Stathmin1 RNA干扰载体的构建及功能检测

目的构建用于stathmin1 RNA干扰的重组质粒并验证其干扰效果，初步探讨stathmin1对于黑色素细胞生长的影响。方法两步PCR法构建含有shRNA编码序列的重组质粒，脂质体转染黑色素细胞。RT-PCR、Western印迹等方法检测stathmin1在黑色素细胞中表达水平改变；MTT、流式细胞仪等方法检测黑色素细胞生长状态。结果成功构建3个stathmin1特异性的RNA干扰载体，在黑色素细胞中能有效降低stathmin1在mRNA水平的表达，蛋白质表达水平分别降低为对照组的6％、17％和15％；黑色素细胞的生长由于stathmin1减弱表达而受到明显抑制，凋亡率提高到约60％。结论干扰质粒能有效介导stathmin1的RNA干扰；stathmin1对于黑色素细胞的生长具有重要意义。

Construction and Functional Analysis of Stathmin 1 RNA Interference Vectors

Objective To investigate the influence of Stathmin 1 on melanocyte growth, three recombinant vectors were constructed and used for research of Stathmin 1 RNA interference. Methods shRNA encoding sequences were amplified with two-step PCR and cloned into the plasmid pBlue-U6. The interfering vectors were transfeeted into melanocytes with Lipofectmin. Stathmin 1 expression was detected at both mRNA and protein levels with RT-PCR and western blotting respectively. Furthermore, growth curve assay was performed by MTT and apoptotic ratio was determined with flow cytometry. Results Three obtained Stathmin 1 RNA interfering vectors showed inhibitory effect on Stathmin 1 mRNA in melanocytes. Protein expression of Stathmin 1 was decreased to 6% , 17% and 15% respectively for pBlue-U6-sil, pBlue-U6-si2 and pBlue-U6-si3 vectors, compared to control groups. Melanocyte growth was remarkably inhibited due to the decrease of Stathmin 1 whereas apoptotic induction was increased to about 60%. Conclusion The constructed recombinant vectors displayed effective and specific Stathmin 1 RNA interference, indicating the important effect of Stathmin 1 on melanocyte growth.