Transendothelial Migration Of Peripheral Blood CD14~+ Cells And The Changes On The Process Of It's Induced By Corneal Endothelial Cell

Objective:To explore the model of transendothelial migration of peripheral blood CD14+cells in vitro, identify the cell's (got from the model) biological characteristic and study the changes of the cells (got from the model) after they co-cultured with cornea endothelial cells (CECs).Methods:The human umbilical vein endothelial cells(HUVECs) were isolated from human umbilical cord by digested with collagenase. After serial subcultivation in vitro, the stem cells were passaged. Morphologic appearance of umbilical vein endothelial cell(UVEC) was observed by an optical microscope. The proliferation rate was measured by MTT assay. Cell cycle and surface antigens were measured by Flow cytometry. CD14+cells were isolated from peripheral blood cells by stratified with different densities of'Percoll'. CD14+cells content was measured by Flow cytometry. To explore the model of transendothelial migration of peripheral blood CD14+cells, Morphologic appearance of the cells got from model was observed by an optical microscope and atomic force microscopy (AFM). The osteogenic differentiation were tested and evaluated by specific stain methods. Cells which were collected from the model were co-cultured with CECs, the morphology changes were studied by HE staining and AFM, and the cell surface antigens were measured by immunofluorescence.Results:The HUVECs were digested with collagenase. The similar growth curves of passage3 and 5 were showing the HUVECs have a large proliferation potential. Flow cytometry analysis revealed that CD34 was highly expressed on the surface of passages 3 HUVECs. After stratified with different densities of'Percoll', the positive rate of CD 14 cells is 73.2%. The appearance of the cells come from model of transendothelial migration of peripheral blood CD14+cells is fibroblast-like cells morphologically. After cultured in inducing medium, these fibroblast-like cells were successfully induced into osteogenic. They were high positive for alkaline phosphate(ALP) staining and also showed mineralization demonstrated with mineralized nodules staining. And after co-cultured with CECs, these fibroblast-like cells'morphology gradually changed from long to triangle or round, and the contact between cells and intracytoplasmic vacuoles increased. There is no NSE expressed in the cells after co-cultured with CECs by immunofluorescence.Conclusions:A method for the isolation and purification of HUVECs from human umbilical cord has been tried. The cultured cells were only composed of undifferentiated cells and biological properties was stabilization. During the test, we practiced model of transendothelial migration of peripheral blood CD14+cells and got the fibroblast-like cells. Culture expanded the fibroblast-like cells maintained their ability to undergo osteogenic differentiation. After co-cultured with CECs, the cell morphology of the fibroblast-like cells changed. Although cells after co-cultured with CECs do not express NSE which is endothelial cells'specific antigen, but their atomic force microscopy showed morphological changes and cytoplasmic vacuolization increased. This Phenomenon remind us to consider the possibility of the cytoskeleton Changes, which is a necessary groundwork for our follow-up experimental study.