Investigation Of Sox9and Collagen Ⅱ MRNA Expression With Space-time Changes During Rabbit’s BMSCs Chondrogenic Pellet Culture

Objucive Isolating mesenchymal stem cells from rabbits’ bone marrow and cultured in intro. To investigate the expression of Sox9and COL2A mRNA during pellet chondrogenesis with the time changes and do some preliminary study of the cartilage tissue engineering on gene lever.Methods3New Zealand white rabbits were involved in this experiment. Rabbits MSCs were obtained by cultured bone marrow aspirated from rabbits’femoral trochanteric in plastic bottle. The third passage cells were harvest, growth curvature were drawed. The experiment are divided into two groups, the experiment group (pellet chondrogenesis group) and control group (non-chondrogenesis pellet group). The experiment group use chondrogenessis medium with lOng/ml rhTGF-β1and the control group use the same medium without rhTGF-β1.Total RNA of two groups was isolated at4day,8day,12day and16day respectively. Sox9and Col2a were detected by reverse-transcription polymerase chain reaction (RT-PCR).Results The primary MSCs adhere to the plastic bottle and with the culture medium changed, the cells get pure gradually during the process and had a similar spindle-like morphology. The third passage of MSCs grew well, and underwent latent period, latent period and plateau phase. Experiment group formed cell pellet at the second day after pellet culture and grew up gradually, but the control group cells couldn’t form pellet and disseminated under the centrifugal tube bottom. The relative expression of Sox9and Co12a mRAN increased in experiment group with an increase in time, and the two gene had direct positive correlation. Col2a was not observed in control group. There were significant differences in expression of the Sox9and Col2a between two groups.Conclusion The combination of the whole bone marrow culture and the adhere culture is an effective method to isolate BMSCs. The culture medium contained TGF-β1could induce BMSCs to chondrocytes during pellet culture in vitro, but the medium without TGF-Pi couldn’t. The expression of Sox9and Col2a in MSCs chondrogenic pellet culture are obviously increased and show direct positive correlation. Because the Sox9is transcription factor of Co12a the chondrogenesis is achieved by raising the expression of Sox9.