重组PcDNA3-hBMP3转染兔关节软骨细胞和对其增殖的影响

Effect of recombined PcDNA3 - hBMP3 transfection on rabbit articular chondrocytes proliferation

目的研究将人骨形成蛋白３（ＢＭＰ３）的ｃＤＮＡ构建于真核，表达载体ＰｃＤＮＡ３，形成重组ＤＮＡ ＰｃＤＮＡ３－ｈＢＭＰ３，转染兔关节软骨细胞，以探讨基因转染对关节软骨细胞增殖的影响。方法利用重组ＤＮＡ和基因克隆技术构建重组ＤＮＡ；用细胞培养和基因转染技术体外转染兔关节软骨细胞；用核酸杂交和蛋白电泳检测细胞ＢＭＰ３的表达，最后通过ＦＣＭ、ＤＮＡ和葡糖醛酸含量检测以及Ⅱ型胶原探针原位杂交分析其对增殖的影响。结果软骨细胞６次传代后，Ⅱ型胶原原位杂交的灰度值降低，同样条件下转染的软骨细胞仍保持较高水平Ⅱ型胶原的表达；转染的软骨细胞经流式细胞仪的分析，软骨细胞Ｓ期细胞比例增多，说明细胞ＤＮＡ的合成增加；经ＤＮＡ和葡糖醛酸含量测定，转染的软骨细胞ＤＮＡ和葡糖醛酸含量较转染前有明显提高。结论ｈＢＭＰ３对维持软骨细胞的表型具有十分重要的作用，在脂质体的介导下，ＤＮＡ ＰｃＤＮＡ３－ｈＢＭＰ３转染兔关节软骨细胞获得成功。

The recombined DNA PcDNA3-hBMP3 was transfected into rabbits articular condrocytes in vitro under liposome to study the instantaneous and steady mRNA or its protein expression of hBMP3 in rabbit's articular chondrocytes, and make further study on the effects of transfection on proliferation of these cells. Methods in order to learn whether there is expression of hBMP protein in these cells after transfection, the protein electrophoresis has been applied. In situ hybridization by type Ⅱ collagen cDNA probe was used to make sure that whether the cell still has its phenotype after transfection. The measurements of the FCM, DNA and GAG have been used to explore the changes in all these fields in the cells. Results Through many generations, chondrocytes after transfection still remain type Ⅱ collagen expression. This shows the important role of the transfection of hBMP3 to maintain the phenotype of chondrocytes. Cellular proportion in S period increased, which indicates the increase of synthesis and propagation of cell DNA. DNA and PG contents of chondrocytes increase remarkably after transfection. Conclusion hBMP3 plays a great role in maintaining the phenotype of chondrocytes. With the help of lipofectamine, the transfection of PcDNA3－ hBMP3 to chondrocyte was carried out successfully.