The Adipogenic Capability Of RBMMSCs In Vitro And The Effects Of Rosiglitazone

Objective To investigate the proliferative and adipogenic capability of rabbit bone marrow mesenchymal stem cells(rBMMSCs) in vitro,also to observe the changs of Peroxisome Proliferator-Activated Receptor-γ(PPARγ) gene expression during cell differentiation and the effects of Rosiglitazone on cell differentiation and PPARγgene expression.Methods New Zealand rabbits were general anesthetized by sumianxinⅡ0.2ml/kg i.m.Two millilitres of bone marrow were isolated from right femoral bone cavitas with a myeloid puncture needle.Rabbit BMMSCs were then obtaind by combination with density gradient centrifugation and adherent culture.The 6th generation of rBMMSCs were used in the following experiments.Cell growth curve was measured by MTT.Then cells were randomly divided into 2 groups:1,Control group,cells were cultured in normal culture medium;2,adipogenic inducing group,cells were cultured in adipogenic inducing medium containing 10%FBS,dexamethasone 1μM,insulin 170 nM,isobutylxanthine 0.2 mM and 3-isobutyl-1-xanthine 0.5mM.After induction for 21 days,the group two was again randomly divided into 2 groups:One was cultured in adipogenic inducing medium to 28 days,and the other was cultured in adipogenic inducing medium containing 2μM rosiglitazone to 28 days.Adipocytes were calculated under microscope and stained with Oil Red O on the 7th,14th,21st and 28th day respectively.After staining , Oil Red O was washed using dimethylcarbino and the content of Oil Red O was measured by spectrophotometer.Gene expression of PPARγwas measured by Reverse transcription-polymerase chain reaction(RT-PCR)assay in each group on the upper time points.Results The rBMMSCs showed stable proliferative capability in primary and passage cultures.The adipogenic rates were 23.1±4.1% , 57.4±3.0%,70.3±3.6%,68.3±3.9% and 84.5±4.2% on the 7th, 14th, 21st, 28th day in adipogenic inducing groups and rosiglitazone group respectively,which means that adipogenic rates increased as time past in the first 21 days'induction and rosiglitazone could enhance the adipogenic rates,P<0.01.The values of OD measured by spectrophotometer were 0.07±0.01,0.21±0.02,0.31±0.06,0.29±0.04 and 0.46±0.05 on the 7th,14th,21st,28th day in adipogenic inducing groups and rosiglitazone group respectively,which means that fat content increased as time past in the first 21 days'induction and rosiglitazone could enhance the fat content,P<0.01.PPARγ/GAPDH mRNA ratioes were 0.16±0.008,0.28±0.02,0.42±0.04,0.60±0.09,0.58±0.09 and 0.87±0.08 on the 0,7th,14th,21st,28th day in adipogenic inducing group and rosiglitazone group respectively,which means that the degrees of PPARγmRNA expression increased as time past in the first 21 days'induction and rosiglitazone could enhance the expressions of PPARγ,P<0.05.Conclusion The cells isolated by the methods adopted in our literature have stable reproductive activity and adipogenic capacity. These cells may be useful in the study of mechanisms about dipocytes differentiation. Rosiglatazone may facilitate the adipogenic capacity of rBMMSCs.