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
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摘要
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),10,20,40mmol/L),a specificity protein inhibitor of glycogen synthase kinase 3β(GSK3β),to the purification of the dairy cow mammary epithelial cells(DCMECs),after 24h,this study detected the effects of GSK3βon cell proliferation and cell cycle in DCMECs,and using the Real-time quantitative PCR to test the effects of different concentrations Licl on GSK3βand Cyclin D1mRNA level and Western blotting to test the effects of GSK3β/phosphorylated GSK3β(p-GSK3β)and Cyclin D1protein expression levels in DCMECs.The results showed that Licl could improve proliferation in DCMECs,Licl inhibiting GSK3βcertainly promote DCMECs proliferation and the optimal concentration of Licl was 20mmol/L.Compared with the control group,GSK3βprotein expression was restrained after adding Licl, p-GSK3βprotein expression level up-regulation,Cyclin D1protein expression level increased at the same time.These dates demonstrated that GSK3βwas a negative regulatory factor of DCMECs on proliferation ability,the deactivation GSK3βthrough Cyclin D1pathway could promote the process of cell cycle.
Adding different concentrations Licl(0(control group),10,20,40mmol/L),a specificity protein inhibitor of glycogen synthase kinase 3β(GSK3β),to the purification of the dairy cow mammary epithelial cells(DCMECs),after 24h,this study detected the effects of GSK3βon cell proliferation and cell cycle in DCMECs,and using the Real-time quantitative PCR to test the effects of different concentrations Licl on GSK3βand Cyclin D1mRNA level and Western blotting to test the effects of GSK3β/phosphorylated GSK3β(p-GSK3β)and Cyclin D1protein expression levels in DCMECs.The results showed that Licl could improve proliferation in DCMECs,Licl inhibiting GSK3βcertainly promote DCMECs proliferation and the optimal concentration of Licl was 20mmol/L.Compared with the control group,GSK3βprotein expression was restrained after adding Licl, p-GSK3βprotein expression level up-regulation,Cyclin D1protein expression level increased at the same time.These dates demonstrated that GSK3βwas a negative regulatory factor of DCMECs on proliferation ability,the deactivation GSK3βthrough Cyclin D1pathway could promote the process of cell cycle.
引文
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12 Shimura T,Kakuda S,Ochiai Y,et al.Acquired radioresistance of human tumor cells by DNA-PK/AKT/GSK3β-mediated Cyclin D1overexpression[J].Oncogene,2010,29(34):4826~4837.
13 Stambolic V,Ruel L,Woodgett J R.Lithium inhibits glycogen synthase kinase-3activity and mimics wingless signalling in intact cells[J].Current Biology,1996,6(12):1664~1669.
14 Takahashi-Yanaga F,Sasaguri T.GSK-3βregulates Cyclin D1expression:A new target for chemotherapy[J].Cellular Signalling,2008,20(4):581~589.
2 史琳琳,赵锋,高学军,等.催乳复合物、胰岛素样生长因子-Ⅰ对奶牛乳腺上皮细胞中AKT-1和mTOR表达的作用[J].中国畜牧兽医,2013,40(3):27~31.
3 李建莎,朱敏,田单,等.锂对A549细胞增殖及细胞周期的影响[J].华中科技大学学报,2007,36(2):145~148.
4 陈太忠,张艳,熊弢,等.脂多糖预处理对GSK-3功能活性的影响[J].第三军医大学学报,2012,34(13):1304~1309.
5 杨泽然.胰岛素经PI3K-GSK3β信号通路调节内皮细胞内皮素-1基因表达[D].杭州:浙江大学,2007.
6 Diehl J A,Cheng M,Rousse M F,et al.Glycogen synthase kinase-3βregulates Cyclin D1proteolysis and subcellular localization[J].Genes Dev,1998,12:3499~3511.
7 Farago M,Dominguez I,Landesman-Bollag E,et al.Kinase-inactive glycogen synthase kinase 3βpromotes Wnt signaling and mammary tumorigenesis[J].Cancer Res,2005,65(13):5792~5801.
8 Hamelers I H L,van Schaik R F M A,Sipkema J,et al.Insulinlike growth factorⅠtriggers nuclear accumulation of Cyclin D1in MCF-7Sbreast cancer cells[J].Journal of Biological Chemistry,2002,277(49):47645~47652.
9 Karlovic D,Jakopec S,Dubravcic K,et al.Lithium increases expression of p21(WAF/Cip1)and survivin in human glioblastoma cells[J].Cell Biology and Toxicology,2007,23(2):83~90.
10 Rao A S,Kremenevskaja N,Resch J,et al.Lithium stimulates proliferation in cultured thyrocytes by activating Wnt/β-catenin signaling[J].European Journal of Endocrinology,2005,153(6):929 ~938.
11 Ryves W J,Harwood A J.The interaction of glycogen synthase kinase-3(GSK-3)with the cell cycle[J].Progress in Cell Cycle Research,2003,5:489~495.
12 Shimura T,Kakuda S,Ochiai Y,et al.Acquired radioresistance of human tumor cells by DNA-PK/AKT/GSK3β-mediated Cyclin D1overexpression[J].Oncogene,2010,29(34):4826~4837.
13 Stambolic V,Ruel L,Woodgett J R.Lithium inhibits glycogen synthase kinase-3activity and mimics wingless signalling in intact cells[J].Current Biology,1996,6(12):1664~1669.
14 Takahashi-Yanaga F,Sasaguri T.GSK-3βregulates Cyclin D1expression:A new target for chemotherapy[J].Cellular Signalling,2008,20(4):581~589.