β-Catenin、CyclinD1、c-myc、Dvl-1在氟化钠诱导心肌细胞中的表达

Expression of β-Catenin, CyclinD1, c-myc, and Dvl-1 in Cardiomyocytes Following Induction by Sodium Fluoride

摘要: 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 < 0.0001)，染毒组细胞存活率均低于对照组(P < 0.0001)；qRT-PCR结果显示，Wnt信号通路中关键性分子mRNA均有不同程度的表达，对照组、680 umol/LNaF组、1360 umol/LNaF组、2720 umol/L。NaF组中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 < 0.001)；经两组间t检验，与对照组相比，低剂量组差异均无统计学意义(t = 1.741, t = 1.866, t = 1.108, t = 3.708, P > 0.05)，中、高剂量组差异有统计学意义(P < 0.05)；中剂量组与低、高剂量组差异有统计学意义(P < 0.05)。结论：AC16细胞经染氟后成功造模，不同NaF浓度可干扰心肌细胞存活率；低、中剂量的氟可能通过Wnt/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。

Abstract: 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 < 0.0001). The survival rates of the infected group were lower than those of the control group (P < 0.0001). The results of qRT-PCR showed that the mRNA of key molecules in the Wnt signaling pathway was expressed in different degrees. The expression levels of 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 < 0.001); After t test between the two groups, compared with the control group, there was no statistical significance in the low-dose group (t = 1.741, t = 1.866, t = 1.108, t = 3.708, P > 0.05), and there was statistical significance in the medium-dose and high-dose groups (P < 0.05). There was significant difference between medium dose group and low and high dose group (P < 0.05). Conclusion: AC16 cells were successfully modelled after fluoride staining, and the survival rate of cardiomyocytes could be affected by different concentrations of NaF. Low and medium doses of fluoride may cause myocardial cell injury through Wnt/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.