Establishment and identification of the rat models of chondrocyte apoptosis induced by tumor necrosis factor-alpha / 中国组织工程研究

ABSTRACT

BACKGROUND:Tumor necrosis factor-α(TNF-α), a main cytokine inducing chondrocyte apoptosis of osteoarthritis, plays a regulatory role in the process of osteoarthritis. OBJECTIVE:To compare the rat models of chondrocyte apoptosis induced by different mass concentrations of TNF-α, thus providing theoretical basis for further study on the regulation of drugs on chondrocyte apoptosis. METHODS:Chondrocytes were isolated from the knee cartilage of 4-week-old Sprague-Dawley rats of clean grade by mechanical l col agenase digestion and were then incubated with different mass concentrations of TNF-αto induce apoptosis. The morphology of chondrocytes was observed under inverted phase contrast microscope, cel s were identified using immunohistochemical staining of type II col agen, as wel as the cel viability and apoptosis were detected by MTT and DAPI, respectively. RESULTS AND CONCLUSION:(1) In vitro, the cytoplasm of chondrocytes was stained brown-yel ow by using immunohistochemical staining of type II col agen. (2) At 48 hours, the apoptosis rate of chondrocytes induced by 10, 20 and 30μg/L TNF-αwas significantly higher than that of the 0μg/L TNF-α(P<0.01), and the apoptosis rate of chondrocytes induced by 40μg/L TNF-αwas significantly higher than that of the 10μg/L TNF-α(P<0.01). (3) The viability of chondrocytes induced by 10, 20 and 40μg/L TNF-αwas significantly lower than that of the 0μg/L TNF-α(P<0.01). In detail, the viability of chondrocytes induced by 20μg/L TNF-αwas lower than that of the 10μg/L TNF-α(P<0.05);the viability of chondrocytes induced by 40μg/L TNF-αwas significantly lower than that of the 10 and 20μg/L TNF-α(P<0.01, P<0.05). (4) These results suggest that 20μg/L TNF-αcan successful y induce the chondrocyte apoptosis model.