糖原合成酶激酶3β对奶牛乳腺上皮细胞增殖及细胞周期的影响

Effect of Glycogen Synthase Kinase 3β on Cell Proliferation and Cell Cycle in Dairy Cow Mammary Epithelial Cells

本试验通过向纯化的奶牛乳腺上皮细胞中添加不同浓度(0(对照组)、10、20、40mmol/L)的糖原合成酶激酶3β(glycogen synthase kinase 3β,GSK3β)特异性蛋白抑制剂氯化锂(Licl),作用细胞24h,研究其对奶牛乳腺上皮细胞增殖及细胞周期的影响,并利用qRT-PCR和Western blotting分别检测不同浓度Licl对奶牛乳腺上皮细胞中GSK3β、细胞周期蛋白D1(Cyclin D1)mRNA水平及GSK3β、磷酸化GSK3β(p-GSK3β)、Cyclin D1蛋白水平表达的影响。结果显示,Licl能促进奶牛乳腺上皮细胞的增殖活性,Licl抑制GSK3β后促进奶牛乳腺上皮细胞增殖的最佳浓度为20mmol/L。与对照组相比,添加Licl后GSK3β蛋白表达受到抑制,p-GSK3β蛋白表达上调,同时提高了Cyclin D1蛋白表达。表明GSK3β对于奶牛乳腺上皮细胞增殖的能力是负调控因子,失活的GSK3β通过Cyclin D1途径促进细胞周期的进行。

Adding different concentrations Licl （0 （control group）, 10,20,40 mmol/L）, a specificity protein inhibitor of glycogen synthase kinase 3β （GSK3β） ,to the purification of the dairy cow mammary epithelial cells （DCMECs） ,after 24 h,this study detected the effects of GSK3β on cell proliferation and cell cycle in DCMECs,and using the Real-time quantitative PCR to test the effects of different concentrations Licl on GSK3β and Cyclin D1 mRNA level and Western blotting to test the effects of GSK3β /phosphorylated GSK3β （p -GSK3β） and Cyclin D1 protein expression levels in DCMECs. The results showed that Licl could improve proliferation in DCMECs, Licl inhibiting GSK3β certainly promote DCMECs proliferation and the optimal concentration of Licl was 20 mmol/L. Compared with the control group,GSK3β protein expression was restrained after adding Licl, p-GSK3β protein expression level up-regulation,Cyclin D1 protein expression level increased at the same time. These dates demonstrated that GSK3β was a negative regulatory factor of DCMECs on proliferation ability, the deactivation GSK3β through Cyc lin D1 pathway could promote the process of cell cycle.