Study Of Synergistic Effect Of BMP-14and Chondrocytes On Chondrogenesis Of ADSCs

ADSCs were transfected by Ad-CMV-BMP-14-IRES-hrGFP-1and thenco-cultured with chondrocytes, to compare the chondrogenesis effect withADSCs were transfected by BMP-14or only ADSCs co-cultured withchondrocytes, in order to evaluate the synergistic effect of BMP-14co-culturewith chondrocytes on chondrogenesis of ADSCs, Optimize the source ofchondrocytes.Methods1. Chondrocytes were isolated and cultured from articular cartilage ofrabbits; ADSCs were isolated and cultured from subcutaneous fat of rabbits.2. The cells which isolated and cultured from subcutaneous fat wereidentified to prove if they were ADSCs. ADSCs were identified by osteoblastinduction(alizarin red staining)、chondroblast induction(alcian blue staining) andlipoblast induction(oil red O staining).3.Calculate the optimum MOI of transfection of Ad-CMV-BMP-14-IRES-hrGFP-1and then ADSCs were transfected by the optimum MOI.4. Establish co-culture model and experiment group: Transwell modelswere adopted as co-culture model, ADSCs were seeded on Transwell models indensity of3×10~4/mL, chondrocytes were seeded on6hole plates in samedensity, two kinds of cells ratio of1:1.Group A: ADSCs were transfected by Ad-CMV-BMP-14-IRES-hrGFP-1and then co-cultured with chondrocytesGroup B: ADSCs co-cultured with chondrocytes Group C: ADSCs were transfected by BMP-14Group D: chondrocytesGroup E: ADSCs5. GAG content were detected by alcian blue staining method, expressionof type II collagen and BMP-14protein were detected by Western blotting, geneexpression of sox-9and Agreecan were detected by RT-PCR. Chondrogenesiseffect were detected by these indexes.Results1. The isolated and cultured cells were proved to be ADSCs byidentification.2. Inverted flurescence microscopy observed optimum transfection effectwhen MOI was150.3. Testing results of alizarin red staining、alcian blue staining、oil red Ostaining show that: GAG content、expression of type II collagen and BMP-14protein、 gene expression of sox-9in group A and C were significantly higherthan those in other3groups, group A higher than group C,(P＜0.05), group Band D were significantly higher than group E(P＜0.05), no significant differencebetween group B and D(P＜0.05).Conclusions1. ADSCs can differentiate into chondrocytes both by transfected byBMP-14and co-culture with chondrocytes.2. Synergistic effect of BMP-14and chondrocytes existence in the processof chondrogenesis of ADSCs.