【作者】 刘美萍；

【导师】 曹仁贤；

【作者基本信息】 南华大学 ， 内分泌， 2011， 硕士

【摘要】 目的:检测并观察PRMT2在甲状腺癌SW579细胞株中的表达情况;构建稳定表达PRMT2和空载体细胞系SW579-PRMT2-V5、SW579-V5,研究PRMT2对人甲状腺癌SW579细胞生长的影响,并探讨其作用机制。方法:应用Lipofectamine 2000将真核表达载体和pcDNA3.2 /V5-PRMT2空载体pcDNA3.2 /V5转染入SW579细胞中,经G418抗性筛选得到稳定的克隆并扩大培养成细胞系,观察转染后细胞形态学的变化,采用RT-PCR和Western blot鉴定稳定表达PRMT2的SW579细胞。MTT法检测稳定表达PRMT2对SW579细胞生长的影响,流式细胞仪分析细胞凋亡的变化,Western blot检测转录因子NF-κB及相关基因蛋白表达的变化,PRMT2对信号NF-κB转导通路的影响。结果:PRMT2在甲状腺癌SW579细胞中有低表达。成功建立了稳定表达PRMT2和空载体的细胞系SW579-PRMT2-V5、SW579-V5,转染后细胞形态学发生了变化,PRMT2显著抑制SW579细胞体外增殖能力,而且在诱导SW579细胞发生凋亡的同时,PRMT2可下调NF-κB活性及其下游靶基因cyclin D1的表达。结论:1.稳定表达PRMT2和空载体细胞系SW579-PRMT2-V5、SW579-V5构建成功。2. PRMT2能下调NF-κB及其下游靶基因cyclin D1的表达,促进甲状腺癌细胞凋亡。更多还原

【Abstract】 Objectives: The Study aims to detect the expression of protein arginine methyltransferase 2 (PRMT2) in the human Thyroid Cancer SW579 cell, to construct the vector of SW579-PRMT2-V5, and to investigate the effect of PRMT2 on the growth of SW579 cells.Methods: the eukaryotic expression plasmid pcDNA3.2 /V5-PRMT2, and empty vector pcDNA3.2 /V5 were transfect into SW579 cells. The cells are selected by G418. After being transfected, proliferation of SW579 cells was observed by MTT assay. Apoptosis of SW579 cells are tested by using Hoechst 33342 staining and flow cytometry. NF-κB activation and its related genes are detected by Western blot. The expression of PRMT2 mRNA and protein are then detected by reverse transcription-polymerase chain reaction ( RT-PCR) and Western blot.Results: A low expression level was detected in human Thyroid Cancer SW579 cells. Cells with SW579-PRMT2-V5 expression were successfully established. After infection of PRMT2, the growth of Thyroid Cancer cells was inhibited significantly. Morever, PRMT2 is induced apoptosis in Thyroid Cancer cells. Also, down -regulation of cyclin D1, companied by decrease in NF–κB activation, are obseved in SW579-PRMT2-V5 cells.Conclusion: 1.Cells with SW579-PRMT2-V5 expressing were successfully established. After transfection of PRMT2, the growth of Thyroid Cancer cells is inhibited significantly.2.PRMT2 induces apoptosis in Thyroid Cancer cells. Down-regulation of cyclin D1, companied by decrease in NF–κB activation, was also obseved in SW579-PRMT2-V5 cells.更多还原