Recombinant Pcdna3-the Hbmp3 Transfection Of Rabbit Articular Cartilage Cells And Its Proliferation

Chondrocyte represents the low mitotic activity and the poor repair ability. The limited proliferation of chondrocyte, its dependence on the inoculation density and labial phenotype are really a difficult problem in chondrocyte culture in vitro, which usually is the preset condition for setting up chondrocyte bank .In non-protein medium, the chondrocytes inoculated by the density of 10⁴ cells/cm² can not survive more than several days, while the chondrocyte with the density of 10⁵ cells/cm² can survive Mary weeks in vitro, which implies that they can survive only when they receive cell active factor released by the nearby cells. The research of Edward indicated that after many generations, the chondrocyte which are grown monolayer in vivo may lose the ability of producing PG and collagen II, while some cell active factor, such as BMP3, FGF, IGF, play critical role in improving its ability of producing PG and collagen II. So it's critical for forming steady, mature chondrocyte that chondrocyte itself release active cell factors. The experiments in vivo or in vitro have confirmed that BMP-3 can stimulate the proliferation of chondrocyte and improve maintenance of cell phenotype. Frank has discovered that BMP3 from cox and people share the autoploidy after measuring sequence of BMP3, but BMP3 of people can only induce the formation of chondrocyte with the increasing release of GAG and collagen II, and Carringto verified that BMP3 can stimulate the proliferation of chondrocyte obviously.The authors aim at constructing recombined gene of PcDNA3-hBMP3, which can translate and transcript in eucaryotic cells and exploring the possibility of stable expression of PcDNA3-hBMP3 in cultured articular chondrocytes of rabbit. It's a new way and a new thought to resolve the chondrocyte proliferation and modification of phenotype using gene transfection method.Part I Investigation of the growth characteristics of cultured rabbit articularchondrocyteSeparation and culture of rabbit articular chondrocyte in vitro is the basis of the study of gene transfection of recombinant PcDNA3-hBMP3 into cell. We investigated the growth characteristics of cultured rabbit articular chondrocytes to provide necessary experimental condition.Articular cartilage were removed from the distal end of humerus,tibia and femurs of one 28 days fatal New Zealand white rabbit, primary chondrocytes were digested with collagenase, trypsin and hyaluronidase. The total number of the chondrocytes after digestion were dilated to 10⁵cells/ml with DMEM, and primary culture was made in CO₂ incubator...