| PurposeTo analysis the effect of high glucose on the the activity of corneal limbal stem cells and corneal epithelium using cultivated corneal limbal stem cells and the model of Type I diabetes mellitus (DM) of Ins2Akita.Methods1. Using mouse corneal epithelium stem/progenitor cell lines as vitro model,adding final30Mmol glucose in the medium to simulate high glucose micro environment of diabetic keratopathy. By phase contrast microscopy, MTT, stem cell clone formation, Annexin V-PI and immunostaining experiments of8-OHdG (DNA single-strand breaks), and observing the effect of high glucose on the morphology, colony forming ability, cell proliferation, cell apoptosis, the stability of DNA of corneal epithelium.2. To choose each10of the same same age adult male C57BL/6mouse (the control group) and Ins2Akita (the diabetic group), and to compare the weight, blood sugar levels, Mouse corneal epithelial central was scraped2mm area. Observe and compare corneal epithelium repair speed at Oh、24h48h、64h、76h、88h、100h、136、172h between the two groups. To detect and analyse the change of morphology, the apoptosis and the DNA stability of the corneal epithelium cells by the transmission electron microscopy, HE stain TUNEL assay (apoptosis) label dyeing,8-OHdG (DNA single strand break dyeing).Results1. The mouse corneal epithelium stem/progenitor cell lines showed following changes, TKE2that processed with high glucose showed long spindle, the status of cells were bad with time prolonging. Cell clones group of high glucose group become small, the number of cloning group reduced, the colony formation rate of the normal group was28.333±5.508, colony formation rate of the high glucose group was6.333±1.155, and there was statistical significance (P<0.05) between the normal group and the high glucose-3generations. MTT method (cell proliferation) shows the there was statistically significant between the normal group and high glucose-1generation (P72h=0.013), there was obvious statistically significant between the normal group and the high glucose-3generation (P72h=0.000), the ability of cell proliferation weakened with the extension of high-sugar processing time. The apoptotic detection of cell shows the proportion of early apoptotic cells of the normal group was8.02±3.14, the proportion of early apoptotic cells of high glucose group of was33.13±2.63, high glucose-3generation had more early apoptotic cells than the normal group, the difference was statistically significant (P<0.001). the8-OhdG marked positive cells (DNA single-strand breaks mark) of high glucose-3generation were more than normal group.2. The weight gain of Ins2Akita mice diabetic model at the detection period (8weeks) significantly reduced, and the blood sugar maintained at a relatively stable high level. The corneal epithelial healing time of the diabetic group was after172h corneal epithelial scraped, the healing speed were significantly delayed compared the normal group (48h), and the expansion of the area of injury in88h. Electron microscopy show the corneal epithelial cell surface microvilli and micro-folds of the diabetic group were significantly reduced, the arrangement of corneal basal epithelial cells were still rules, but the cell volume increased, the vacuolization of basal cells was obvious, the tight junctions was loose between cells, the hemidesmosomes were significantly reduced between the corneal epithelium and basement membrane. HE staining showed that the corneal epithelial cell surface of the diabetic mice was not flat, irregular in shape, loose connections between cells, cells numbers decreased, unclear boundaries, corneal basal epithelial cell vacuolization, stroma edema. TUNEL results show the TUNEL marked staining of corneal basal epithelial cell increased significantly compared the normal group;8-OHdG staining results show the8-OHdG marked positive cells of corneal basal epithelial cell increased significantly compared the normal group.conclusion1. In high glucose environment, the morphology of the corneal epithelial stem cell changed, the colony formation ability and proliferation of corneal epithelial stem cell were significantly inhibited. Early apoptosis increased significantly and the stability of the genome decreased.2. The corneal epithelial repair speed of Ins2Akita diabetic mice slowed down significantly, and the emergence of repeated exfoliation; the form of cornea basal epithelial cells changed significantly, apoptosis of the corneal basal epithelial cells increased and DNA stability decreased. |
PDF Full Text Request