The Experimental Study On Conjunctival Reconstruction By Induced Differentiation Of Human Amniotic Epithelial Cells

Objective:To trans-differentiate to conjunctival epithelium from human amniotic epithelial cells as a new kind of seed cells under the condition of denuded conjunctival matrix and conjunctival homogenate,then to study the biological characteristics of differentiated cells and the tissue engineering conjunctival for ocular surface reconstruction.Methods:(1) Human amniotic epithelial cells were cultured under the inducing conditions of denuded conjunctival matrix and conjunctival homogenate,immunohistochemistry with cytokeratin4,cytokeratin8,cytokeratin13,cytokeratin18,muc5ac and PAS staining were performed respctively;the concentrations of muc5ac in different time were measured by ELISA;the gene expressions of intracellular muc5ac were tested by RT-PCR.(2) Biological characteristics of differentiated cells was been studied by MTT, population doubling time and trace labelling with quantum dots.(3) The differentiated cells with quantum dots were shifted onto denuded amniotic membrance used as a carrier and were cultured with air-lifting cultivation by transwell culture plate to contruct tissue engineering conjunctival.It was removed in 7^th day and 14^th day respectively,differentiated cells were indentified to whether compound layer by HE staining and transmission electron microscope;the growth state of differentiated cells with quantum dots were frequently studied by confocal laser scanning microscopy;cytokeratin4, cytokeratin8,cytokeratin13 and muc5ac were performed by immunohistochemistry in 14^th day.(4) Tissue engineering conjunctival labelling with quantum dots was transplanted to rabbit model with conjunctival defect and fresh amniotic membrance as the control in the opposite eye.the graft were observed by slit lamp after surgery;tissue engineering conjunctival and fresh amniotic membrance were removed respectively in14^th day after surgery,HE staining,PAS staining,transmission electron microscope,immunohistochemistry and confocal laser scanning microscopy were performed.Results:(1) After induction of human amniotic epithelial cells,the expression in immunohistochemistry was positive for cytokeratin4,cytokeratin8,cytokeratin13, cytokeratin18 and muc5ac,the positive cells of purplish red color were detected in PAS staining,trace amount of mu5ac in culture medium was measured by ELISA and it increased gradually in 1-7days;gene expression of muc5ac in differentiated cells was positive by RT-PCR.(2) MTT showed an increasing trend of differentiated cells activities in 1-4days and then decreased in 5^th day during1-5days;cell growth curve and population doubling time from 1^th day to 5^th day simultaneously indicated differentiated cells had favorable proliferation abilities;quantum dots successfully labelled differentiated cells after 2 hour culture,the decay of fluorescence in cells wasn't detected,cellular morphology was normal and showed a good state of growth during following 7 days.(3) The differentiated cells with quantum dots inoculated onto denuded amniotic membrance and exposed to an air-liquid interface(air-lifted),2-3 layers of cells in 7^th day and 4-5 layers in 14^th day were detected by HE staining and transmission electron microscope,differentiated cells and denuded amniotic membrance connected tightly,which indicated it successfully established tissue engineering conjunctival;no decay of fluorescence in cells and regular cell morphology was observed by confocal laser scanning microscopy;immunohistochemistry indicated the cellular phenotype didn't change.(4) Clinical manifestations in ocular surface both the control with fresh amniotic membrance and the experimential with tissue engineering conjunctival hadn't existed obvious difference during the postoperative 2 weeks,conjunctival grafts survived and were integrated;2 weeks after surgery,2-5 layers of cells were observed by HE staining, some positive cells of purplish red color were detected in PAS staining, immunohistochemistry showed positive for cytokeratin4,cytokeratin8,cytokeratin13 and muc5ac,orderly arrangements and regular morphologies of cells with quantum dots were found.Conclusions:(1) Human amniotic epithelial cells directionally differentiated to conjunctival epithelium-like cells and globet cells which synthesized and secreted muc5ac under induction of denuded conjunctival matrix and conjunctival homogenate,which proved amniotic epithelial cells possessed multi-differentiation potentials of stem cells.(2) The differentiated cells had good biological characteristics,quantum dots were firstly used to label and trace conjunctival epithelium,which verified quantum dots was perfect as a trace marker and it fited long-term observations of living cells. (3) Denuded amniotic membrance was a ideal carrier of tissue engineering; differentiated cells inoculated onto this carrier and it successfully constructed tissue engineering conjunctival which had similar organization structures of normal conjunctival; differentiated cells maintained a favorable physiological state and cellular phenotype didn't change in the process of construction.(4) Tissue engineering conjunctival and rabbit model had a superior biocompatibility during experimental observation,transplantation rebuilt ocular surface integrity of rabbit model.it foretelled tissue engineering conjunctival should provide a alternative approach to cure many severe ocular surface disease.