中文摘要：

目的构建表达细胞转录因子PPARγ的RNA干扰（RNAi）质粒，为研究PPARγ参与的细胞信号通路以及PPAR吖为靶点的基因治疗提供稳定转染的RNA干扰质粒。方法选取PPARγ基因的19nt特异性序列，设计针对PPARγ的shRNA序列，应用基因重组技术克隆到pSilencer1．0-U6表达载体中，用EcoRI、ApaI进行双酶切和DNA测序鉴定重组克隆，在脂质体的介导下将包含PPARγ基因的RNAi重组载体转染前列腺癌细胞Pc．3。转染48h后，收集细胞裂解液，Westem blotting分析对照组与转染组PPARγ蛋白的表达水平。结果双酶切证实shRNA正确插入pSilencer1．0-U6质粒，DNA测序证实插入的序列正确；含PPARγ基因的RNAi载体转染前列腺癌细胞PC-3细胞成功；Western blotting结果显示，与空质粒对照组相比，转染组细胞PPARγ表达下调。结论成功构建含人PPARγ基因的RNAi载体，为研究PPARγ参与的细胞信号通路提供了稳定转染的RNA干扰质粒。同时，阻断PPARγ的信号通路将为基因治疗提供一个较好的靶点，研究结果为以PPARγ为靶点的基因治疗提供了有利工具。

英文摘要：

Objective To construct RNA inferference plasmid targeting PPARγ transcription factor, which provided stable transfection RNA interference plasmid for the study of PPAR γ participation in cell signaling pathway and PPAR γ-targeting gene therapy. Methods We selected 19 nt specific sequence of PPAR γ gene. PPAR γ -targeting shRNA sequence were designed and cloned into the expression vector pSilencer 1.0-U6. The recombinants were identified by using EcoR I ＋ Apa I double digestion and DNA sequencing, then were transfected into prostate cancer cells PC-3 under the mediation of Lipofectamine 2000. Collecting cell lysate after 48 h, PPARγ expression of the transfection group and control group was detected by Western blotting. Results shRNA was inserted to plasmid pSilencer 1.0-U6 correctly, confirmed by double digestion and DNA sequencing. RNA interference plasmid targeting PPAR 51 was transfected into prostate cancer cells PC-3 successfully. PPARγ expression was down-regulated in the transfection group, compared with control group by the results of Western blotting. Conclusion RNA interference plasmid targeting PPARγ transcription factor was constructed successfully and it provided stable transfection RNA interference plasmid for the research of PPAR γ participation in cell signaling pathway. Meanwhile, blocking the PPARγ participation in cell signaling pathway provided a good target for gene therapy. It offered favorable tools for PPAR γ-targeting gene therapy.