Cathepsin K基因抑制后对软骨细胞功能的影响研究

Study of Cathepsin K Gene Inhibition on Cartilage Cell Function

目的研究应用RNA干扰技术早期抑制人Cathepsin K(CTSK)基因的表达对延缓软骨细胞的去分化过程及维持软骨细胞表型的影响。方法pGCsilencerTMH1/Neo/GFP/CTSK RNAi质粒体外转染人软骨细胞,并用G418筛选3周,再通过RT-PCR检测CTSK、Ⅱ型胶原和Aggrecan cDNA转录的表达,Western-Blot、免疫荧光检测转染后软骨细胞的CTSK、Ⅱ型胶原和Aggrecan在蛋白水平的表达。结果装入质粒载体转染第1代的软骨细胞,在体外通过细胞形态观察可发现,抑制CTSK基因后3周软骨细胞仍大多数保持多角形,而对照组则向成纤维样细胞形态变化。RT-PCR结果显示,在mRNA水平抑制了CTSK的表达,而不影响对软骨细胞Ⅱ型胶原和Aggrecan的mRNA表达。免疫荧光和Western-blot的结果证实了在早期抑制了软骨细胞CTSK基因表达并维持3周左右,软骨细胞的特异性基质Ⅱ型胶原和Aggrecan在蛋白水平明显增加。结论早期抑制了软骨细胞CTSK基因的表达,可使软骨细胞去分化过程中其软骨特异性基质Ⅱ型胶原和Aggrecan增加,说明可以维持软骨细胞的表型。

Objective To study the application of RNA interference inhibits the early Cathepsin K （CTSK） gene expression in delaying the process of chondroeyte dedifferentiation and maintaining the chondrocytes phenotype. Methods pGCsilencerTM H1/Neo/GFP/CTSK RNAi plasmid was transfected into human chondrocytes in vitro, three weeks later, CTSK, collagen Ⅱand Aggrecan cDNA expression were analyzed by RT-PCR CTSK, collagen Ⅱ and Aggrecan expression at the protein level in transfected chondrocytes were analyzed by Western-Blot, immunofluorescence assay. Results The plasmid vector was transfected into the first generation of cartilage cells in vitro, 3 weeks after CTSK gene suppression chondrocytes can be found with most of polygonal by cell morphology, compared with the control group to the fibroblast-like morphological changes. By RT-PCR results CTSK was at inhibited while the type Ⅱ collagen and Aggrecan of the mRNA without affecting. Immunofluorescence and western-blot results confirmed cartilage-specific matrix of type Ⅱcollagen and Aggrecan in the protein level increased significantly. Conclusion Early inhibition of CTSK gene expression in chondroeytes, the cartilage- specific matrix type Ⅱ collagen and Aggrecan significantly increased during the dedifferentiation process, the cbondrocyte phenotype can be maintained.