软骨细胞退变模型 z-bo 2020-12-01 374

　　Objective: To express human Wnt7b gene in eukaryotic cells and observe its degenerative effect on primary rat chondrocytes.

　　Method: Take out the cartilage from the joints of SD rats at 24 h, and obtain primary chondrocytes after multiple digestion with type Ⅱ collagenase. Take P1 generation cells for experiment. Amplify the human Wnt7b gene and clone it into PCDH-GFP, transfect PCDH-GFP and PCDH-Wnt7b into 293ft cells, collect the cell supernatant and transfected cells after 48 hours, Western blot to identify the expression of Wnt7b in 293ft cells. The collected supernatant was diluted 10-fold and 50-fold to culture rat chondrocytes. After 24 hours, the cell morphology was observed and cell protein and RNA were collected. Western blot and quantitative PCR were used to detect cartilage degeneration indicators MMP13, MMP3, type II collagen, Expression of A-can, ADAMTS5, ColⅩ and SOX9.

　　Result: The human Wnt7b gene was successfully cloned into the PCDH-GFP vector and effectively expressed in 293ft cells. The cartilage cell morphology changed from the original polygonal shape to a long fusiform shape after the Wnt7b-containing medium intervened with the cartilage cells for 24 h. The expression of MMP13 and MMP3 in the Wnt7b treatment group was significantly up-regulated, and the expression of type II collagen was down-regulated. PCR results showed that the expression of A-can and Sox9 decreased, and the expression of ColⅩ and ADAMTS5 increased.

　　 Conclusion: Effectively express Wnt7b gene in eukaryotic cells and promote the degeneration of rat chondrocytes, and establish an in vitro model of chondrocyte degeneration.