中文摘要：

目的：构建hsa-miR-26b沉默载体，与其靶基因PTEN共转染HEK-293T细胞验证其沉默效果。 方法：用miRNA海绵体技术设计并合成与hsa-miR-26b成熟序列反向互补的3个重复序列的双链DNA，将短发卡状RNA（shRNA）克隆入LV3-GFP-Puro载体，与靶基因PTEN共转染HEK-293T细胞，于24 h后收集细胞RNA，用real-time PCR检测hsa-miR-26b表达水平，同时用双荧光素报告系统检测其对靶基因的作用。 结果：成功构建了有效的hsa-miR-26b沉默载体，经测序验证与设计序列一致；real-time PCR验证hsa-miR-26b表达水平降低了75%；双荧光素报告系统验证其对靶基因无抑制作用（P〉0.05）。 结论：成功构建hsa-miR-26b沉默载体，并验证其对靶基因PTEN无抑制作用。

英文摘要：

Objective：To construct a recombinant vector carrying hsa-miR-26b sponges and co-transfect with its target gene PTEN into HEK293T cells to validate the silencing efficiency. Methods：The double-stranded DNAs of three repetitive sequences which were antisence complementary to mature sequence of hsa-miR-26b were designed and synthesized by miRNA sponge. The short hairpin RNA （shRNA） was cloned into the LichV3-GFP-Puro vector and validated exactly by sequencing. The expressed plasmid and target gene PETN plasmid were co-transfected into HEK-93T cells. The cellular RNA was collected after 24 hours. The hsa-miR-26b silencing level and the effects on target gene were respectively detected by real-time quantitative PCR and dual-Luciferase reporter assay system. Results：The constructed hsa-miR-26b sponge vector was validated to be consistent with the designed sequence. The expression level of hsa-miR-26b reduced by 75%. No inhibitory effect of hsa-miR-26b on its target gene was shown by dual-Luciferase reporter assay （P〉0.05）. Conclusion：The silencing vector carrying hsa-miR-26b sponge was successfully constructed. It should be helpful for the further study on the function and mechanism of hsa-miR-26b in human adipocytes.