| Background and objective: Arteriosclerosis and its complications (heart attack and stroke) are the major causes of death in developed and developing countries, and the change in vascular smooth muscle cells (SMC) from a differentiated to a dedifferentiated state is the critical phenotypic response that promotes occlusive arteriosclerotic disease. Despite its importance, we know little about the mechanisms that regulate SMC differentiation. One of the biggest technical difficulties in studies of SMC differentiation has been the lack of good in vitro differentiation system. Although these years several groups have developed in vitro culture systems in which animal multipotent cells are induced to differentiate into SMC. But the research findings coming from these animal cells cannot be used in clinical medicine directly, because of the species differences. For these reasons, to establish an in vitro differentiation system of human SMC is badly needed in this area. Blood vessel assembly during embryogenesis begins with clustering of primitive vascular cells into tube like endothelial structures, while mural cells (SMC or pericytes) become associated with the forming vessels at later stages of development, which has led to the suggestion, having been proved in animal cell experiments, that the vascular endothelial cells (EC) may recruit mural cell precursors and induce their differentiation into mural cells. In this study, we intend to in vitro recapitulate the SMC differentiation process during vasculogenesis, use human umbilical vein endothelial cells (HUVEC) to induce the differentiation of the multipotent human embryonic mesenchymal stem cells (HEMSC) into SMC, and then examine the underlying mechanisms, putting emphasis on the role of the contact, including gap junctions and adhesive molecules (e.g. integrins), between vascular EC and mural cell precursors in this process. HUVEC are important cellular experiment models being widely used in many bio-medical fields. But they are difficult to proliferate in vitro, tend to become senescent quickly, andalways need precious growth factors supplemented in culture medium to support their growth. We also encountered these problems in the previous studies. Thus we aims to construct human vascular endothelial growth factor (hVEGF)-121 protein eukaryotic expression plasmid, and transfect it into HUVEC to speed up their growth in vitro through a paracrine way.Section One: In Vitro Differentiation Model of Smooth Muscle Cells from Human Embryonic Mesenchymal Stem CellsMethods: (1) To characterize the cells: Using immunocytochemistry and/or immunofluorescence methods to detect smooth muscle(SM)-a-actin, calponin and SM-myosin heavy chain(MHC), the early, middle and late state makers respectively of SMC differentiation, in the primary culture HUVEC and the 4-8 passaged HEMSC. (2) To establish in vitro differentiation model of SMC from HEMSC: Use the porous membrane of the cell culture inserts as a substitute for the vascular basal membrane and plate HUVEC and HEMSC on each side of it respectively, with direct cellular contacts may formed between cells of each side through the pores (like the myoendothelial junctions through the fenestras on the basal membrane in vivo). Plate HEMSC alone on the porous membrane serving as control group. In 1, 2, 4, 8d, the SMC differentiation makers mentioned above were detected by immunocytochemistry method; In 1, 2, 3, 4d, SM-MHC mRNA and protein were assayed by RT-PCR and western blotting. Results: (1) The primary culture HUVEC did not express SM-a-actin, calponin and SM-MHC; the 4-8 passaged HEMSC expressed SM-a-actin and calponin, but not SM-MHC. (2) Immunocytochemistry experiments showed that in 4d of co-culture, SM-MHC began to be detectable in HEMSC of the contact co-culture group, weaker or stronger, almost in all cells, and filament-like distributing along the long axis of cell; and in 8d, the expression level of it became stronger. The expression levels of SM-a-actin and calponin in HEMSC of contact co-culture group did not vary...
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