磷脂酰肌醇-3激酶-蛋白激酶B信号通路在哺乳期乳腺细胞钠碘转运体表达中的作用

Role of phosphatidylinositol 3-kinase-protein kinase B signaling pathway in Na±I^- symporter expression for lactating breast cells

目的探讨磷脂酰肌醇-3激酶（phosphatidylinositol 3-kinase，P13K）．蛋白激酶B（protein kinase B，AKT）信号通路在调节哺乳期乳腺细胞钠碘转运体（Na＋-I^-symporter，NIS）表达中的作用，以及不同碘水平对该通路的影响。方法将原代培养的小鼠哺乳期乳腺细胞分为3组：①对照组[0μmol／LP13K抑制剂LY294002＋0μg／L胰岛素样生长因子I（insulin—like growth factorI，IGF—I）]；②刺激组（50μg／LIGF—I）；③抑制组（40μmol／LLY294002＋50μg／LIGF—I）。此外，将原代培养的小鼠哺乳期乳腺细胞按不同碘水平（0、5、50、1000、3000μg／L）处理分为低碘1、2组，适碘组，高碘1、2组，并加入IGF-I（50μg／L）刺激。采用实时荧光定量PCR及蛋白免疫印迹法检测上述各组细胞中AKT、NISmRNA及蛋白表达水平。结果刺激组AKTmRNA表达水平（1．497±0．550）高于抑制组（0．777±0．108，P〈0．05），而刺激组NISmRNA及蛋白表达水平（0．783±0．187、0．618±0．103）均低于抑制组（2．430±1．423、1．417±0．250，P均〈0．05）。随着碘水平的增加，除高碘1组（1．090±0．356）外，低碘1、2组，适碘组，高碘2组AKTmRNA表达水平（1．758±0．893、1．320±0．538、1．003±0．006、0．745±0．307）为逐渐下降趋势；低碘1、2组，适碘组，高碘1、2组NISmRNA（2．259±0．682、1．823±0．332、1．409±0．366、1．321±0．405、1．150±0．454）及总AKT蛋白（0．640±0．106、0．601±0．081、0．583±0．089、0．555±0．097、0．532±0．023）表达水平均呈下降趋势；除低碘2组（0．484±0．179）外，NIS蛋白表达水平（0．556±0．199、0．502±0．179、0．455±0．126、0．435±0．138）呈下降趋势；除低碘2组（0．076±0．045）外，p-AKT蛋白表达水平（0．078±0．049、0．079±0．040、0．085±0．055、0．095±0．051）呈上升趋势。结论PI3K—AKT信号通路在调节哺乳期乳腺细胞NIS表达方面起抑制作用。

Objective To elucidate the function of phosphatidylinositol 3-kinase （PI3K）-protein kinase B （AKT） signaling pathway underlying the regulation of Na±I- symporter （NIS） and the influence of different levels of iodine on PI3K-AKT signaling pathway in lactating breast cells. Methods The primary cultured mammary gland cells were divided into three groups： （1）control group [0 μmol/L LY294002 ＋ 0 μg/L insulin-like growth factor I （IGF- I ）]; （2）stimulation group （50 μg/L IGF- I ）; （3）inhibition group （40 μmol/L LY294002 ＋ 50 μg/L IGF- I ）. In addition, the cells were treated with different iodine contents （0, 5, 50, 1 000, 3 000 μg/L） for low iodine groups 1 and 2, iodine group, high iodine groups 1 and 2, and IGF-I （50 μg/L） was used to stimulate PI3K-AKT signaling pathway. The expressions of AKT and NIS mRNA and protein were determined by real-time quantitative PCR and Western blotting, respectively. Results The expression of AKT mRNA （1.497 ± 0.550） in stimulation group was higher than that in inhibition group （0.777 ± 0.108, P 〈 0.05）, while the expression of NIS mRNA and protein in stimulation group （0.783 ± 0.187, 0.618 ± 0.103） was lower than those in inhibition group （2.430 ± 1.423, 1.417 ± 0.250,all P 〈 0.05）. With the iodine concentration increasing, except high iodine group 1 （1.090 ± 0.356）, the expression of AKT mRNA in low iodine groups 1 and 2, iodine group, high iodine group 2 （1.758 ± 0.893, 1.320 ±0.538, 1.003 ± 0.006, 0.745 ± 0.307） tended to decline; total AKT protein （0.640 ± 0.106, 0.601 ＋ 0.081, 0.583 ± 0.089, 0.555 ± 0.097, 0.532 ± 0.023） and NIS mRNA （2.259 ± 0.682, 1.823 ± 0.332, 1.409 ± 0.366, 1.321 ± 0.405, 1.150 ± 0.454） tended to decline in low iodine groups 1 and 2, iodine group, high iodine groups 1 and 2; except low iodine group 2 （0.484 ± 0.179）, NIS protein expression tended to decline （0.556 ± 0.199, 0.502 ± 0.179, 0.455 ± 0.126, 0.435 ± 0.138）; however, except low iodine group 2 （0.076 ± 0.045）, the p-AKT protein expressions （0.078 ± 0.049, 0.079 ± 0.040, 0.085 ± 0.055, 0.095 ± 0.051） were on the rise. Conclusion PI3K-AKT signaling pathway may play an inhibition role in the expression of NIS in lactating breast cells.