摘要
目的探讨外源性基因ｈＢＭＰ３在软骨细胞获得稳定表达的可能性。方法将人ＢＭＰ３的ｃＤＮＡ构建于真核表达载体ＰｃＤＮＡ３，形成重组真核表达载体ＰｃＤＮＡ３ｈＢＭＰ３，转染培养状态下兔关节软骨细胞。利用重组ＤＮＡ和基因克隆技术构建重组真核表达载体ＰｃＤＮＡ３ｈＢＭＰ３；利用细胞培养和细胞基因转染技术体外转染ｈＢＭＰ３基因至关节软骨细胞；利用核酸提取（ＮｏｕｔｈｅｒｎＢｌｏｔ）和核酸杂交技术检测细胞转染后４周ｈＢＭＰ３基因的表达情况。结果兔关节软骨细胞属贴壁生长，呈多角形，细胞一代时间５天，指数生长期为接种后２～４天。ＰｃＤＮＡ３ｈＢＭＰ３用ＥｃｏＲⅠ和ＸｂａⅠ双酶切下的片段和λＤＮＡ／ＨｉｎｄⅢＭａｒｋｅｒｓ于１５％琼脂糖电泳，为５４ｋｂ和１４ｋｂ，符合各自的物理图谱，表明构建重组基因成功。细胞转染后用Ｇ４１８筛选至４周，仍有新的细胞克隆形成，提取克隆细胞的ｍＲＮＡ做斑点杂交，结果显示阳性。结论在脂质体的介导下，重组真核表达载体ＰｃＤＮＡ３ｈＢＭＰ３转染兔关节软骨细胞且获得了稳定表达。
bjective To explore the possibility of stable expression of PcDNA3hBMP3 in cultured articular chondrocytes of rabbit.WT5HZMethods PcDNA3hBMP3 was constructed using gene clone technique and recombined DNA technique. With the help of profectamine, the cultured articular chondrocytes were transfected with PcDNA3hBMP3, and the evidence of successfully stable transfection in these cells could be obtained by positive northern blot.T5HZResults The cultured articular chondrocytes of rabbits seemed to be polygonal, and its logarithmic growth phase was 24 days after cell inoculation. The two fragments cut from PcDNA3hBMP3 by EcoR and Xba represented 54 kb and 14 kb by electrophoresis, which were confirmed to be the carrier and the fragment inserted originally, indicating that the construction of PcDNA3hBMP3 was successful. The RNA extracted from cultured chondrocytes was screened for 4 weeks by G418 hibrided with the fragment cut from hBMP3 positively. Conclusions With the help of profectamine, the cultured articular chondrocytes can be transfected by recombined gene of PcDNA3hBMP3 successfully, and their stable expression at 4 weeks after transfection is obtained.
出处
《中华外科杂志》
CAS
CSCD
北大核心
1999年第7期391-394,I026,共5页
Chinese Journal of Surgery
