| Purpose:Diabetic keratopathy has been a frequently encountered diseasewith the incidence of diabetes increased year by year; Its pathogenesis is associated with many aspects such as disorders of inflammation and stem cells function. The mesenchymal stem cells(MSCs) are one kind of adult pluripotent stem cells with anti-inflammatory and immune regulation function. Through subconjunctival transplantationof MSCs to the streptozocin(STZ)-induced type1 diabeticmice, this study researched the molecular mechanismin promotional effect of MSCs on diabetic mice corneal epithelium healing.Method:1. By intraperitoneal injection of STZ100 type 1 diabetic micewere built;andwithbone planting method, bone marrow mesenchymal stem cells of mice were separated and subcultured. 2. MSCs weretransplantedunderconjunctiva in14-16 weeks old diabetic mice after their corneal epithelium was shaved, and took C57 mice and PBS-injection diabetic mice as controls. The corneal epithelial healing was observed through the slit lamp; homingof CM-Dillabeled MSCs to the corneaandimmunofluorescence stainingof neutrophils marker Ly-6g on cornea were observed by immunofluorescence,so as the anti-inflammatory tumor necrosis factor alpha(TNF-α) stimulatingprotein 6(TSG-6) in cornea. Immunofluorescencestaining of Ki67 on cornea was taken to observe proliferation of corneal epitheliumi; Further analysis by ELISA measured TSG-6 in cornea before and after shaving of corneal epithelium. 3. TSG-6 was subconjunctival injectedinto diabetic mice after cornealepithelium damage, and difference of corneal repair was observed among groups. By slit lamp regenerationof corneal epithelium was observed, and by immunofluorescence staining, expression of inflammatory factors such as cyclooxygenase-2(COX2), neutrophil marker of Ly-6g and two subtypes markers of macrophages( CD68 and CD206)were dected. Through q RT-PCRlevels ofinflammatory factors in cornea were quantitatively checked at later period of corneal epithelium repairment.Result: 1.In diabetic mice with subconjunctival transplationof MSCs, regenation of corneal epithelium was faster than that in the control diabetic mice; in MSCs transplanted mice, a small amount of MSCs homed into the anterior corneal stroma, and the proliferation of epithelial cellwas much more activatedand less Ly-6g immunofluorescence staining in cornea was detected compared to the control diabetic mice. After damaging of corneal epithelium, the expression of TSG-6in cornea of C57 raised significantly, but in contrast compare to C57 it wassignificantly lower in diabetic mice; and MSCs- injection could recover theexpression of TSG-6;2.With subconjunctival injection into diabetic mice,TSG-6 helped in epithelial repairing, and inhibited infiltration of neutrophils granulocyte into cornea,and decreased expression of proinflammatory factor COX2; q RT-PCR detected that TSG-6 could decrease the proinflammatory factors such as induced nitric oxide synthase(i NOS) and COX2 significantly, and increase the expression of anti-inflammatory factors such as IL-10 and Arg-1. 3. Flow cytometry indicated that, CD45(+) immune cells homing to cornea increased significantly, and the proportion of CD45(+)/F4/80(+)CD86(+) proinflammatory M1 macrophage raised significantly, and proportion of CD45(+)/F4/80(+)CD206(+) anti-inflammator M2 macrophagedecreasedsignificantly in diabetic mice compare to C57.Injection of MSCs or TSG-6could inhibited remarkedly the infiltration of immune cells and raised the proportion of M2 macrophage.Conclusion:1. Transplantion of MSCs could significantly promote restoration of corneal epithelium after damaging in diabetic mice:MSCs could home to the injured part of cornea, and increase expression of TSG-6, and inhibit infiltration of neutrophils granulocyte, and promote proliferating ofepithelial cells in cornea; and finally promoted the regeneration of corneal epithelium. 2.TSG-6 could significantly promote restoration of corneal epithelium after damaging in diabetic mice:TSG-6could inhibit significantly infiltration of neutrophils granulocyte, and decrease expression of proinflammatory factors, and promote the anti-imflammatory cytokines; finally it promoted the regeneration of corneal epithelium. 3. The function of macrophage in diabetic mice was abnormal, such as after damaging of corneal epitheliumthe proportion of M1 type increased; but injection of MSCs or TSG-6 could promote transformingof macrophages from proinflammatory M1 to anti-inflammatory M2 type.
|
PDF Full Text Request