Effects Of DangGui Injection On Proliferation And Metabolism Of Rabbit Chondrocytes In Vitro

Objective:To investigate the method of isolation and culture of rabbit articular chondrocytes for biological characteristic studies, To observe the effect of DangGui injection on proliferation of rabbit articular chondrocyte and the protective effect of DangGui injection on chondrocyte lesion induced by TNF-α, and discuss the mechanism of DangGui injection for treating Osteoarthritis.Methods:Articular chondrocytes were isolated from the cartilages of 3 week-old New Zealand rabbits were cultured in vitro. The morphological changes and growth feature of primary culture and subculture of chondrocytes were observed under the inverted microscope each day. Toluidine blue staining was used to indentify the chondrocytes .The first subculture chondrocytes were inoculated into 96- hole culture plate and divided into the experimental groups DangGui A - F group and the G group, which were cultured in the 10% serum DMEM media containing 10%, 5%, 2.5%, 1.25 %, 0.625%, 0.312%and 0% of DangGui injection, respectively. G Group is the blank control group were cultured in 10% serum DMEM medium, H Group were cultured in serum-free DMEM medium and no chondrocyte. After cultured for 48 hours,chondrocytes proliferation was assessed by MTT chromometry. The second subculture chondrocytes were inoculated into two 24-hole culture plates. After incubation for five days, chondrocytes were divided into 8 groups randomly with different medium. DangGui A-F groups, which were cultured in the 10% serum DMEM media containing 10%, 5%, 2.5%, 1.25%, 0.625%, 0.312% of Dang Gui injection, with100U/ml TNF-α, respectively. Experimental group were cultured in 10% serum medium with 100U/ml TNF-α, Blank control group were cultured in 10% serum DMEM only, after incubation for 72 hours, culture supernatants were collected for SOD and NOS measurement.Results:The primarily cultured chondrocytes was in polygonal shape, and became dedifferentiation after 3 passage. The chondrocytes maintained the morphology and immunochemical staining pattern within the first 3 passages . Compared with the control, 5%, 2.5%, 1.25%, 0.625%, 0.312% of DangGui injection could promote the proliferation of chondrocytes with significant difference. 0.625% DangGui injection had the greatest function in the six doses with a significant difference from other groups. 10% DanShen injection had no significant promotion effect difference. TNF-αcould promote the NOS activity and depress SOD activity in chondrocytes. DangGui injection could altered the above mentioned results in a concentration dependent manner.Conclusions:The method used in this work for isolation and culture of chondrocytes is simple and feasible. The chondrocytes cultured in vitro maintained the specific chondrocytes phenotype in the first 3 passages. The following experiment suggested that DangGui injection can effectively promote the proliferation of chondrocytes and prevent and protect against chondrocytes lesion induced by TNF-α, and regulate anabolism of chondrocytes, thus it can defer the degeneration of osteoarthritis cartilage. It demonstrated that DangGui injection was effective to prevent and cure osteoarthritis.