The Experimental Research On Trans-differentiation Of Human Peripheral Blood Monocytes Into Corneal Epithelial Cells By Monocytes Migration Model

ObjectiveTo establish the monocyte cells migration model in vitro.Let the migration cells be induced under the micro-condition of fresh limbal stem cell and corneal epithelial cells for 7 days, study the possibility of trans-differentiation of monocytes.Methods1, The monocyte cells migration model in vitro:Top wells(inters) with 8-μm pore filters were coated with typeⅥcollagen and seeded with vein endothelial cells,then,it were put into plastic bottom wells in 650ng/ml MCP-1.Human peripheral blood monocytes were place into the top well.After 48h,the top inserts and nonadherent cells were removed.To To observe the factors affecting the model, we divide into two groups:the first group,add 10%FBS (volumetric fraction) basic medium to the top inter well; the second group,add 0%FBS (volumetric fraction) basic medium to the top inter well, add 650ng/ml MCP-1 and Ong/ml MCP-1 to buttom well. After 48h,remove the top insert well, collect the adherent cells in the buttom well. Observe the pattern and growing course of cells under inverted microscope.detect the CD14 which is the special marker of m with monocytes with flow cytometry.2 Put the migration cells into the micro-condition of fresh limbal stem cell and corneal epithelial cells for 7 days, collect the cells, detect the change of cytokeratin and vimentin which is the special marker of the cell before being induced and after that with flow cyeometry.Results1 With flow cyeometry, the CD14 positive rate of mononuclear cells is 19.6% before the migration model, and the figure is 1.5% after that. under inverted microscope. the shape of the migration cell are regular cultured under the codition of FBS (0%),Ⅳcollagen(1μg),MCP-1 (600ng/ml).2 With flow cyeometry, the positive rate of cytokeratin is 1.64% before being induced, the positive rate of Vimentin is 96.29%.Their positive rate is 11.30%,31.55% respectively after being induced.Conclusion1 To stimulate migration model in vitro, human peripheral monocytes are much esay to migrate under the condition of FBS (0%),Ⅳcollagen(1μg),MCP-1(600ng/ml).2 The change of the CK,Vimentin positive rate of monocytes bfore and after being induced indicate monocytes possibly can trans-differentiation into corneal epithelial cells by monocytes migration model...