γ-secretase inhibitors on the efficiency of the hair follicle stem cell differentiation into vascular endothelial cells

Abstract

Objective To study the specificity of the Notch signal pathway blockers-γ-secretase inhibitors (DAPT) on the efficiency of the hair follicle stem cell (rHFSCs) differentiation into vascular endothelial cells (ECs). Methods Applying the modified method of rHFSCs separation, culture, purification, identification for seed cells, and using transmission electron microscope to observe the internal structure. With 10 μg/Lvascular endothelial growth factor 165 (VEGF165) as inducing factor to carry on the inductio, and flow cytometry instrument to detect differentiation efficiency. Then divided them into induction and inhibition group. With different concentrations of DAPT (0.5, 1.0 μmol/L) to suppress, Two group of cells are both handled for 1 week, with the method of real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting, respectively detecting the expression of mRNA and protein CD31, vascular endothelial cadherin (VE-cadherin) in the two groups, then conbined with Dil-labeled acetylated low density lipoprotein (Dil-ac-LDL) phagocytosis test and Lumen formation experiment test, finally comparing the result of two group. To comprehensive evaluate DAPT on the influence of rHFSCs differentiation ECs after it inhibited Notch signaling pathway. Results The rHFSCs with modified of isolation, culture, purification has strong cloning ability and vitality. Flow cytometry instrument testing shows β1, α6 integrin and CK15 with a high expression, respectively, 98.9%, 97.9%, 68.1% and 98.5%; and Low weakly expressed CD31, VE-cadherin, respectively, 13.6% and 17.9%, Transmission electron microscopy (sem) show that cells in original condition. Flow cytometry instrument testing shows CD31, VE-cadherin with high expression afer induced Cells for a week, respectively, 61.5% and 95.9%. After inhibition of Notch signaling pathway, according to the results of FQ-PCR: there is no obvious difference of variation of mRNA of CD31 (P=0.136) and VE-cadherin (P=0.003) between two groups, the protein expression of CD31 and VE-cadherin significantly reduced through Western blotting method (P=0.000), Dil-ac-LDL phagocytosis and lumen formation ability in vitro decreased significantly. Conclusion After interdicting the Nntch signal pathway, although DAPT has no obvious influence on the mRNA level of CD31 and VE-cadherin, but can inhibit protein expression of CD31 and VE-cadherin and ability of phagocytosis and lumen formation in vitro, thereby reducing efficiency of rHFSCs differentiation for ECs. It lays a experimental basis on further research of the mechanism of Notch signaling pathway in inducing vascular endothelial cells. Key words: The hair follicle stem cells; γ-secretase inhibitors DAPT; Notch signaling pathway; Vascular endothelial cells; Induction; Inhibition