Abstract:

Objective： To discuss the effect of Ghrelin combined with LY294002 on neural differentiation of the adipose-derived mesenchymal stem cells （ADSCs）， and to clarify its mechanism. Methods The morphology of ADSCs was observed under inverted phase contrast microscope， and the positive expression rates of surface antibodies of the ADSCs in various groups were identificated with flow cytometry； the fourth-generation ADSCs were divided into control group （given non-intervention）， LY294002 group ［given 40 μmol·L^－1 phosphatidylinositol-3-kinase （PI3K）/protein kinase B （Akt） pathway specific inhibitor LY294002］，Ghrelin group （given 0.1 μmol·L^－1 Ghrelin）， and Ghrelin+LY294002 group （given 40 μmol·L^－1LY294002+0.1 μmol·L^－1 Ghrelin）.Inverted phase contrast microscope was used to observe the morphology of the ADSCs in various groups after the neural differentiation， immunofluorescence staining was used to detect the percentages of neuron specific enolase （NSE）， nestin antibody，and glial fibrillary acidic protein （GFAP） positive cells in the ADSCs in various groups， and Western blotting method was used to detect the expression levels of PI3K/Akt pathway proteins in the ADSCs in various groups，and the ratios of phosphorylated PI3K（p-PI3K）/PI3K and phosphorylated Akt（p-Akt）/Akt were calculated. Results The expressions of CD90 and CD13 of ADSCs surface antibodies were high，and the expressions of CD34，CD45，and CD106 were low.Compared with control group，parts of the cells in LY294002 group changed from long spindle shape to round shape and the protuberance was short；compared with LY294002 group，the transformation speed of the cells in Ghrelin+LY294002 group from long spindle shape to round shape was quick，and the number of protuberance was increased.Compared with control group， the percentages of NSE and Nestin positive cells and the ratios of p-PI3K/PI3K and p-Akt/Akt in the ADSCs in LY294002 group were decreased significantly（P<0.05）， the percentages of NSE and Nestin positive cells and the ratios of p-PI3K/PI3K and p-Akt/Akt in the ADSCs in Ghrelin group were increased （P<0.05）； compared with LY294002 group， the the percentage of NSE and Nestin positive cells and the ratios of p-PI3K/PI3K and p-Akt/Akt in the ADSCs in Ghrelin+LY294002 group were increased （P<0.05）； there were no significant differences in the percentage of GFAP positive cells in the ADSCs among various groups （P>0.05）. Conclusion Ghrelin promotes the neural differentiation of the ADSCs by activating the PI3K/Akt pathway.

Key words: Ghrelin, LY294002, Adipose-derived mesenchymal stem cells, Neural differentiation, Phosphatidylinositol-3-kinase/protein kinase B signaling pathway