Study On Function Of Chick Vascular Endothelial Zinc Finger-1 In Endothelial Cells Differentiation And Vessel Formation

The vascular system starts with the endothelial precursor cells(hemangioblasts/ angioblasts) differentiation from the forming mesoderm.Subsequently,these precursor cells migrate to the destinated position,proliferate,differentiate,and coalesce to form a primitive vascular network.This process is termed vasculogenesis. The primitive vascular network is then remodeled into the hyrachical mature vasculature through a process termed angiogenesis.Moreover,during this time the vascular tree also becomes successively invested by the mesenchymal cells,which give rise to pericytes,SMCs(smooth muscle cell),and fibrocytes.The interactions between endothelial cells and their supporting cells as well as the surrounding matrix of different sources result in the functional vasculature seen in various organs and tissues.In these processes endothelial cells are key cells.In order to study the mechanism of vascular endothelial cell differentiation,we cloned some vascular endothelial(precursor) cells specific expression genes from the three-somites chick embryos.I chose mouse vezf1(vascular endothelial zinc finger transcription factor-1) homolog as the aim of our research.This newly discovered gene has been reported to specific expression in the vascular endothelial cells and their precursors during early mouse development.We expect the study of Vezf1 function will offer some positive information for unveiling the cellular and molecular mechanism of endothelium differentiation and vessel formation.RNAi technology was first applied in functional analysis on chicken embryo, through isolation of chicken angioblats cells with immunomagnetic beads,we researched on the influence of vezf1 on these cells.Targeting to the Vezf1 mRNA sequence,an RNAi construct pPRIME-CMV-GFP-cVEZF1-microRNA was built and packed into virus with a three plasmids system in 293T.We infected chick endothelial cells in vitro and early chick embryos in vivo.The results are as follows:1.We successfully constructed RNAi lentivirus vector pPRIME-CMV-GFP-cVEZF1 -microRNA.The Vezf1 gene silencing efficiency is about 75%in chicken fibroblast cells,. 2.By applying whole-mount in situ hibrydization,we have got the expression pattern of Vezf1 during early chick embryos development.3.The rabbit anti-chick VEZF1 polyclonal antibody serum was successfully produced.And the antibody titer was up to 10000 through Elisa test.4.We successfully isolated primary chicken angioblasts cells using Dynal immuno -magnetic beads.The purity could reach 90%.5.Double immunocytochemical staining shows that cVezf1 expresses in chicken angioblasts cells.6.Silencing Vezf1 on chick embryos in vivo suggests that Vezf1 plays an important role in development of blood vessel and heart.