ABSTRACT
Objective:To investigate the impacts of astragaloside Ⅳ (AS-Ⅳ) on in- vitro proliferation and angioblastic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUCBMSCs), providing a basis for further research about the effects of AS-Ⅳ on mesenchymal stem cells (MSCs)-mediated angiogenesis. Methods:The hUCBMSCs were extracted from umbilical cord blood of normal full-term infants and subcultured. Osteoblasts, chondroblasts, and lipoblasts were induced, differentiated and identified. At the same time, the surface antigens CD44, CD73, and CD105 on hUCBMSCs were determined by flow cytometry. The successfully identified hUCBMSCs were cultured and treated with a series concentrations of AS-Ⅳ (0, 50, 100, 200, 300, and 400 mg/L). The optimum concentration of AS-Ⅳ for cell proliferation in hUCBMSCs was confirmed. In another experiment, hUCBMSCs were randomly divided into the experimental group and the control group. The cells in the experiment group were treated with the optimum concentration of AS-Ⅳ, and those in the control group were treated with equal volume of PBS. The impact of AS-Ⅳ on the proliferation of hUCBMSCs was detected using the cell counting kit (CCK-8). Besides, the impact of AS-Ⅳ on the angioblastic differentiation of hUCBMSCs was examined using the matrigel in- vitro tube formation assay. CD31 and von willebrand factor (vWF) expressions were determined using immunofluorescence after hUCBMSCs differentiated towards endothelial cells. Results:Under the light microscope, hUCBMSCs had clear edges and arranged orderly, showing a typical long fusiform structure. Flow cytometry confirmed that hUCBMSCs had surface markers of mesenchymal stem cells. The optimum concentration of AS-Ⅳ for the proliferation of MSCs was 300 mg/L. The OD values of the control and experimental groups were (0.51±0.01) and (0.98±0.05), respectively, with statistical significance ( t=15.96, P<0.05), indicating that the proliferation ability of the experimental group was enhanced. Compared with the control group, the tube density and the length of the tube network in vitro in the experimental group were higher, with statistically significant difference [(629.80±52.94)mm vs (110.36±13.19)mm, P<0.05]. Compared with the control group, the expression of CD31 and vWF increased in the experimental group after AS-Ⅳ induced hUCBMSCs differentiation ( t=13.64, 13.18, P<0.05). Conclusions:AS-Ⅳ has no toxicity to human umbilical cord blood mesenchymal stem cells, and can improve their proliferation function, and induce hUCBMSCs to differentiate into endothelial cells.
ABSTRACT
AIM: To establish a reversed-phase HPLC method of determining three kinds of berberine type alkaloids(jatrorrhizine,palmatine and berberine) in Rhizoma Coptidis and Jinqi Jiangtang Tablet(Flos Lonicerae japonicae,Rhizoma Coptidis and Radix Astragali) METHODS: RP-HPLC was used for determination.The separation was carried out on Diamonsil C_(18)column(4.6 mm?250 mm,5 ?m) with the mobile phase containing acetonitrile-0.1 mol/L potassium dihydrogen phosphate(30(∶)70) and a detective wavelength was at 345 nm.The flow rate was 1.0 mL/min. RESULTS: The average contents of jatrorrhizine,palmatine and berberine in Rhizoma Coptidis and Jinqi Jiangtang Tablet were 1.34%,1.61%,7.12% and 0.35%,0.64%,1.54% respectively. CONCLUSION: The method is simple,accurate with a good recovery and repeatability and can be used to control the quality of Rhizoma Coptidis and Jinqi Jiangtang Tablet.