SiRNA抑制小鼠皮肤成纤维细胞TGF-β1基因表达的实验研究  

SiRNAInhibits Gene Expression of TGFβ1 in Mouse Skin Fibroblast

作　　者：任润健 方磊[1] 贾传龙 赵虎[1] 陈亮[2] 秦登科 周慧敏[1] 毕波[1] Ren Runjian Fang Lei Jia Chuanlong Zhao Hu Chert Liang Qin Dengke Zhou Huimin Bi Bo(Laboratory Department, Huadong HospitaIAffiliated to Fudan University, Shanghai, 200040, P. R. China Department ofplastic sur- gery, Huadong Hospital Affiliated to Fudan University, Shanghai, 200040,P. R. China)

机构地区：[1]复旦大学附属华东医院检验科,上海200040 [2]复旦大学附属华东医院整形外科,上海200040

出　　处：《老年医学与保健》2017年第3期193-197,共5页Geriatrics & Health Care

基　　金：国家自然科学基金资助项目(81301642)

摘　　要：目的设计、合成及筛选光老化相关基因—转化生长因子-β1(transforming growth factor-β1,TGF-β1)小分子干扰RNA(siRNA),了解基因调控效果,用以探索皮肤抗衰老基因治疗可能的新途径。方法应用RNA设计软件,设计并合成针对小鼠TGF-β1 mRNA的3对21核苷酸siRNA,转染小鼠成纤维细胞,检测转染效率及细胞活性;分别在基因和蛋白水平检测TGF-β1表达。结果 TGF-β1基因siRNA工作浓度为150nmol/L,Lipofectamine2000脂质体4μL/孔(24孔板)时转染率最高(84.0%。综合考虑转染率与细胞活性,选用0.5μL/孔(转染率为72.4%。与对照组相比,转染TGF-β1 siRNA的小鼠皮肤成纤维细胞TGF-β1表达下调,其差异有统计学意义(P<0.05),以siRNA-S1最显著,基因水平抑制率达96.9%,蛋白水平抑制率达62.0%。结论:TGF-β1siRNA转染小鼠成纤维细胞最佳条件是Lipofectamine2000脂质体0.5μL/孔,siRNA-S1有效抑制TGF-β1蛋白表达时间可达72h。Objective To design,synthesize and screen small interfering RNA（siRNA） targeting at transforming growth factor-β1 TGF-β1） and to understand the effect of gene regulation so as to explore a possible way for the gene therapy of skin anti-aging.Methods 3 pairs of 21 nucleotide siRNAs directed to mouse TGFβ1 mRNA were designed and synthesized by utilizing RNA design software;these siRNAs were respectively transfected into fibroblastsby oligofectamine package;transfection efficiency and cell activity were detected;the expression of TGFβ1 was detected in gene and protein level.Results When the work concentration of siRNA was 150nmol/L and that of Lipofectamine2000 was 4μL/well（24-well plates）,transfection efficiency of fibroblasts was highest（84.0%）;considering cell viability and transfection efficiency,0.5μL/well（72.4%）was chosen;compared with bland controls,the expression of TGFβ1 protein level was markedly down-regulated in siRNA transfected fibroblasts and the difference was of statistical significance（P〈0.05）;the inhibitory rate of the siRNA of the target-1was highest up to 96.9%in mRNA level and 62.0%in protein level.Conclusions The optimal condition of TGF-β1 siRNA transfecting fibroblasts was Lipofectamine2000 0.5μL/well;siRNA-β1 can effectively inhabit the protein expression of TGF-β1 for 72 hours.

关 键 词：TGF-Β1 小干扰RNA 小鼠 皮肤成纤维细胞 

分 类 号：R450[医药卫生—治疗学] R751[医药卫生—临床医学]