| Stem cells have long been regarded as undifferentiated cells capable of proliferation, self-renewal, production of a large number of differentiated progeny, and regeneration of tissues. Generally, it has been thought that only embryonic stem cells are pluripotent. Indeed, extensive data support that stem cells exist in many adult tissues, including liver, brain, skin, cornea and so on. These stem cells have a high proliferative potential. They can be activated by wounding or disease. Limbal stem cells can maintain corneal epithelium long-term by proliferation and differentiation. It is more important that they like a barrier to prevent the transfer of conjunctival epithelium. These are very important to maintain normal physiology function and transparency of cornea. Cornea is located in the forepart of eyeball, and has elaborate structure. It's very easy to be damaged or infected. If cornea is harmed, the vision will reduce or lose. So the corneal diseases are the second cause of the blindness. With the development of ophthalmology, many patients with cornea disease can be healed by corneal transplantation. This method, however, is effectless to treat ocular surface disease with limbal stem cell deficiency such as Stevens-Johnson syndrome, chemical and thermal burns, radiation injury, extensive microbial infection, and aniridia. If using limbal stem cell transplantation first, The ocular surface structure can be improved, and the successful rate of corneal transplantation will be promoted. So many scholars pay their attention to limbal stem cell culture, special markers, proliferation and differentiation. This experiment used immunohistology and different methods of limbal stem cell culture to find a better way for stem cell culture and a special marker, make a deeper understand to human limbal stem cell. Objective: To look for a better methods to culture human limbal stem cells, detective E-cadherin's expression in human central, fringe epithelium and cultured limbal stem cells in low Ca2+, analyze it's relationship with limbal stem cell's transference, proliferation and differentiation and the probability to be a markers, and analyze the specialty to be a markers of E-cadherin, K19 and P63.Through these experiments, grasp more basic theory to human limbal stem cell culture in vitro. So we can extensively use gene therapy and stem cells transplantation to treat the patients with diseases that have the character of limbal stem cells deficiency and function disorder.MethodsUsing or not using DispaseⅡ digest limbal tissues, analyze the time from culture to outgrow and their growing rate of two groups.Low Ca2+ culture media is used to culture human limbal stem cells. The cell expressions of E-cadherin are observed by immunocytochemistry.Record the time from culture to outgrow and their growing rate of two groups.The expression of K19, E-cadherin, P63 are observed by immunohistochemistry. The specialty is contrasted. ResultsThe group digested by DispaseⅡ has slower speed of cell outgrow and lower growing rate than the other group. The numbers of tissues that can grow are 108 and 116. The times from culture to outgrow are 5.48±0.12days and 6.26± 0.18days.Only few of cells cultured by low Ca2+ culture media express E-cadherin.Few of limbal epithelium expresses P63, but a lot of cells express E-cadherin and K19. ConclusionIt's more beneficial to the human limbal stem cell culture if the tissue dose not digested by DispaseⅡ.E-cadherin play a important role in the process of human limbal stem cell's transference, proliferation and differentiation.As a marker, P63's specialty is higher than K19.
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