ABSTRACT
Objective To induce human-induced pluripotent stem cells(iPSCs)to differentiate into spinal motor neuron precursor (MNP)and compare the induction efficiency in systems of feeder and feeder-free. Methods iPSCs cultured on mouse feeder cells or in feeder-free condition were induced into neuroepithelial progenitors (NEP) on the sixth day and MNP on the twelveth day.Their morphology was observed under inverted micro-scope,and the markers of iPSCs,NEP,MNP were detected with immunofluorescence.NEP-related genes SOX1 and HOXA3,MNP-related genes OLIG2 and PAX6,and pluripotency genes SOX2 and OCT4 were detected with real-time quantitative polymerase chain reaction. Results iPSCs expressed pluripotency markers,while NEP and MNP expressed high levels of neural related markers and low levels of pluripotency markers in two systems. The expression of the genes SOX1, HOXA3, OLIG2 and OCT4 was higher in the feeder system,and there was no significant difference in the expression of genes SOX2 and PA X 6. Conclusion iPSCs can differentiate into MNP in culture systems of feeder and feeder-free,and the induction efficiency is higher in the feeder system.
ABSTRACT
This study was designed to investigate the effect of Xiao-Ai-Ping injection on cancer angiogenesis. CCK8 assay and BrdU incorporation immunofluorescence assay were used to detect the effect of Xiao-Ai-Ping injection on HUVECs proliferation; wound healing assay and transwell assay were employed to test the effect of Xiao-Ai-Ping injection on HUVECs migration. The anti-angiogenic effect of Xiao-Ai-Ping injection was examined by tube formation assay, rat aortic ring assay and chicken chorioallantoic membrane (CAM) assay. ELISA assay was used to measure the secretion of vascular endothelial growth factor (VEGF); and the activation of vascular endothelial growth factor receptor 2 (VEGFR2) protein and its downstream signaling pathways were examined by Western blot. Our data demonstrated that Xiao-Ai-Ping injection inhibited HU-VECs proliferation in a time-and dose-dependent manner, and the IC50 (mg·mL-1) values for 24, 48 and 72 h were 48.7±7.14, 29.1±2.25 and 22.0±4.53, individually. Xiao-Ai-Ping injection inhibited HUVECs DNA synthesis and migration. Xiao-Ai-Ping injection suppressed HUVECs tube formation, and reduced microvessel sprouting from rat aortic rings and vessel growth in CAMs. Furthermore, Xiao-Ai-Ping injection attenuated the secretion of VEGF, and inhibited the expression of p-VEGFR2 and phosphorylation of protein kinase B (p-AKT). We conclude that Xiao-Ai-Ping injection inhibits angiogenesis by down-regulation of VEGF signaling and AKT pathway.