Experimental Research On The Proliferation Of Corneal Endothelial Cells And Tissue Reconstruction In Vitro

Objective: (1) By investigating the effect of laminin on cytoskeleton and proliferation of the corneal endothelial cells, the correlation of cytoskeleton and cellular proliferation was considered; (2) To investigate the relative effect of single and multiple applications of laminin (LN) and recombinant human epidermal growth factor (rhEGF) on cell cycle of cultured rabbit corneal endothelial cells; (3) In order to search the optimal carrier material and better tissue culture methods, the morphology and structure of the cultured cat corneal endothelium were observed.Methods: (1) After 36hs'culturing in the medium with and without laminin, the cell cycle was detected by flow cytometry and the cytoskeleton stained by Coomassie blue G250 was observed by microscope; (2) The change of morphology and cell number was surveyed by the inverted microscope; Counting cell numbers on cultured 0,12,24 36 and 48hs obtained cell growth curve; The change of cell cycle were examined by flow cytometry; (3) The cat corneal endothelial corneal cells were seeded on the Descemet's membrane of the dehydrated swine corneal stroma, and then cultured in the medium with epidermal growth factor and laminin for 7ds. The morphology and structure of reconstructed tissue was detected by microscope and scanning electron microscope.Results: (1) Compared with the control group, the cellular proportion of laminin group increased in 62 ~M phase, and decreased in Go~Gi phase significantly. As shown by the microscope, the cells of control group were in low density. The cells in mass connected tightly. The microfilament appeared reticular formation. The nucleus were the same in size. The cells of laminin group were in high density and put out so many lamellipodia, filopodia, which connected with the surrounding cells. The microfilament increased, elongated, and changed from reticulodromous to sarciniform, which reached to the pseudopods. The nucleus were different in size . (2) As shown by the inverted microscope and the cell growth curve, comparing with the controlgroup, cells of each test group increased evidently. The cellular proportion of each test group increased in S phase and G2 ~M phase, and decreased in Go~Gi phase significantly, but there was no considerable interations between LN and EGF; (3) As shown by the morphological observations, the cultured cat corneal endothelial cells formed an integrated membrane, and attached to the Descemet's membrane closely, which was similar to the natural tissue. The cells connected tightly to each other, and some of them arranged in hexagon approximately.Conclusions: (1) Laminin improves the proliferation and change of the microfilament of the corneal endothelial cells, and they are closely correlated with each other; (2) Single and multiple applications of LN and EGF could prompt the corneal endothelial cells proliferation and division marketedly, but there is no considerable interaction between them; (3) The cat corneal endothelial tissue could be rebuilt on the dehydrated swine corneal stroma, which is similar to the nature tissue.