Abstract:

Objective To discuss the promotive effect of nerve growth factor （NGF）， which is highly expressed in the adipose-derived stem cell （ADSC）-induced Schwann-like cells （SCLCs）， on the growth of dorsal root ganglion （DRG） cell processes in the rats， and to clarify its mechanism. Methods The ADSCs were extracted from the epididymal adipose tissue of the SD rats， and their multidirectional differentiation potential was identified through osteogenic， adipogenic， and chondrogenic induction. The ADSCs were induced to differentiate into the SCLCs， and the expression levels of glial fibrillary acidic protein （GFAP） and S100 calcium-binding protein β （S100β） protein in the ADSCs and SCLCs were detected by immunofluorescence staining and Western blotting methods. The DRG cells were isolated and cultured， and immunofluorescence staining was used to detect the βⅢ-tubulin expression in the DRG cells for identification. The SCLCs were co-cultured with the DRG cells（co-culture group）， the single-culture DRG cells were regared as DRG group and toluidine blue staining was used to observe and measure the length of DRG cell processes under the optical microscope in co-culture group and DRG group. Small interfering RNA （siRNA） transfection was used to knock down NGF， and plasmid transfection was used to over-express NGF. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the NGF mRNA expression levels in the cells in various groups； enzyme-linked immunosorbent assay （ELISA） method was used to detect the NGF protein levels in the cell supernatants. The transfected SCLCs were co-cultured with DRG cells and divided into control group， siNC/vector group， NGF knockdown group （si-NGF group）， and NGF over-expression group （oe-NGF group）. The lengths of DRG cell processes in various groups were observed. Results The primary ADSCs adhered within 24 h after seeding， with a small number of lipid droplets remaining. After 3 d of culture， the cells were mostly short spindle-shaped， fusiform， or polygonal， growing rapidly in a vortex pattern. After passaging， the cells exhibited a uniform morphology， appearing as long spindles arranged in a fish-school pattern. After 14 d of adipogenic induction， the cell morphology changed from spindle-shaped to flat-round， with translucent lipid droplets forming in the cytoplasm， which were stained red by Oil Red O. After 28 d of osteogenic induction， the cells appeared sand-like with blurred morphology， and calcified nodules were observed， which were stained red by Alizarin Red and deposited in the extracellular matrix. After 28 d of chondrogenic induction in a 3D culture system， millet-sized chondrogenic spheres formed. Frozen sections of the spheres were stained with Alcian Blue， and acidic mucopolysaccharides in the cartilage tissue were stained blue under the microscope. Under the fluorescence microscope， the third-passage purified ADSCs showed positive expression of CD29 ［fluorescein isothiocy anate（FITC）-labeled green fluorescence］ and CD44 （Cy3-labeled red fluorescence）. The immunofluorescence staining results showed that GFAP was labeled with FITC （green fluorescence）， and S100β was labeled with Cy3 （red fluorescence）. The Western blotting results showed that compared with ADSCs， the expression levels of S100β and GFAP proteins in the SCLCs were increased （P<0.05）. The primary DRG cells began to adhere 6 h after conventional culture， and after 3 d， the cell bodies appeared round and bright， with two linear processes extending from them. Under fluorescence microscope， the cells positively expressed the neuron-specific marker βⅢ-tubulin， confirming that the isolated cells were DRG cells. Compared with the ADSCs， the NGF protein expression level in the SCLCs was increased （P<0.05）. Compared with DRG group， the length of DRG cell processes in co-culture group was the highest when DRG cells and SCLCs were co-cultured at a 1∶2 ratio （P<0.05）. The RT-qPCR results showed that compared with si-NC group， the expression levels of NGF mRNA in the cell supernatant in si-NGF-1， si-NGF-2， and si-NGF-3 groups were significantly decreased （P<0.05）， with si-NGF-1 showing the highest knockdown efficiency， which was selected for subsequent experiments. The ELISA results showed that compared with si-NC group， the NGF levels in the cell supernatant of si-NGF-1， si-NGF-2， and si-NGF-3 groups were decreased （P<0.05）. Compared with Vector group， the expression level of NGF mRNA and NGF protein level in the supernatant in oe-NGF group were increased （P<0.05）. Compared with control group and siNC/vector group， the length of DRG cell processes in si-NGF group was decreased （P<0.05）， while the length of DRG cell processes in oe-NGF group was increased （P<0.05）. Conclusion ADSCs can be directionally differentiated into SCLCs， and the differentiated cells highly express NGF. Knockdown or overexpression of NGF can affect the growth of DRG cell processes.

Key words: Schwann-like cells, Nerve growth factor, Dorsal root ganglion cells, Adipose-derived stem cells, Peripheral nerve injury