中文摘要：

目的：构建S100A16基因RNA干扰（RNAi）的真核表达质粒,转染3T3-L1小鼠前体脂肪细胞,初步鉴定其干扰效果。方法：以小鼠S100A16基因为靶基因,以PLKO.1-sP6-GFP质粒为载体,根据GenBank数据库提供的S100A16基因核苷酸序列,选择设计2条siRNA干扰序列,构建针对S100A16基因的shRNA真核表达质粒PLKO.1-S100A16-GFP-shRNA1、2,经PCR鉴定和测序分析,确认质粒构建成功后,用脂质体LipofectamineTM 2000将重组质粒瞬时转染小鼠3T3-L1小鼠前体脂肪细胞,在荧光显微镜下观察绿色荧光蛋白表达,计算转染效率,Real-time RT-PCR法检测质粒对S100A16基因的表达抑制效果。结果：构建的shRNA序列经PCR和DNA测序证实与设计完全一致;在荧光显微镜下观察到3T3-L1细胞表达绿色荧光蛋白（GFP）,证实重组质粒已转入细胞,转染效率达到90%;Real-time RT-PCR结果显示转入PLKO.1-S100A16-GFP-shRNA2的3T3-L1细胞中S100A16基因被特异抑制,基因表达抑制率达70%以上,与空白对照组、阴性序列对照组的差异具有统计学意义（P均〈0.05）。结论：成功构建了靶向S100A16基因的shRNA真核表达质粒PLKO.1-S100A16-GFP-shRNA1和2,并筛选出有效抑制S100A16基因表达的质粒,为进一步研究S100A16基因功能奠定了基础。

英文摘要：

Objective：To construct the eukaryotic expression plasmid carried mouse S100A16 gene short hairpin RNA（shRNA）,and to investigate the silencing effect of shRNA on the S100A16 gene in mouse 3T3-L1 cells.Methods：siRNAs were designed according to the S100A16 cDNA sequence in GenBank,and inserted into plasmid PLKO.1-sP6-GFP.The recombinant plasmids were identified by PCR and sequence analysis,and transfected into 3T3-L1 cells with LipofectamineTM 2000.The transfection efficiency was reported by green fluorescence protein expression and gene inhibition efficiency was analyzed by realtime RT-PCR.Results：The correct sequences of recombinant PLKO.1-S100A16-GFP-shRNAs were confirmed by DNA sequencing.Green fluorescence observed by fluorescence microscope showed that shRNAs had been transfected into 3T3-L1 cells（transfection rate was 90%）.And the expression of S100A16 mRNA was effectively down-regulated by PLKO.1-S100A16-GFP-shRNA2（inhibition rate was above 70%）.Conclusion：The eukaryotic expression plasmids of S100A16 shRNAs were constructed successfully.The PLKO.1-S100A16-GFP-shRNA2 could down-regulated the expression of S100A16 gene effectively.This is fundamental for the study on the function of S100A16 gene.