摘要
目的研究多梳蛋白家族成员NSPc1对HeLa细胞增殖的影响。方法运用生物信息学设计并合成敲低NSPc1表达的siRNA序列,用半定量RT-PCR、Real-time PCR及Western blot等检测siRNA序列在HeLa细胞中敲低NSPc1的有效性;将相应有效序列构建到pSUPER载体中,观察其瞬时转染对HeLa细胞BrdU掺入的影响;用G418筛选并建立稳定敲低NSPc1的HeLa细胞系,观察敲低NSPc1对HeLa细胞体外增殖能力的影响。结果(1)设计的siRNA序列可显著敲低NSPc1的表达;(2)瞬时敲低NSPc1阻碍了HeLa细胞BrdU的掺入;(3)成功构建稳定敲低NSPc1的HeLa细胞系,其增殖速度较对照组明显变慢。结论NSPc1的正常表达是维持HeLa细胞正常增殖能力所必需的,稳定敲低NSPc1的HeLa细胞群可作为进一步研究NSPc1相关信号通路的有效模型。
Objective To study the effect of PcG member NSPc1 on proliferation of HeLa cells. Methods Using bioinfomatic analysis to design the siRNA sequence to knockdown NSPc1. Detecting the expression level of NSPc1 in HeLa cell line using semi-quantitative RT-PCR, Real-time PCR and Western blot after transfection of the designed siRNA. Transient transfecting pSUPER-NSPc1 into Hela cells and performing BrdU incorporation assay. Establishing NSPc1 stably knockdown cell line, comparing proliferation abilities with the control cells. Results （ 1 ） The designed siRNA did efficiently knockdown the expression of NSPc1 ; （2） Transient knockdown of NSPc1 could repress BrdU incorporation; （3） The established NSPc1-knockdown cell lines had a signifieantly lower proliferation rate than that of control cells. Conclusion The expression of NSPc1 is necessary for the normal proliferation of HeLa cells. The NSPc1 stably knockdown cell pool is a useful model for further study of pathway related to NSPc1.
出处
《基础医学与临床》
CSCD
北大核心
2009年第5期453-458,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(30600166)
