瘦素对牦牛乳腺上皮细胞(MECs)中瘦素受体及STAT5a蛋白表达的影响

Effect of leptin on the expression of leptin receptor and STAT5a protein in mammary epithelial cells of yak

瘦素受体(leptin receptor, LEPR)为Ⅰ型细胞因子受体家族的单一跨膜信号转导复合物,是瘦素(leptin, LEP)参与调控乳腺上皮细胞生长和凋亡的重要中介物质。信号转导及转录激活蛋白-5a(signal transducer and activator of transcription-5a, STAT5a)介导大约90%的妊娠期乳腺上皮乳蛋白的合成。为了解LEP对牦牛乳腺上皮细胞(mammary epithelial cells, MECs)中LEPR和STAT5a表达的作用特点及催乳素(prolactin, PRL)对LEP的功能发挥可能产生的影响,试验设计有、无PRL(500μg/L)刺激时,不同质量浓度LEP(0,50,100,200,400,800μg/L)作用牦牛MECs 48 h,通过RT-qPCR检测不同质量浓度LEP对牦牛MECs STAT5a mRNA表达水平的影响,Western blot技术检测STAT5a及LEPR蛋白的表达特点。结果显示,随着LEP作用质量浓度的升高,STAT5a mRNA的相对表达水平呈现先上升后降低的趋势,当LEP质量浓度为200μg/L时,其相对表达水平最高(P<0.05)。无PRL存在时,LEPR蛋白表达水平随LEP质量浓度的增大逐渐降低;而当LEP质量浓度为100,200μg/L时,对STAT5a的蛋白水平表现出显著抑制作用(P<0.05)。而有PRL存在时,LEP质量浓度为200μg/L时,LEPR蛋白相对表达量达到最高;而STAT5a在LEP质量浓度为100,200μg/L时蛋白表达水平较高(P<0.05)。LEP在有、无PRL参与下,对LEPR、STAT5a的表达有不同的影响,可能是因为有PRL存在时,结合受体引起相关机制反应,进而导致蛋白表达有差异。

Leptin receptor(LEPR),a single transmembrane signaling transduction complex of type Ⅰ cytokine receptor family, is a vital mediator of leptin(LEP) to regulate growth and apoptosis of mammary epithelial cells(MECs).Signal transducer and activator of transcription-5 a(STAT5 a) mediates about 90% of milk protein synthesis in the BECs during pregnancy.To elicit the effects of the LEPR and STAT5 a expression as well as the function of prolactin(PRL) on LEP in the MECs of the yak, the MECs were cultured in DMEM medium or DMEM medium with 500 μg/L PRL and stimulated with 0,50,100,200,400,800 μg/L of LEP for 48 h.The effects of different concentrations of the LEP on mRNA expression of STAT5 a in the MECs were analyzed by RT-qPCR,meanwhile, the protein expressions of the STAT5 a and LEPT receptor were analyzed by Western blot.The results showed that the mRNA expression of the STAT5 a upregulated firstly, and then downregulated with the increase of LEP concentration;the relative expression level arrived at the peak point when LEP mass concentration was 200 μg/L(P<0.05).In the absence of PRL,the protein expression of the LEPR in the MECs slowly decreased along with the increase of LEP concentration.Importantly, the protein expression of the STAT5 a was significantly inhibited in the MECs treated by 100,200μg/L of LEP(P<0.05).Besides,the protein expression of LEPR reached the highest level when the MECs cultured with PRL were treated with 200μg/L of LEP.However,the protein expression of the STAT5 aremarkably increased in the MECs cultured with PRL at LEP mass concentrations of 100,200μg/L(P<0.05).Thus,the different effects of LEP on the expression of LEPR and STAT5 awith and without the involvement of PRL may be due to the presence of PRL,it induces a relevant mechanistic response depending on the binding receptor,which in turn leads to a difference in protein expression.