大鼠c-ski基因siRNA真核表达载体的构建及其在成纤维细胞中的干扰效果观察  

Construction of eukaryotic expression vector expressing siRNA targeting rat c-ski gene,and the interference effect of the vector on rat fibroblasts

作　　者：李平[1] 刘苹[1] 陈星云[1] 赵艳[1] 彭艳 陈磊[1] 周元国[1] 

机构地区：[1]第三军医大学附属大坪医院野战外科研究所分子生物学中心,创伤,烧伤与复合伤国家重点实验室,重庆400042

出　　处：《解放军医学杂志》2010年第5期551-554,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助(30801195);国家高技术研究发展计划(973计划;G1999054205;2005cb522602)

摘　　要：目的构建大鼠c-ski基因的特异性siRNA沉默质粒,并评价其在大鼠皮肤原代成纤维中的干扰效果。方法采用pSUPER质粒构建大鼠c-ski基因siRNA的重组真核表达质粒,转染大鼠皮肤成纤维细胞,采用荧光纤维镜观察转染效率,BrdUELISA法检测转染后细胞增殖情况并作为其干扰效果的初步筛选,定量PCR、免疫组化法检测c-ski基因和蛋白的表达变化。结果构建3个c-SkisiRNA沉默质粒,按照质粒(μg)/脂质体(μl)1∶2.5的比例转染时效率最高,约为53.5%。转染c-SkisiRNA沉默质粒后,细胞增殖降低,且抑制效果与转染剂量成正比。定量PCR、免疫组化法结果显示转染后c-ski基因表达下调51%(P<0.01),蛋白表达明显降低。结论c-Ski沉默质粒构建成功,瞬时转染大鼠皮肤原代成纤维细胞后可以显著抑制c-ski基因和蛋白的表达。Objective To construct a eukaryotic expression vector expressing the small interfering RNA （siRNA） targeting rat c-ski gene, and investigate the interference effect of the vector on the primary rat skin fibroblasts. Methods The siRNA targeting rat c-ski gene was designed according to the sequence of c-ski mRNA available in GenBank, and the eukaryotic expression vector was constructed using a pSUPER vector. Plasmids containing c-ski target sequence were transfected into primary rat skin fibroblasts, and the efficiency of the cell transfection was detected by fluorescence microscopy. The effect of the plasmid on the fibroblast proliferation was analyzed by BRDU ELISA Kit as a result of preliminary screening of the interference effect. The expressive level of c-ski gene and protein was then detected by real-time quantitative PCR （RT-PCR） and Immunohistochemistry, respectively. Results Three c-Ski siRNA expression vectors were constructed and identified by double endonuclease digestion and sequence analysis respectively. The efficiency of cell transfection was 53.5% after transient transfection of the c-Ski siRNA plasmid in primary rat skin fibroblasts, and the c-Ski siRNA plasmid could inhibit cell proliferation in a dose-dependent manner. The c-Ski siRNA plasmid showed an inhibition effect on the c-ski gene expression by 51%, and the c-Ski protein expression decreased significantly as shown by the results of quantitative PCR and immunohistochemistry detection. Conclusion The c-Ski siRNA expression vector has been successfully constructed. The vector could significantly inhibit the expression of c-ski gene or protein and the cell proliferation after transient transfection in primary rat skin fibroblasts.

关 键 词：基因 c-ski RNA干扰 转染 细胞增殖 

分 类 号：R349.64[医药卫生—基础医学]