| Due to its common,frequently-occurring and high recurrence rate,ocular surface disease is one of main blind eye ailment.Currently,the best way of tr eating corneal epithelial wound caused by various kinds of ocular surface diseases is ocular surface reconstruction,which is usually implemented with autologous or geneic corneal transplantation.However,the materials of autografting of corneal limbus was limited and with the risk of lacking key corneal limbal stem cells.Because of allogeneic cornea was required to be fresh,not enough source,and reject the reaction,researchers turned to the studies of treating the ocular surface disease with amnion due to its special structure and promoting the tissue repair characteristic.Based on the basic research of amniotic membrane transplantation,the amniotic grind extracting solution(amniotic homogenate),which reserved the effective biological components of amnion,achieved a certain progress and got a novel attempt in treating of ocular disease.With the development of biotechnology and other corresponding technologies,constructing the t issue engineering corneal epithelium with the amnion cells(seed cells)reveals a good promising future in ocular surface disease treatment.Taking amnion as raw material,this paper studies the therapeutic action and active mechanism of corneal epithelial wound by using amniotic membrane graft,amniotic homogenate and amniotic epithelial cells,respectively.And then,to support new methods and theoretical basis of solving the problems of ocular surface reconstruction materials shortage and serious ocular surface disease treatment.The main research work and contribution of this dissertation are:(1)To observe the action of amnion to the animal corneal wound healing,the animal model of rabbit corneal epithelial injury was constructed and employed with amniotic membrane transplantation firstly.Results show that the healing of corneal epithelium was faster in amniotic membrane transplantation group than control group.The morphological observation showed that the order level,cell morphology,the number of inflammatory cells were improved compared with those of control group.(2)Based on the animal experiments,25 ocular surface epithelial defect patients caused by different etiologies were employed with amniotic membrane transplantation.The clinic experimental results showed that 21 eye surfaces were reconstructed with corresponding eyesight improvement in different degrees.The epithelium of regenerated corneas was smooth,transparent or translucent,and the sodium fluorescein staining was negative.The regenerated conjunctiva was stable and not infection.Up to 28.3 weeks,all of 21 eyes were not recurrence.The operation of three eyes was failed because of complication.(3)The amnion homogenate with different concentration was applied to epithelial defect rabbit corneal to observe the action of amnion homogenate to the proliferation of corneal epithelial cells.Experimental results showed that the amnion homogenate took a great promotion on the proliferation and repair process of rabbit corneal epithelial cells.The concentration of amnion homogenate is increasing;accordingly,the proliferation action is more obvious.This means that the proliferation of corneal epithelial cell has a dependency trend on amniotic homogenate.Compared with amniotic membrane transplantation group,the amnion homogenate got a same affection but the operation complication can be avoided.(4)The last study is the cytology of the culture of amniotic membrane epithelial cells,corneal epithelial cells,amniotic membrane epithelial cells and corneal epithelial cells.Experimental results showed that amniotic membrane epithelial cells began to grow adherent after 2h primary culture.The cells exhibited with polygon,irregular,paving stones aligned.In vitro can continually translate 4~5 generation.CK3?CK18?CK19 polyclonal antibody staining are positive.Flow cytometer detected that the expression ratio of CK3 ? CK18 ? CK19 is 41.84±3%,46.51±2% and 33.41±3%,respectively.Corneal epithelial cells began to climb out after 3 days culture.The cells exhibited with polygon,evenly aligned.Amniotic membrane epithelial cells cultured by induction exhibited with polygon,flat and cell enlargement.The of expression ratio CK3 detected by flow cytometry was 75.70±0.3%,There wasa significant differece compared with befo re induction(P<0.05),Experimental results were statistical significant.Based on analysis above,this paper studied three research works including animal experiments,clinical research and cell culture based on amniotic membrane.Experimental and clinical studies proved that amniotic membrane can promote corneal epithelium defect repair,to solve the problem of lacking of ocular surface reconstruction materials and offer new idea in the treatment of severe ocular surface diseases. |
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