动物造模,软骨细胞退变模型 z-bio 2022-11-24 246

　　Objective: To express human Wnt7b gene in eukaryotic cells and observe the degeneration of rat primary chondrocytes.

　　Methods: Chondrocytes were obtained from the articular cartilage of 24 h old SD rats. The primary chondrocytes were obtained after multiple digestion with type Ⅱ collagenase, and P1 generation cells were taken for experiment. The human Wnt7b gene was amplified and cloned into PCDH-GFP. It was transfected into PCDH-GFP and PCDH-Wnt7b 293ft cells. The supernatant and transfected cells were collected 48 hours later. Western blot was used to identify the expression of Wnt7b in 293ft cells. The collected supernatant was diluted 10 times and 50 times respectively to culture rat chondrocytes. After 24 hours, cell morphology was observed and cell protein and RNA were collected. Western blot and quantitative PCR were used to detect the expression of cartilage degeneration indicators MMP13, MMP3, type II collagen, A-can, ADAMTS5, Col X and SOX9.

　　Results: Human Wnt7b gene was successfully cloned into PCDH-GFP vector and effectively expressed in 293ft cells. After the intervention of chondrocytes with Wnt7b containing medium for 24 hours, the morphology of chondrocytes changed from polygonal to spindle. The expression of MMP13 and MMP3 in chondrocytes of Wnt7b treated group was significantly up-regulated, while the expression of type II collagen was down-regulated. The results of PCR showed that the expression of A-can and Sox9 decreased, while the expression of Col X and ADAMTS5 increased.

　　Conclusion: Wnt7b gene can effectively express in eukaryotic cells and promote the degeneration of rat chondrocytes, so as to establish an in vitro model of chondrocyte degeneration