重组BMP7真核表达载体的构建及其对关节软骨细胞的转染  

Construction of BMP7 eukaryotic expression recombination plasmid and its transfection in articular cartilages of rabbits

作　　者：曲福军[1] 侯宜[1] 戚良晨[2] 刘丽玲 池光范[1] 侯立中[1] 

机构地区：[1]吉林大学再生医学科学研究所生物化学研究室 [2]吉林大学中日联谊医院胸外科,吉林长春130031 [3]黑龙江省哈尔滨市兽医研究所,黑龙江哈尔滨150086

出　　处：《吉林大学学报（医学版）》2005年第6期819-823,F0002,共6页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30271319)

摘　　要：目的:构建pcDNA3.1-BMP7真核表达载体,并探讨转染关节软骨细胞的脂质体和DNA的最佳比例及BMP7基因表达。方法:构建pcDNA3.1-BMP7真核表达载体。用脂质体将pcDNA3.1-BMP7包裹形成DNA脂质体复合物,转染家兔关节软骨细胞,G418筛选。转染过程中确定脂质体浓度、脂质体与pcDNA3.1-BMP7质粒的比例。原位杂交、PCR、Western blotting测定BMP7表达。结果:构建了pcDNA3.1-BMP7真核表达载体,脂质体与pcDNA3.1-BMP7质粒最佳转染浓度为3 mL培养液中加10μL脂质体和4μg pcDNA3.1-BMP7质粒(2.5∶1);以400 mg.L-1G418的正常培养液筛选28 d,BMP7为阳性表达。结论:在脂质体介导下,pcDNA3.1-BMP7转染家兔关节软骨细胞获得成功,且BMP7在该细胞中稳定表达。Objective To construct the eukaryotic expression vector of pcDNA3.1-BMP7 and transfect it into the articular chondrocytes of rabbit cultured in vitro in order to study the best condition of liposome and plasmid DNA. Methods Expression vector of pcDNA3.1-BMP7 was constructed and was digested by two enzymes and sequenced. pcDNA3.1-BMP7 was transfected into the articular chondrocytes of rabbit mediated with liposome and screened by G418. The best rates of liposome and G418 , liposome and pcDNA3.1-BMP7 were identified. In situ hybridization, PCR and Western blotting were used to detect the expression of BMP7. Results The MP7 gene of pcDNA3.1- BMP7 was same as the BMP7 gene reported. The best concentration was 10 μL liposome and 4μg pcDNA3.1- BMP7 in 3 mL IMDM mediurna including 400mg · L^-1 G41a. Conclusion Madiated with liposome reagent, the articular cartilages of rabbit are successfully transfected with pcDNA3.1-BMP7 and BMP7 express stably in the articular cartilages.

关 键 词：基因转染 软骨细胞 骨形成蛋白-7 真核表达载体 

分 类 号：Q78[生物学—分子生物学]