At-RA Inhibits Its Adipogenic Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells

Objective To investigate the mechanisms of all-trans retinoic acid(at-RA) inhibiting the adipogenic differentiation of rat bone marrowmesenchymal stem cells (rBMSCs).Material and methods rBMSCs were isolated from the bone marrow of4-week-old SD rats. And the cells were incubated and induced to differentiateinto adipocytes. At-RA at high concentration (0.1-5.0mol/L) was added to theadipogenic induced medium. rBMSCs were cultured under the followingconditions: blank control (no adipogenic medium), serum (no at-RA), andat-RA at0.1,0.5,1.0,5.0mol/L, respectively. The dynamic morphologicchanges of rBMSCs during differentiation were observed under microscopewith or without oil red O staining. The effect of at-RA on adipogenicdifferentiation was quantitatively analyzed via oil red O staining. Theexpression of retinoic acid receptors and (RAR, RAR),CCAAT/enhancer binding protein (C/EBP) and peroxisomeproliferators-activated receptor2(PPAR2) was analyzed by real-timequantitative polymerase chain reaction (real-time PCR) at the end of induction.We also determined their mRNA levels at fixed concentration of1.0mol/L ofat-RA at different time, i.e. induction0,1,3,5, and15days.Results (1) Primary and adipogenic cultures: rBMSCs were fibrous,arranged as a palisade. They can be induced to differentiate into adipocytes inadipogenic medium.(2) Cell morphology and oil red O staining observation: After adding at-RA to the adipogenic medium, the appearance of intracellularlipid droplets was delayed, lipid droplet size and the number of cells containinglipid droplets were less than that of serum group. at-RA inhibited rBMSCs todifferentiate into adipocytes, and the effect was in a dose-dependent manner.(3)The lipids of differentiated rBMSCs (i.e. adipocytes) decreased with increasedat-RA concentrations measured by Oil Red O staining semi-quantitativemeasurement (P <0.01).(4) Real-time PCR detection: Compared with controland serum groups, C/EBP and PPAR2expressions were significantlydecreased at the end of induction after adding at-RA,(P<0.05). The mRNAlevel of RAR was significantly increased (serum group vs1.0mol/L：ｘ-±s=0.2259±0.0012vs ｘ-±ｓ=0.0531±0.0023，P<0.005), although RAR hadno significant difference (serum group vs1.0mol/L：ｘ-±ｓ=0.0084±0.0019vs ｘ-±s=0.0091±0.0013，P>0.05). At1.0mol/L of at-RA, C/EBP andPPAR2expressions did not changed too much for the first five days. However,significant inhibitory effect was identified at end of differentiation (p<0.001).Conclusions (1) rBMSCs were obtained successfully from whole bonemarrow, and could be induced into adipocytes.(2) At-RA (0.1-5.0mol/L) caninhibit the adipogenic differentiation of BMSCs by up-regulating theexpression of RAR and down-regulating the expression of C/EBP, PPAR2.