The Research About Rabbit Corneal Limbal Stem Cells In Primary Culture

Objective: Cultured limbal stem cells, the morphology of the cells was observed,and the establishment of limbal stem cells in vitro cultivation and identification methods.Provides a theoretical basis and experimental basis for the clinical application of limbalstem cell therapy-related diseases.Methods: Dalian Medical University Experimental Animal Center of the rabbit.In accordance with the block size of the organization to take, will be divided into twogroups: group I was five eye under the microscope a small piece of limbus of rabbit, thesize is about1x1mm. Ⅱ group of five eyes, and take about the size of2x2mm limbus.Both groups were given the same methods and culture conditions on cell culture.The limbus with0.25%trypsin digest by adding the mixed solution containing20%fetal bovine serum,100u/ml penicillin and100μg/m1of streptomycin dubbedanti-dual (the ratio of1:1) and10ng/ml of EGF DMEM, and culture into the CO2incubator (5%CO2,37°C). Optical microscope to observe the cultured cells as well asits growth, and application of keratin K3monoclonal antibody AE5immunochemicalidentification of cultured cellsIn vitro culture of limbal stem cells, the use of the microscope observation of thecultured cells as well as its growth, the application of keratin K3monoclonal antibodyAE5immunochemical identification of cultured cells.Results: In this study, the first five rabbit eyes in Group I of small organizationsblock limbus above the size of about1x1mm access to the front of the matrix layer(thickness of about100μm to150μm), after removal of epithelial tissue blocks werecultured in vitro, no cellgrowth. Explore the reasons for cell growth, we take five rabbiteyes in group Ⅱ2x2mm size, thickness and the same limbal tissue block to use thesame training, observations indicate that cell growth. 2x2mm limbus growth after2-3days of their cell growth and morphology wereobserved. The first2-3days of cell growth be observed: cell migration from the tissueedge, forming a "beach-like" transition zone between cells arranged more closely.Cultured for six days, the cells were almost covered with the bottom, smaller cells,mainly oval or polygonal nucleus single-core or multi-core, the proportion of nucleartransfer. Application keratin K3monoclonal AE5antibody staining, the cultured cellsstained positive cells for most, that is, corneal epithelial cells, while a small number ofcells staining was negative for limbal stem cells.Conclusion:1. Access to the front of the stroma tissue culture:1x1mm size, remove theepithelium (thickness of about100μm to150μm), almost no cell growth on tissue block,while2x2mm size, thickness and the same limbus after culturewith limbal stem cells,but the relatively small number of stem cells.2. Monoclonal antibody AE5keratin K3-specific combination, can be used for theidentification of one of the limbal stem cells.