脂质体介导RNA干扰质粒转染鸡成骨细胞条件的优化  

Optimization of conditions for transfecting RNAi plasmid into osteoblasts mediated by liposome

作　　者：张杰[1] 马会杰[1] 崔军[1] 孟范勇 侯加法[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《畜牧与兽医》2014年第6期20-22,共3页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31272618)

摘　　要：本试验的目的是优化转染条件,提高在阳离子脂质体LipofectamineTM 2000介导下鸡TRPV6基因RNA干扰(RNAi)质粒转染入成骨细胞的效率。将携带绿色荧光蛋白基因的真核表达载体pSIREN-retroQ-shTRPV6在LipofectamineTM2000介导下转染至鸡成骨细胞,对转染前细胞融合度、质粒质量与脂质体体积比及转染时间进行优化,在荧光倒置显微镜下观察并计算转染效率,MTT法检测在不同质粒质量与脂质体体积比条件下成骨细胞的活性。结果表明,在转染前细胞融合达到90%以上,质粒DNA与脂质体比例为1∶2.5时转染效率最高,并且在转染后48 h转染效果最好,对细胞的毒性作用较小。本试验优化了脂质体介导转染鸡成骨细胞的条件,提高了转染效率,为用RNA干扰技术研究鸡TRPV6基因功能奠定了基础。The aim of this study is to optimize the transfection condition and improve the efficiency of transfecting TRPV6 gene RNAi plasmid into chicken osteoblasts mediated by LipofectamineTM 2000. Chicken osteoblasts were transfected with eukaryotic expression vector pSI- REN-retroQ-shTRPV6 carrying green fluorescent protein gene in mediation of LipofectamineTM 2000. The cell fusion level before transfection, the ratio of plasmid mass to LipofectamineTM 2000 volume, transfection time were optimized. The transfection efficiency was calculated by observing the expression of green fluorescent protein with inverted fluorescence microscope. The osteoblast activity was determined by using MTT assay. The results showed that when the cell fusion was more than 90% before transfection and the ratio of plasmid mass to Lipo- fectamineTM 2000 volume was 1 ： 2. 5, the transfection efficiency was high, and reached a peak value at 48 h after transfection; when the ratio of plasmid mass to LipofectamineTM 2000 volume was 1 ： 2. 5, the cytotoxicity was low. The resuh indicated the conditions for transfection of osteoblasts in mediation of LipofectamineTM 2000 was optimized, and the transfection efficiency was increased. This study laid a foundation for investigating the function of TRPV6 gene by RNAi technology.

关 键 词：成骨细胞 鸡 转染 RNA干扰 脂质体 

分 类 号：S852.2[农业科学—基础兽医学]