伴肌动蛋白相关锚定蛋白特异性siRNA筛选及效能评价  

Screening and evaluation of a newly designed siRNA targeting nebulin related anchoring protein

作　　者：张竞[1] 张颖[2] 王良哲[3] 孙其志[2] 袁文[2] 

机构地区：[1]上海交通大学附属新华医院,上海200092 [2]第二军医大学附属长征医院骨科,上海200003 [3]第二军医大学附属长征医院病理科,上海200003

出　　处：《生物骨科材料与临床研究》2013年第6期5-7,11,共4页Orthopaedic Biomechanics Materials and Clinical Study

基　　金：2010年度"上海市卫生局科研课题"(基金编号:2010138)

摘　　要：目的设计、合成人伴肌动蛋白相关锚定蛋白(Nebulin related anchoring protein,NRAP)的特异性siRNA,转染人成纤维细胞筛选最佳干扰序列。方法设计并合成4条针对NRAP的siRNA,以不同浓度转染人成纤维细胞。通过Westernblot、RT-PCR法检测其对NRAPmRNA及蛋白表达的抑制效果。结果siRNA50mol/L及Lipofectamine2000 1 l有较高的转染效率。4种siRNA干扰成纤维细胞24小时后,NRAP的mRNA及蛋白表达量均有显著下降,其中siRNA47抑制效果最为显著。结论成功筛选出可有效抑制成纤维细胞NRAP表达的siRNA,为观察阻断NRAP表达后应力诱导OPLL细胞成骨的变化奠定了基础。Objective To design and synthesize specific siRNA targeting Nebulin related anchoring protein(NRAP), and to screen the most effective sequence by transfecting human fibroblasts. Methods Four pairs of siRNA sequence targeting NRAP were designed and synthesized and transfected into human fibroblast with different concentrations. mRNA and protein expression of NRAP were detected by RT-PCR and Western blot respectively. Results 50mol/L siRNA and 1 l Lipofectamine 2000 were the optimum concentration of NRAP siRNA.mRNA and protein levels of NRAP decreased significantly 24 hours after transfecting fibroblasts by four pairs of siRNA. The inhibitory effect of N-RAP siRNA47 was the most significant than that of the others. Conclusion The newly designed NRAP siRNA can effectively silencing the expression of NRAP in fibroblast, which help to observe change of stress-induced ossifcation after siencing NRAP.

关 键 词：后纵韧带骨化症 伴肌动蛋白相关锚定蛋白 小干扰核糖核酸 

分 类 号：R686[医药卫生—骨科学]