摘要：
目的：观察不同浓度17β-雌二醇(17β-estradiol，E2)或甲基乙基酮(MEK)信号通路抑制剂U0126对小鼠哺乳期乳腺细胞MEK、细胞外信号调节蛋白激酶(ERK)和钠/碘同向转运体(NIS)mRNA和蛋白表达，探讨MEK-ERK信号传导通路在哺乳期乳腺细胞摄碘中的作用。方法采用细胞体外培养的方法，对Balb/C小鼠哺乳期乳腺细胞进行传代培养，观察不同含量(0.0008、0.0040、0.0200、0.1000、0.5000 mg/L)E2刺激后，小鼠哺乳期乳腺细胞MEK、ERK和NIS mRNA表达；细胞分组为U0126+ E2组[加入MEK的抑制剂U0126(20μmmol/L)30 min后，再加入E2(0.1 mg/L)]、E2组和对照组，培养24 h后，收集样本。用实时荧光定量PCR方法检测小鼠乳腺原代细胞中MEK、ERK和NIS mRNA表达；蛋白质免疫印迹(Western blot)方法检测小鼠乳腺原代细胞总ERK、磷酸化ERK和NIS蛋白表达。结果在E2刺激试验中，随着E2含量的不断升高，哺乳期乳腺细胞ERK、NIS mRNA的表达量逐渐增加(F=28.23、18.37，P均<0.05)。在MEK抑制实验中，E2组NIS mRNA的表达量(1.90±1.36)较U0126+E2组(0.90±0.39)和对照组(0.76±0.18)高，组间比较差异均有统计学意义(t=3.218、2.737，P均<0.05)；在总ERK蛋白表达中，E2组(1.62±0.30)最高，与U0126+E2组(1.19±0.32)和对照组(1.25±0.27)比较，差异均有统计学意义(t=2.401、2.246，P均<0.05)；ERK磷酸化后，E2组磷酸化ERK蛋白表达(0.97±0.02)比U0126+E2组(0.76±0.18)高，组间比较差异有统计学意义(t=-2.840，P<0.05)；E2组NIS蛋白表达(0.49±0.08)比U0126+ E2抑制组(0.37±0.04)和对照组(0.41±0.03)高，组间比较差异均有统计学意义(t =3.286、2.294，P均<0.05)。结论 E2激活MEK-ERK信号通路，上调哺乳期乳腺细胞ERK、NIS mRNA及蛋白的表达。抑制该通路后，ERK、NIS mRNA与蛋白表达均呈明显降低的趋势；MEK-ERK可能是哺乳期乳腺摄碘的一条重要信号传导通路。%Objective Balb/c mouse mammary gland primary cells were cultured, and the expression of methyl ethyl ketone (MEK), extracellular signal-regulated protein kinase (ERK) and Na+/I-symporter (NIS) in mouse mammary gland cells was observed after stimulation with different concentrations of l7β-estradiol (E2); after application of MEK signaling pathway inhibitor U0126, the stimulation effect of E2 on mice lactating mammary cell ERK and NIS mRNA transcription level and protein expression were studied, and the role of MEK-ERK signaling pathway in iodine uptake of lactating mammary gland cell was observed. Methods Balb/c mouse mammary gland primary cells of normal primary generation were sub cultured, and then the stimulation effect of different concentrations of E2 (0.000 8, 0.004 0, 0.020 0, 0.100 0, 0.500 0 mg/L), mouse mammary gland cells MEK, ERK and mRNA NIS expression were observed;MEK inhibitor U0126 (20μmmol/L) was applied and after 30 minutes E2 (0.1 mg/L) was administered. Cells were sub grouped to inhibitor U0126 + E2 group, E2 group and control group, cultured for 24 h, and samples were collected. Mouse mammary primary cell MEK, ERK and NIS mRNA expression levels were detected by real-time quantitative PCR; Western blotting was used to detect primary mouse mammary cell phosphorylated ERK, total ERK and NIS protein expression levels. Results In the E2 stimulating test, with increasing of E2 concentration, the expression of ERK and NIS mRNA in breast cancer cells increased gradually (F=28.23, 18.37, all P< 0.05). In the MEK inhibition test, NIS mRNA expression level of E2 group (1.90 ± 1.36) was higher than that of U0126 + E2 inhibitor group (0.90 ± 0.39, P< 0.05), and that of control group (0.76 ± 0.18, P<0.05), the differences were statistically significant(t = 3.218, 2.737). In total ERK protein expression, ERK protein expression of E2 group was the highest. ERK protein expression of E2 group (1.62 ± 0.30) compared with that of U0126+E2 inhibitor group (1.19 ± 0.32) and control group (1.25 ± 0.27), the differences were statistically significant respectively (t = 2.401, 2.246, all P< 0.05). After ERK phosphorylation , ERK phosphorylated protein expression of E2 group (0.97 ± 0.02) was higher than that of U0126+E2 inhibitor group (0.76 ± 0.18, t = - 2.840, P< 0.05). NIS protein expression of E2 group (0.49 ± 0.08) was higher than that of U0126 + E2 inhibitor group (0.37 ± 0.04) and the control group (0.41 ± 0.03), and the differences were statistically significant (t = 3.286, 2.294, all P<0.05). Conclusions E2 has raised the MEK, ERK, NIS mRNA and protein expression of lactating mammary gland cells. When MEK-ERK signaling pathway is activated, ERK and NIS mRNA and protein expression show a rising trend;when this pathway is inhibited, ERK and NIS mRNA and protein expression show a significant decreasing trend. We speculate that MEK-ERK pathway is an important signal transduction pathway of iodine uptake in lactating mammary gland.
