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AIM: To investigate the changes of ultrastructure in early diabetic rat cornea and the pathogenesis of diabetic keratopathy. METHODS: 20 Sprague-Daxley rats were sacrificed 6, 8, 10 and 12 weeks after induction of diabetes mellitus by streptoxotocin. 20 untreated rats at the same age were used as normal controls and were sacrificed at the same intervals. The ultrastructures of cornea were observed with transmission electronic microscopy and scanning electron microscopy. RESULTS: During the experimental period, the corneal ultrustructure of diabetic rats showed that epithelial and endothelial cells were swollen, the mitochondrions in the cytoplasm were swollen and increased. The collagen fibers appeared in disarrangement 10 weeks after streptoxotocin treatment. The endothelial of cornea was damaged from the periphery to the center gradually. CONCLUSION: The ultrastructural changes of cornea leads to dysfunction in streptoxotocin-induced diabetic rats, which may be related to the abnormal metabolism in hyperglycemia condition.
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AIM: To investigate the influence of laminin (LN) on the expression of bcl-2 in rabbit cornea endothelial cells. METHODS: Cultured rabbit corneal endothelial cells were divided into three groups: normal control group, LN group and negative control group. Immunohistochemical technique and ELISA were used to measure the staining and A value to assess the levels of bcl-2 expression. RESULTS: Bcl-2 expression score showed that LN group had a strong positive expression. Control group only showed a weak expression. Negative control group showed a negative expression by immunohistochemical staining. The mean A value of each group were 1.21?0.18 (LN group), 1.05?0.14 (normal control group) and 0.04?0.01 (negative control group). CONCLUSION: LN promotes bcl-2 gene expression in rabbit cornea endothelial cells, and protects the cells from apoptosis.
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AIM: To investigate the influence of the cornus officinalis glycosides (COG) on immunological function of corneal transplantation model of rats, and to clarify the immunosuppressive mechanism of COG through observing the activation of lymphocytes in blood. METHODS: Wister rats were used as recipients and SD rats were used as corneal graft donors, then the corneal allografts transplantation model on the closed colony rats were set up. Splenocytes proliferation and mixed lymphocyte reaction of Wister rats activated by ConA were observed. The phenotype change of CD4, CD8, CD25 in blood in different time postoperatively were observed by the di-sign flow cytometry, and the rate of CD4/CD8 was calculated. RESULTS: 1. The COG suppressed the proliferation of T lymphocytes and one-way mixed lymphocyte reaction on the corneal allografting. 2. The phenotype change of lymphocytes in boold was as follows: there was no significant difference between the different time of the CD4, CD8 expression and the CD4/CD8 rate in blood of the control group. The CD4 positive cells expressed CD25 postoperatively increased obviously. The CD4/CD8 rate of medicine group had the tendency to decrease. The CD4 positive cells expressed CD25 postoperation in the medicine group were less than that in the control group obviously. CONCLUSION: The suppression of the T lymphocyte proliferation, mixed lymphocyte reaction, CD molecule expressed by the activated T lymphocytes and the IL-2 receptor expression may be the main immunosuppressive mechanisms of Cornus officinalis glycosides on the cell-mediated immunity.
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AIM: In order to search carrier material and better tissue culture method, the morphology and structure of the cultured cat corneal endothelium was observed. METHODS: The cat corneal endothelial cells were seeded on the Descemet's membrane of the dehydrated swine corneal stroma, and then cultured in the medium with epidermal growth factor and laminin for 7 days. The morphology and structure of reconstructed tissue was tested by the inverted microscope and the scanning electron microscope. RESULTS: As shown by the morphological observations, the cultured cat corneal endothelial cells formed an integrated membrane, and attached to the Descemet's membrane, which was similar to the nature tissue. The cells connected tightly to each other, and some of them arranged in hexagon approximately. CONCLUSION: The cat corneal endothelial cells could be rebuilt on the carrier of the dehydrated swine corneal stroma successfully, which is similar to the nature tissue.
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AIM:To investigate the feasibility of using human amniotic epithelial cells(HAECs) sheets,which are cultured by using a biodegradable fibrin sealant to re-establish corneal surface layer.METHODS:Human amniotic membrane(AM) harvested at the time of elective caesarean section was digested by collagenase and trypsin respectively to obtain HAECs.The HAECs were planted on Millicell culture dishes coated with biodegradable fibrin glue,and cultured by using the air-lifting cultivation.The HAECs sheets were investigated morphologically by Hematoxylin-Eosin staining,scanning electron microscopy(SEM) and were identified by cytokeratin immunohistochemistry.RESULTS:HAECs sheets were transparent and successfully cultured by using a biodegradable fibrin sealant.Most of the cultured cells were round or polygon and typical slabstone-like appearance.Many microvilli were observed on cell surfaces by SEM.Cytokeratin staining was positive.HAECs had stratificated growth tendency and became corneal-like stratificating epithelial cells.CONCLUSION:A commercially available fibrin sealant was an effective means of tissue engineering to create a carrier-free,transplantable HAECs sheets.The sheets were potential graft to re-establish corneal surface layer.
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AIM:To investigate the effects of rhTGF-?1 and TGF-?1 gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro.METHODS:Cell growth induced by various concentrations of rhTGF-?1 was determined by MTT proliferation assay.Under the induction of liposomes,recombinant pSecTag2-TGF-?1MP vectors were transferred into the corneal endothelial cells.Morphological changes of transfected cells were observed by HE staining.The expression levels of TGF-?1 were assessed by ELISA.Cell cycle analysis was assessed by flow cytometry.DNA fragment analysis was used to confirm the presence of apoptosis.RESULTS:rhTGF-?1 in concentrations of 5-20 ?g/L showed a significant suppressive effect on the proliferation of corneal endothelial cells,0.5-1 ?g/L had no effect,0.05-0.1 ?g/L facilitated cell growth,as compared with negative controls.The morphous of transfected corneal cells had no significant abnormality compared with normal cells.According to the result of ELISA,the concentration of TGF-?1 in the supernatant was calculated to be(98?3)ng/L.Flow cytometry assay showed that S and G2/M phase of transfected cells decreased significantly compared with that of control group,but the cell cycle recovered normally after adding 10 ?g/L EGF into the culture medium.Agarose electrophoresis didn't show marked ladders in transfected group.CONCLUSION:Effects of rhTGF-?1 on the proliferation of corneal endothelial cells are different with various concentrations.TGF-?1 gene transfection shows suppressive effect on the proliferation of cultured corneal endothelial cells,but does not induce cell apoptosis.EGF is the antagonist of this suppressive effect.
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0.05), the expression of CD4+ was increased in rabbit corneal stroma (P
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AIM: To observe the effect of cornus of ficinalis glycosides(COG) ophthalmic solution on the corneal allograft rejection by topical instillation. METHODS: The corneal transplantation model on the closed colony rats was established. The rejection time of all animals was recorded and compared by slit-lamp microscope. The pathologic changes were measured by immunohistochemistry and scanning electron microscope.RESULTS: The histopathological and immunohistochemistry findings showed that the lymphocytes, neovascularity and the expression of ICAM-1 in COG-treated group were significantly fewer than that in control group at 15 d after operation.CONCLUSION: COG ophthalmic solution prevents and suppresses the corneal allograft rejection.