摘要
目的:构建并鉴定糖皮质激素受体β(GRβ)RNA干扰稳定表达胶质瘤细胞株,研究GRβ对胶质瘤细胞周期及增殖的影响。方法:根据GRβmRNA序列设计特异性小干扰RNA(siRNA),以LipofectamineTM 2000转染人胶质瘤细胞,RT-PCR及Western blot鉴定其有效性;体外合成该siRNA插入序列小发卡RNA(shRNA)并定向克隆至真核表达载体pGPH1/GFP/Neo,转染后,通过G418筛选稳定表达细胞株(G0306),Western blot检测GRβ表达、MTS和流式细胞术检测GRβ干扰对胶质瘤细胞增殖及细胞周期的影响。结果:GRβsiRNA能有效抑制GRβ的表达,DNA测序证实合成并克隆入真核表达载体pGPH1/GFP/Neo的shRNA序列完全正确;GRβshRNA稳定表达人胶质瘤细胞株阻滞于G1期、细胞增殖能力显著下降。结论:成功构建人GRβshRNA稳定表达胶质瘤细胞株(G0306),初步表明GRβ影响胶质瘤细胞的增殖,为后期研究GRβ在中枢神经系统肿瘤中的作用及机制奠定了基础。
Objective：To construct and indentify glucocorticoid receptor β（GRβ） shRNA stably transfected human glioma cell lines and investigate the effect of GRβ on glioma cell cycle and proliferation. Methods：The specific siRNA was designed according to human GRβ sequence and transfected into human glioma cells by LipofectamineTM2000 reagent. Knock down of GRβ by siRNA was measured by RT-PCR and Western blotting assays. The in vitro synthesized siRNA was inserted into shRNA sequence and directionally cloned into eukaryotic expression vector pGPH1 / GFP / Neo. After transfection,the stable G0306 cell lines were selected by G418. Western blotting assays,MTS and flow cytometry were applied to detect the effect of GRβ interruption on glioma cell proliferation and cycle,respectively. Results：GRβ siRNA effectively inhibited the expression of GRβ. The inserted sequence of shRNA cloned into eukaryotic expression vector pGPH1 / GFP / Neo was confirmed by DNA sequencing. The stable GRβ shRNA cell lines showed growth inhibition in G1 period,and the cell proliferation ability was significantly decreased. Conclusion：Stable GRβknockdown glioma cell line（G0306） was constructed successfully and GRβ affected glioma cell proliferation,which laid a foundation for further study of the role and mechanism of GRβ in the central nervous system tumors.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2014年第7期883-888,共6页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金(81000527)
无锡市科技发展指令性计划(CSEY1N1101)
