| Objective: It was considered that corneal limbal stem cells deficiency caused by sorts of diseases and would result in many serious ophthalmological pathological changes. It is the difficulty and emphases in ophthalmological researches and need be resolved immediately. Transplantation of corneal limbal stem cells maybe the only effective method to treat the severe cornceal limbal stem cells deficiency. Transplantation of corneal limbal stem cells between different people would cause exclusion, and if within the same one the quantity of corneal limbal cells is too limited to meet the demand of the transplantation. The key matter is to prepare abundant corneal stem cells which have the normal feature and function and can amplify. The test make cell-suspension culture and explant culture for expanding corneal limbal epithelial cells in vitro and do some comparison in order to get enough corneal limbal stem cells.Method: Got eyeballs axenicly (24 eye) from 12 health rabbit. Amniotic membrane made by scraped epithelium and put in 96- hole plate or 35mm culture dish. The cell-suspension group was A group and explant culture was B group. The morphology and growth feature of conceal limbal epithelial cells in A and B group were observed by inverted microscope everyday. The number of desmosome was quantified under transmission electron microscope (TEM) in the fifth day after fixed in 0.25g/ml glutaric dialdehyde. Cells of A group divided to A1 group (with amniotic membrane) and A2 group (without amniotic membrane) and cells of B group divided to B1 and B2 group at the same way. The cell proliferation of the 1st, 2nd, 5thand 7th day calculated after the OD value at 590nm was measured through MTT. The cell proliferation = (the OD value of experimental group-the OD value of control group)/the OD value of control group. Compared the proliferous ability and the proliferation of those groups.Results: A monolayer of cultured corneal limbal epithelial cells was observed by inverted microscope in both A group and B group. The cells of A group growed evenly, compactly arranged and divided a new generation in 4-5 days. The cells of B group crawled outward from the tissue inoculation in 2-3 days and the cells were polygen or rotundity and contacted each other. The cells of B group got a generation in 7-9 days and some fibroblast mingled. Blank areas appeared and no cellular layer observed when the tissue patch moved. The gaps between cells of A group were smaller than those of B group. Desmosome observed in both groups and the number was 3.71±0.94(n=10) or 0.24±0.28(n=10) in each group.The cell proliferation compared by MTT. The cell proliferation of A1 group was 23.2 %,29.4 %,45.7 %,42.7 % and was higher than those of B1 group which was 20.9 %,25.7 %,41.5 %,40.8 % corresponding in the 1st, 2nd, 5thand 7th day(p<0.05). The cell proliferation of A2 group was19.4 %,22.3 %,37.8 %,31 % and higher than that of B1 group which was 15.8 %,20.5 %,33.5 %,31 % corresponding in the 1st, 2nd, 5thand 7th day(p<0.05).Conclusion:Corneal limbal epithelial cells had gained in the experiment. The cell-suspension method was easier to get corneal limbal epithelial cells with better quality and higher cell proliferation. Amnion membrane would promote the growth of corneal epithelium and was suitable for the culture of corneal limbal stem cell. Culture corneal limbal cells on amnion membrane by cell-suspension method could obtain plentiful cells and the cellular layer was better for transplantation. The method deseaved researching furthermore. |
