PPARγ基因shRNA真核表达载体的构建及其干扰效果鉴定  

Construction of shRNA eukaryotic expression vector of PPARγgene and identification of its interference efficiency

作　　者：辛婧[1] 沈亚非[1] 邓飞[1] 谢亚争 张日华 刘云 XIN Jing;SHEN Ya-fei;DENG Fei;XIE Ya-zheng;ZHANG Ri-hua;LIU Yun(The Central Hospital of Luohe,Department of endocrinology,Henan Luohe 462000,China;不详)

机构地区：[1]漯河市中心医院内分泌科,河南漯河462000 [2]南京医科大学第一附属医院老年医学内分泌科

出　　处：《江苏预防医学》2021年第3期255-258,共4页Jiangsu Journal of Preventive Medicine

基　　金：河南省教育厅高等学校重点科研项目(21A320011);河南省科技攻关项目(212102310199)。

摘　　要：目的构建过氧化物酶体增殖物激活受体γ(PPARγ)基因RNA干扰的真核表达载体,转染小鼠3T3-L1前体脂肪细胞,并鉴定其干扰效果。方法选择设计2条针对小鼠PPARγ基因的干扰靶序列,构建真核表达载体PLKO.1-PPARγ-GFP-shRNA1/2,以PCR鉴定并进行序列分析。证实质粒构建成功后,转染小鼠3T3-L1前体脂肪细胞,荧光显微镜下观察绿色荧光蛋白(eGFP)的表达,计算转染效率;采用实时定量PCR法和Western印迹检测载体对PPARγ基因的表达干扰效果;采用油红染色观察PPARγ在脂肪细胞分化过程中对脂滴形成的作用。结果成功构建PPARγ干扰质粒PLKO.1-PPARγ-GFP-shRNA1/2,PCR和DNA测序证实序列与设计完全一致;荧光显微镜下观察到3T3-L1细胞绿色荧光蛋白的表达,证实重组质粒成功转入细胞,转染效率(92.67±1.53)%;实时荧光定量PCR和Western Blot印记试验均显示PLKO.1-PPARγ-GFP-shRNA1/2转染的3T3-L1细胞PPARγ基因分别被特异性抑制;靶点1干扰效率[(78.2±2.1)%]高于靶点2[(55.8±4.3)%],差异有统计学意义(P<0.05),靶点1初步鉴定为有效靶点。油红染色显示PLKO.1-PPARγ-GFP-shRNA1/2转染均可抑制3T3-L1脂肪细胞分化和脂滴聚集。结论成功构建了靶向干扰PPARγ基因的shRNA真核表达质粒PLKO.1-PPARγ-GFP-shRNA1/2,并筛选出有效抑制靶基因的表达质粒,初步验证PPARγ基因对脂肪细胞分化的作用,为进一步研究提供了有效的分子生物学工具。Objective To construct the eukaryotic expression vectors to interfere the expression of peroxisome proliferator activated receptorγ(PPARγ)gene;to transfect mouse 3 T3-L1 cells and to study the silencing effect.Methods Two interference target sequences were designed for PPARγgene silencing in mice.The eukaryotic expression vectors PLKO.1-PPARγ-GFP-shRNA1/2 were constructed and identified by PCR and sequence analysis.The confirmed plasmids were transfected into mice 3 T3-L1 preadipocytes.The transfection efficiency was calculated by observing the green fluorescence protein expression under the fluorescence microscope;the gene inhibition efficiency was analyzed by real time PCR and Western blot analysis for PPARγgene expression.The lipid droplets were observed by oil-red O staining to explore the role of PPARγin 3 T3-L1 preadipocyte differentiation.Results The recombinant plasmids PLKO.1-PPARγ-GFP-shRNA1/2 were constructed successfully,both PCR and DNA sequencing confirmed that the sequences were consistent with the design.The expression of green fluorescent protein in 3 T3-L1 cells was observed under fluorescence microscope,which confirmed that the recombinant plasmids were successfully transfected into 3 T3-L1 cells,resulting the transfecting efficiency of(92.67±1.53)%.The real time fluorescence quantitative PCR and Western blot analysis showed that PPARγgene expression levels in 3 T3-L1 cells transfected with PLKO.1-PPARγ-GFP-shRNA1/2 were specifically inhibited.The interference efficiency of target 1[(78.2±2.1)%]was higher than that of target 2[(55.8±4.3)%],the difference was statistically significant(P<0.05);target 1 was preliminary identified as effective target.Oil red staining showed that PLKO.1-PPARγ-GFP-shRNA1/2 trasfection could inhibit 3 T3-L1 preadipocytes differentiation and lipid droplet accumulation.Conclusion The eukaryotic expression vectors of PLKO.1-PPARγ-GFP-shRNA1/2 were constructed successfully.The effective target silencing plasmid was identified,the effect of PPARγon adipocyte

关 键 词：小鼠3T3-L1前体脂肪细胞 RNA干扰 PPARΓ基因 

分 类 号：R392[医药卫生—免疫学]