| BackgroundCorneal epithelial cells have been reported to be self-renewal and the limbal stem cell (LSC) can help repairing damaged corneal tissues by cell proliferation, differentiation and migration. The repairing ability is severely compromised when LSC is absent, which will lead to conjunctivalization, chronic inflammation and persistent epithelial damages, even vision lost. Transplantation of LSC and peripheral corneal tissues can ameliorate corneal injury by their proliferation, differentiation and migration into the damaged sites, where the expression of epithelial markers are induced. The molecular mechanism and signaling pathway between LSC and peripheral tissues, however, remain unknown. One previous study on corneal tissues after mechanical or chemical injury revealed the expression of growth fator and related cytokines, including transforming growth factor a (TGF-a), transforming growth factor ? (TGF-?), epidermal growth factor (EGF), heparin-like growth factor (HGF), fibroblast growth factor-? (FGF-?), all of which can be synthesized by corneal epithelial cells, these factors may regulate cell proliferation and growth. Besides, insulin-like growth factor 2 (IGF-2) has been demonstrated to be able to facilitate both the proliferation of keratinocytes and the synthesis of protein N-cadherin,which contribute to the generation of extracellular matrix and keratin collagen. IGF related signaling pathway has been known to be involved in cell proliferation and differentiation, which plays a crucial role in mediating cell growth at both cellular and organ level. Therefore IGF and other cytokines may also be related to the proliferation, differentiation and migration of LSC, which can be identified from other corneal cells by cell surface markers such as keratin K3, K12 or connexin 43.ObjectiveThe key regulatory molecules involved and their mechanisms during corneal wound healing play an important role in reducing scarring and promoting corneal wound healingIn is study, we make a preliminary exploration on the interation molecular mechanism of various kinds of cytokines between corneal epithelial cells and limbal stem cells in the rat model of corneal mechanical damage.MethodsMachinery unilateral cornea injury rat model were constructed, the contralateral cornea was taken as control group, tissues were collected at different time points on both sides of the central cornea and LSC, cell culture and genetic testing were employed to detect the changes of these cytokines such as EGF, FGF-b, KGF, HGF, TGF-b1, IGF-1 andIGF-2 cytokines, such as different levels of reactive changes, at same time we also explored the main factors on the differentiation of LSC and the transformtion of fibroblasts. And we also explored the the regulation of mTOR/STAT3 signaling pathway in the proliferation and differentiation of corneal cell stimulated by IGF-2Results1.The symptoms of blink and photophobia aggravated, and irregular patchy structure appeared after the rat cornea injury. The damaged corneal tissue stained by hematoxylin-eosin (HE), demonstated that the corneal epithelium was damaged, while strom was undamaged, therefore the model of cornea mechanical damage has a good feasibility.2.2. At 0,3,6,12 and 24h after the injury of the central cornea, tissues of the damaged cornea was taken. The gene expression of EGF, FGF-b, KGF, HGF, TGF-bl, IGF-1 and IGF-2 were dectected. By real-time PCR,we found that the level of IGF-2 increased significantly at 3h,and the level of IGF-2 was significantly higher than other factors (p <0.05).3.The gene expression of IGF-2 receptor increased more apparently in LSC than that in corneal epithelial cells, the differences were statistically significant at 3h after cornea injury (p<0.05).4.IGF-2 can promote the proliferation of corneal epithelial cells with the incubation time, and the ability of proliferation increased more significantly than the control group. At 24h, the ability of cell migration in IGF-2 group was apparently higher than that in the control. It come to the highest level at 48h.We observed the stained cytoskeleton and found that IGF-2 had a stronger adhesion rate than the control group.5.After cornea injury,IGF-2 expressed abundantly and rapidly, which lead to the increased expression of IGF receptor increased in LSC, and induced the differentiation of LSC into corneal epithelial cells labeled with K12.6.Immunofluorescence analysis showed that ?-smooth muscle actin levels were significantly increased in the damaged corneal epithelial cells, suggesting that the transformation of fibroblasts into myofibroblasts.7.We detected protein expression of phosphorylated mTOR and phosphorylated STAT3, we can found that both increased with the concentration of IGF-2 increased.8. We detected the expression of damage associated protein such as EGF, FGF-b, KGF, HGF and TGF-b1 after using IGF-2 and found that the expression of these proteins had increased significantly by the stimulation of IGF-2 compared to the control group, but they were significantly inhibited by given RAPA 50 ? molL,the inhibitor of mTOR/STAT3 signaling pathway.Conclusion1. Unilateral corneal mechanical damage in rats is reproducible and the damaged corneal epithelium can restore, this model can be used for research on the process of corneal wound healing.2. The expression of IGF-2 in corneal epithelial cell was upregulated significantly after mechanical corneal damage, and expression of IGF-2 receptor increased in LSC,they both promoted the transformation of LSC to corneal epithelium and fibroblasts to muscle fibroblasts. IGF-2 plays a certain role in corneal wound healing.3.In vitro state, specifically blocks the mTOR/STAT3 signaling pathway can block IGF-2 cells to repair the cornea, this effect may be completed through the activation of mTOR/STAT3 signaling pathway. |
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