Understanding the mechanisms of vascular remodeling could lead to more effective treatments for ischemic conditions. We aimed to compare between the abilities of both human Wharton jelly derived mesenchymal stem cells (hMSCs) and human cord blood endothelial progenitor cells (hEPCs) and CD34+ to induce angiogenesis in vitro. hMSCs, hEPCs, and CD34+ were isolated from human umbilical cord blood using microbead (MiniMacs). The cells characterization was assessed by flow cytometry following culture and real-time PCR for vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand factor (vWF) to prove stem cells differentiation. The study revealed successful isolation of hEPCs, CD34+, and hMSCs. The hMSCs were identified by gaining CD29+ and CD44+ using FACS analysis. The hEPCs were identified by having CD133+, CD34+, and KDR. The potential ability of hEPCs and CD34+ to differentiate into endothelial-like cells was more than hMSCs. This finding was assessed morphologically in culture and by higher significant VEGFR2 and vWF genes expression (p<0.05) in differentiated hEPCs and CD34+ compared to differentiated hMSCs. hEPCs and CD34+ differentiation into endothelial-like cells were much better than that of hMSCs.
Fetal Blood ; Flow Cytometry ; Humans ; Mesenchymal Stromal Cells* ; Microspheres ; Real-Time Polymerase Chain Reaction ; Stem Cells* ; Vascular Endothelial Growth Factor Receptor-2 ; von Willebrand Factor ; Wharton Jelly

OBJECTIVETo establish a highly sensitive screening method for phytoestrogen active constituents and to primarily screen the phytoestrogenic active constituents from the chickpea extractions by the method.

METHODHuman ERalpha cDNA was cloned using MCF-7 total RNA as the template by RT-PCR and then was constructed into a pcDNA3 and named as pERalpha. The cell line MCF-7 was co-transfected with pERalpha and the reporter plasmid pERE-Luc which carrying the estrogen response element (ERE) plus the luciferase reporter gene. The luciferase activity was then assayed. The model was optimized by changing the ratio of two plasmids. The feasibility of the optimized model was further proved by the several known phytoestrogen compounds including fermononetin, biochanin A and genistein, et al. As an application of the model, the phytoestrogen activity of the extracts of the chickpea was assayed.

RESULTThe recombinant plasmid (pERalpha) can enhance luciferase activities of pERE-Luc transfected MCF-7 cells. The highest transfection efficiency and luciferase activity were found at the ratio of 10:1 (pERE-Luc: pERalpha), the luciferase activity was improved five times as high as the unique pERE-Luc transfection. The co-transfection screening model also indicated that fermononetin, biochanin A and genistein could induce ERE-driven luciferase activity and ICI 182,780 suppressed the induced transcription. As the application of the model, the results showed that the ethanol (70%) total extraction, the ethyl acetate extraction and the ligarine extraction of the chickpea can induce ERE-driven luciferase activity. Concurrent treatment with ICI 182,780 abolished the induced luciferase activity.

CONCLUSIONA phytoestrogen active constituent screening mode have been established based on co-transfection method. It is sensitive to assay the phytoestrogen active constituents and can be applied to screen the active component of phytoestrogens.

Cell Line, Tumor ; Cicer ; chemistry ; metabolism ; Drug Evaluation, Preclinical ; methods ; Estrogen Receptor alpha ; genetics ; metabolism ; Genes, Reporter ; drug effects ; Genetic Vectors ; metabolism ; Genistein ; chemistry ; pharmacology ; Humans ; Luciferases ; drug effects ; metabolism ; Phytoestrogens ; analysis ; pharmacology ; Plant Extracts ; chemistry ; metabolism ; pharmacology ; Plasmids ; drug effects ; metabolism ; Transfection ; methods