重组pcDNA3.1-BMP7转染兔膝关节软骨细胞及其表达  

Transfection and expression of recombinant pcDNA3.1-BMP7 in knee articular chondrocytes of the rabbit

作　　者：曲福军[1] 侯宜[1] 周余来[1] 刘丽玲 颜炜群[1] 杨同书[1] 侯立中[1] 

机构地区：[1]吉林大学再生医学科学研究所 [2]哈尔滨兽医研究所,黑龙江省哈尔滨市150001

出　　处：《中国临床康复》2006年第17期79-81,i0005,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金(30271319);国家高技术研究发展计划资助(2004AA205020)~~

摘　　要：目的:将重组pcDNA3.1-BMP7真核表达载体转染至培养的兔膝关节软骨细胞中,观察BMP7基因在兔关节软骨细胞中的表达。方法:实验于2003-08/2004-08在哈尔滨兽医研究所完成。①选取清洁级健康成年家兔1只,兔膝关节软骨切碎后取2.0～2.5kg软骨组织,进行关节软骨细胞的分离与培养。②脂质体与pcDNA3.1-BMP7DNA质粒形成脂质体-DNA质粒复合物,复合物的最佳比例为脂质体10μL、pcDNA3.1-BMP7质粒4μg。用含G418400mg/L的正常培养液筛选转染pcDNA3.1-BMP7DNA质粒的软骨细胞,显微镜观察细胞的形态及活性。③软骨细胞转染后7d和28d,分别用聚合酶链式反应、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳、Westernblot、免疫荧光、原位杂交检测等方法测定BMP7蛋白在软骨细胞中的表达。结果:①家兔膝关节软骨细胞的分离与培养情况:家兔膝关节软骨切碎后用胰蛋白酶/Ⅱ型胶原酶进行消化,能使细胞与细胞外基质很好地分离。接种24h,部分细胞开始贴壁生长,72h后分裂增殖,7d时细胞融合率为90%～100%。贴壁初期细胞呈圆形或三角形,以三角形为主,每7d传代1次。②脂质体介导下pcDNA3.1-BMP7质粒转染兔软骨细胞情况:转染后的软骨细胞用G418筛选,4d转染效率约15%,阳性克隆细胞七八天融合率约为90%。G418连续筛选28d,阳性克隆细胞仍然存活,细胞融合率为100%。未转染pcDNA3.1-BMP7质粒的细胞,G418筛选4d时细胞全部死亡。③转染后兔软骨细胞聚合酶链式反应检测结果:聚合酶链式反应产物进行琼脂糖凝胶电泳分离,结果在1.3kb处可见DNA条带,作为对照的软骨细胞未见此DNA条带。④转染后软骨细胞BMP7蛋白表达电泳检测结果:在十二烷基磺酸钠-聚丙烯酰胺凝胶上可见相对分子质量约为18000的电泳条带。⑤转染后软骨细胞BMP7蛋白表达的Westernblot检测结果:可见相对分子质量约为18000的特异性条带。⑥转染后软骨细胞蛋白表�AIM： The recombinant eukaryotic expression vector pcDNA3.1-BMP7 is transfected into the cultured knee articular chondrocytes of the rabbits. Then we observe the expression of BMP7 gene in the articular chondrocytes of the rabbits. METHODS： This experiment was conducted at the Veterinarian Institute of Harbin from August 2003 to August 2004. ①One healthy adult rabbit of clean degree was chosen. 2.0 to 2.5 kg cartilaginous tissue was chosen to perform the isolation and culture of articular chondrocytes following cutting the k nee articular chondrocytes into pieces. ②Lipesome and pcDNA3.1-BMP7 DNA plasmid formed lipesome -DNA plasmid compound , and the best proportion of the compound was 10 pL liposome and 4 μg pcDNA3.1-BMP7 plasmid. 400 mg/L normal culture medium containing G418 was used to screen the chondrocytes with transfected pcDNA3.1-BMP7 DNA plasmid, and cellular morphology and activity were observed under the microscope. ③Expression of BMP7 protein in the chondrecytes was measured with polymerase chain reaction （PCR）, sod.dedecyl sulfate-pelyacrylamide gel electrophoresis （SDS-PAGE）, Western blot, immunofluorescence and in situ hybridization 7 and 28 days after transfection of chondrocytes. RESULTS： ①Isolation and culture of knee articular chondrocytes of the rabbits： trypsin / type Ⅱ collagenase was used for digestion following cutting the knee articular chondrocytes into pieces, which could well isolate the cells from the extracellular matrix. After inoculation for 24 hours, some cells began to grow; proliferated 72 hours later and cellular fusion rate was 90%-100% On day 7. In the initial stage of the adherence, the cells presented round or triangle, and triangle mainly; passage was conducted once every 7 days.② Chondrocytes trahsfected by peDNA3.1-BMP7 plasmid under the induction of lipesome： The chondrocytes after transfection were screened with G418, and the transfection rate was 15% on the 4^th day. The fusion rate of positive clone cells was about 90% 7 or 8 days l

关 键 词：生物医学工程 骨形态发生蛋白质类 分析 转基因 软骨细胞 遗传载体 质粒 

分 类 号：R318[医药卫生—生物医学工程]