Molecular Mechanism of Adipose Tissue-derived Mesenchymal Stem Cells Differentiating into A Nucleus Pulposus-like Phenotype
|School||Huazhong University of Science and Technology|
|Keywords||Fat -derived mesenchymal stem cells (ADSCs) Nucleus pulposus (NP) cells Co-culture Transforming growth factor- β1 (TGF-β1)|
[Objective] To investigate the co-culture environment with SD rats sources nucleus pulposus ( T nucleus of space of huge nucleus , NP ) cells promote fat mesenchymal stem cells (adipose tissue-derived mesenchymal stem cells, ADSCs) molecular mechanisms directed differentiation to NP -like cells , as human intervertebral disc cell transplantation in the treatment of degenerative diseases of the basis for . [ Method ] ( 1 ) 150g SD female rats were sacrificed , taken groin perirenal adipose tissue , 0.1% collagenase digestion vitro training of inoculation , ADSCs were purified by limiting dilution method ; rats nucleus pulposus , the use of tissue blocks NP cell culture method . ( 2 ) the use of flow cytometry of ADSCs were identified . NP cells collagen type II antibody staining . ( 3) are respectively the third generation ADSCs and primary NP cell digestion count to adjust the cell density was adjusted to 1 × 105/ml ADSCs were seeded in six-well plates , NP cells were inoculated in the BD culture pool ; two cell 1:1 ratio through non - cell contact co-culture . ( 4 ) were cultured for 2 weeks to do the TGF-β1 receptors immunofluorescence assay, extracted the ADSCs RNA do RT-PCR and real-time quantitative PCR . Results ( 1) flow cytometry results show adipose mesenchymal stem cells Sca- 1 positive of CD44 were 95.2 % , 99.7% , CD45 , CD11b positive rate of 0.4% , 1.5% . NP cells type II collagen antibody positive immunostaining . (2) the immunofluorescence detection results show : the co-culture group fluorescence intensity was significantly higher than ADSCs group . The the NP cells secrete cytokines role of TGF-β1 in ADSCs, promote the ADSCs surface TGF-β receptor 1 expression . (3) PCR test results show : ADSCs co-cultured environment focusing NP cells induced proteoglycan ( aggrecan ) , type II collagen (Collagen II ) , sox- 9 bands were significantly brighter than the simple ADSCs group ; co-culture group Smad3 and Smad4 gene expression levels higher than the the ADSCs group alone . [Conclusion] The results show that : the co-culture environment NP cells can induce ADSCs differentiation to NP - like cells .