The Effect Of Brain-derived Neurotrophic Factor On The Proliferation And The Osteogenic Differentiation Of Human Adipose-derived Stem Cells

Objective1. To investigate the expression of TrkB receptor and p75NTR receptor in hASCs.2. To define the effect of BDNF on the proliferation of hASCs and on the cell-cycle-related gene expression of hASCs.3. To understand the impact of BDNF on the ALP activity of hASCs and on bone-related protein gene expression of hASCs in order to clarify the role of BDNF in the osteogenic differentiation of hASCs.Methods1. Cells were isolated from human adipose tissue by collagenase digestion and were purified by extension. hASCs were monitored by flow cytometry and further cultures in osteogenic and adipogenic medium for directed differentiation, followed by staining experiments for identification.2. hASCs were treated with BDNF of various concentration and were detected by MTT assay at 12h,24h, and 48h, respectively. Futhermore, c-myc and ki-67 mRNA level were confirmed by real-time PCR at 48h.3. hASCs were cultured in osteogenic medium which contained BDNF with various concentration and the ALP activity were tested at 14d. Real-time PCR were employed to monitor the mRNA level of Runx2, ALP, and OCN at different time point.Results1. hASCs expressed the surface markers of mesenchymal stem cells but did not express the surface markers of hemopoietic stem cells. hASCs could be directedly induced toward osteogenic and adipogenic differentiation in vitro.2. hASCs expressed the TrkB receptors but didn’t express the p75NIR receptors.3. BDNF promoted the proliferation of hASCs in dose-dependent manner, and up-regulated the expression of c-myc and ki-67.4. There was an inverse relationship between the ALP activity of hASCs and the concentration of BDNF. The suppression effect was the most significant in the group of 100ng/ml. The mRNA level of ALP decreased at 7d during osteogenic induction. At 14d, the gene expression of Runx2, ALP, and OCN decreased with the increase of BDNF concentration, and the suppression was also the most notable in the group of 100ng/ml. ConclusionhASCs can be effectively isolated from human adipose tissue by collagenase digestion and purified by extension. BDNF with high concentration promotes the proliferation of hASCs while inhibits the osteogenic differentiation.