摘要
目的:探讨PTEN基因对甲状腺髓样癌(medullary thyroid carcinoma,MTC)细胞生长、周期的影响以及作用机制。方法:使用酶切酶连的方法构建真核表达载体pcDNA3.1-PTEN,采用脂质体转染法将其转入pA10RP8细胞中。实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT-qPCR)检测细胞内PTEN表达水平的变化,Western印迹法检测PTEN,Ki-67,p21,cyclinD1蛋白表达水平的变化;CCK-8法和平板克隆试验检测PTEN对pA10RP8细胞增殖的影响;细胞流式术检测pA10RP8细胞周期的变化。结果:成功构建了PTEN真核表达载体。转染pcDNA3.1-PTEN质粒的pA10RP8细胞PTEN和p21表达上调,Ki-67和cyclin D1蛋白表达下调;转染pcDNA3.1-PTEN质粒的pA10RP8细胞生长增殖受到抑制;细胞在G0/G1期比例增加,发生周期阻滞。结论:过表达PTEN能够抑制pA10RP8细胞的增殖,诱导细胞周期阻滞,其机制可能与周期蛋白p21上调和cyclin D1下调表达有关。
Objective:To investigate the effect of over-expression of PTEN gene on the growth and cell cycle of medullary thyroid carcinoma cells and to explore its mechanism.Methods:The eukaryotic expression vector pcDNA3.1-PTEN was constructed by restriction enzyme linked method transferred into pA10RP8 cells by liposome transfection.The mRNA expression level of PTEN in the cells were detected by RT-qPCR,the protein expression level of PTEN,Ki-67,p21 and cyclin D1 were detected by Western blot;the effect of PTEN in pA10RP8 cells on the proliferation was detected by CCK-8 and flat clones;and the changes of cell cycle were detected by flow cytometry.Results:The PTEN eukaryotic expression vector was successfully constructed;the expression of PTEN and p21 in cells with pcDNA3.1-PTEN was significantly up-regulated;the expression of Ki-67 and cyclin D1 protein in pA10RP8 cells with pcDNA3.1-PTEN was down-regulated;the proliferation of pA10RP8 cells with pcDNA3.1-PTEN was inhibited;the PTEN gene induced cell cycle arrest of pA10RP8 cells with a significant increase of the proportion of pA10RP8 cells in G0/G1 phase.Conclusion:Over-expression of PTEN could inhibit the proliferation and induce cell cycle arrest of pA10RP8 cells,whose mechanism may be related to p21 and cyclin D1 protein.
作者
翟明慧
袁殿宝
赵峻峰
张敬
ZHAI Minghui;YUAN Dianbao;ZHAO Junfeng;ZHANG Jing(Department of Medical Oncology,First Affiliated Hospital of Hebei North University,Zhangjiakou Hebei 075000,China;Department of Gastroenterology,First Affiliated Hospital of Hebei North University,Zhangjiakou Hebei 075000,China)
出处
《临床与病理杂志》
2019年第3期464-469,共6页
Journal of Clinical and Pathological Research
