinD1 (F = 6.48, P = 0.016);Dvl-1 (F = 41.90, P 0.05), and there was statistical significance in the medium-dose and high-dose groups (P β-catenin signaling pathway, and increase mRNA expression of β-catenin, CyclinD1, c-myc and DVL-1. The mRNA expressions of fluoro-β-catenin, CyclinD1, c-myc and DVL-1 were decreased at high doses, which may be due to the over-activation of Wnt signaling pathway, but the damage to cardiomyocytes was reduced.展开更多
文摘目的:氟化钠(NaF)染毒人心肌细胞(AC16),实时荧光定量PCR (qRT-PCR)法检测Wnt/β-catenin信号通路中β-连环蛋白(β-catenin)、细胞周期蛋白(CyclinD1)、myc癌基因(c-myc)、蓬乱蛋白1 (Dvl-1) mRNA的表达情况,为氟中毒所致心肌损伤机制提供理论依据。方法:体外培养AC16细胞,设置不同浓度梯度NaF进行染毒,将其分为0 umol/LNaF组(对照组)、680 umol/L NaF组、1360 umol/L NaF组、2720 umol/LNaF组,染毒24 h后置于倒置荧光显微镜下观察其细胞形态,CCK-8法检测细胞存活率;qRT-PCR检测染毒24 h细胞中Wnt/β-catenin信号通路β-catenin、CyclinD1、c-myc、Dvl-1 mRNA的表达水平。结果:AC16细胞成功造模后CCK-8结果显示,不同浓度NaF染毒AC16细胞24小时,对照组细胞存活率为100% ± 0.08%,染毒组细胞存活率分别为83.66% ± 0.06%、42.75% ± 0.07%、31.75% ± 0.02%。经单因素的方差分析得出(F = 168.97, P β-catenin表达量分别为(1.00 ± 0.73, 1.02 ± 0.23, 5.48 ± 0.72, 2.94 ± 0.10)、c-myc (1.00 ± 0.78, 1.96 ± 0.94, 3.34 ± 0.75, 3.83 ± 0.25)、CyclinD1 (1.00 ± 0.95, 0.98 ± 0.24, 12.36 ± 0.97, 1.05 ± 0.13)以及Dvl-1 (1.00 ± 0.64, 1.51 ± 0.24, 37.54 ± 0.33, 18.96 ± 0.26)。对照组、低、中剂量组随染毒浓度升高,Wnt/β-catenin信号通路β-catenin、CyclinD1、c-myc、Dvl-1 mRNA表达升高,高剂量组反而降低;经单因素方差分析可得,β-catenin (F = 8.16, P = 0.008);c-myc (F = 4.34, P = 0.043);CyclinD1 (F = 6.48, P = 0.016);Dvl-1 (F = 41.90, P 0.05),中、高剂量组差异有统计学意义(P β-catenin信号通路导致心肌细胞的损伤,β-catenin、CyclinD1、c-myc、Dvl-1 mRNA表达升高,高剂量氟β-catenin、CyclinD1、c-myc、Dvl-1 mRNA表达反而降低,可能由于Wnt信号通路过度激活反而降低了对心肌细胞的损伤。Objective: Sodium fluoride (NaF) infected human cardiomyocytes (AC16);Real-time quantitative fluorescent PCR (qRT-PCR) was used to detect the expression of β-catenin (β-catenin), CyclinD1, myc oncogene (c-myc) and Dvl-1 mRNA in Wnt/β-Catenin signaling pathway, to provide a theoretical basis for the mechanism of myocardial injury caused by fluorosis. Methods: AC16 cells were cultured in vitro and exposed with different concentration gradients of NaF. They were divided into 0 umol/LNaF group (control group), 680 umol/LNaF group, 1360 umol/LNaF group and 2720 umol/LNaF group. 24 h after exposure, the cell morphology was observed under inverted fluorescence microscope, and the cell survival rate was detected by CCK-8 method. The expression levels of Wnt/β-catenin signaling pathways β-catenin, CyclinD1, c-myc, Dvl-1 mRNA were detected by qRT-PCR in 24 h infected cells. Results: After AC16 cells were successfully modeled, CCK-8 results showed that the survival rate of AC16 cells in the control group was 100% ± 0.08% after 24 hours of exposure to NaF. The survival rates of the infected group were 83.66% ± 0.06%, 42.75% ± 0.07% and 31.75% ± 0.02%, respectively, according to one-way analysis of variance (F = 168.97, P β-catenin in control group, 680 umol/LNaF group, 1360 umol/LNaF group and 2720 umol/LNaF group were (1.00 ± 0.73, 1.02 ± 0.23, 5.48 ± 0.72 and 2.94 ± 0.10, respectively), c-myc (1.00 ± 0.78, 1.96 ± 0.94, 3.34 ± 0.75, 3.83 ± 0.25), CyclinD1 (1.00 ± 0.95, 0.98 ± 0.24, 12.36 ± 0.97, 1.05 ± 0.13), Dvl-1 (1.00 ± 0.64, 1.51 ± 0.24, 37.54 ± 0.33, 18.96 ± 0.26);control group, low dose group and medium dose group increased with the concentration of exposure. The expression of β-catenin, CyclinD1, c-myc, Dvl-1 mRNA in Wnt/β-catenin signaling pathway increased;The high dose group decreased;By one-way ANOVA, β-catenin (F = 8.16, P = 0.008) was obtained. c-myc (F = 4.34, P = 0.043);CyclinD1 (F = 6.48, P = 0.016);Dvl-1 (F = 41.90, P 0.05), and there was statistical significance in the medium-dose and high-dose groups (P β-catenin signaling pathway, and increase mRNA expression of β-catenin, CyclinD1, c-myc and DVL-1. The mRNA expressions of fluoro-β-catenin, CyclinD1, c-myc and DVL-1 were decreased at high doses, which may be due to the over-activation of Wnt signaling pathway, but the damage to cardiomyocytes was reduced.
