Experimental Study Of Human Umbilical Cord-derived Mesenchymal Stem Cells Against Corneal Injury By Subconjunctival Injection

Corneal blindness is an eye disease that resulting in permanent loss of visual function.The cornea is located in front of the eye,when the trauma,the cornea is most vulnerable to injury,resulting in rupture,infection or turbidity.The most common cause of ocular surface tissue damage is chemical burns and thermal burns,which often due to corneal structure damage and corneal neovascularization,led to serious visual impairment.Corneal transplantation is still the primary means of treatment.However,high immunological rejection rate after corneal transplantation and low long-term graft survival are the thorny issue of clinical practice.Human umbilical cord-derived mesenchymal stem cells(hUCMSCs)are a unique,accessible,and non-controversial source of embryonic stem cells that can be widespread availability.The hUCMSCs can differentiate into several mesenchymal cells.During the inflammation and tissue trauma,hUCMSCs can secrete cytokines to promote tissue regeneration.Thus,it has great application value in the cell and tissue replacement therapy and regenerative medicine.In this study,we used two experimental stages of in-vitro cell culture and in-vivo animal research to discuss the role of hUCMSCs repair and cure corneal epithelial defects,corneal neovascularization and etc in the early of corneal alkali injury.Part ? Isolated culture and Identification of Human Umbilical Cord-derived Mesenchymal Stem Cells in VitroObjective: To establish the standard methods of isolation and purification,large scale cultivation and differentiative capacity of mesenchymal stem cells from human umbilical cord;to resolve the technical problems in cryopreservation and recovery of hUCMSCs.Preliminarily establish the quality control standards of hUCMSCs suspension,so as to obtain a sufficient amount of hUCMSCs to meet the storage and post-animal experimental treatment needs.Method: Human umbilical cords were collected to isolate and culture human umbilical cord-derived mesenchymal stem cells(hUCMSCs)by tissue mass suspension method at 37? temperature.During the whole culturing and passaging period,morphologic characteristics of hUCMSCs were observed under inverted microscope and HE staining.Have or not mycoplasma contamination of hUCMSCs was detected by PCR.The immunophenotyping of hUCMSCs was detected by flow cytometry.The hUCMSCs were differentiated into osteoblasts and adipocytes in the specific-induction medium.Alizarin red and oil red O staining were used to observe calcium deposition and lipid droplet formation to identify the hUCMSCs.The hUCMSCs were amplified by gelatin microcarrier culture system.On the basis of large-scale culture,we tried to explore the technological conditions of 5L culture system.Through the establishment of hUCMSCs cryoprotective agent,cryopreserved way and recovery standard process,so as to solve the corresponding technical problems in the process of cryopreservation and recovery.The hUCMSCs were marked through CM-Dil dyeing in vitro.Results: We successfully isolated and cultured mesenchymal stem cells from human umbilical cord by using tissue mass suspension method.The hUCMSCs had negative result of mycoplasma contamination after PCR detection.The hUCMSCs expressed the marker of mesenchymal stem cells,such as CD73?CD90 and CD105;but not expressed hematopoietic and graft rejection phenotypes,like CD11b?CD19?CD34?CD45 and HLA-DR.The hUCMSCs had positive results of Alizarin red and Oil Red O staining after induction in the specific-induction medium.A large number of hUCMSCs were successfully amplified under gelatin microcarrier culture system;and further established the standard process of hUCMSCs cryopreservation and recovery;hUCMSCs were stained with CM-Dil in vitro and could be highly expressed red fluorescence.Conclusions: The hUCMSCs can be isolated and cultured from umbilical cord by using tissue mass suspension method.They had the immunephnotype of mesenchymal stem cells and multilineage differentiation potentials.Through the gelatin microcarrier culture system,hUCMSCs were largely amplified in vitro.By the establishment of hUCMSCs cryoprotective agent,cryopreserved way and recovery standard process,the corresponding technical problems in this process are solved.Part ? Protective and Anti-apoptotic Effect of Human Umbilical Cord-derived Mesenchymal Stem Cells against H2O2-induced Human Corneal Epithelial Cells Oxidative DamageObjective: To investigate the protective and anti-apoptotic effect of human umbilical cord-derived mesenchymal stem cells against H2O2-induced human corneal epithelial cells oxidative damage through co-culture system.Method:The cultured human corneal epithelial cell lines were removed from the liquid nitrogen tube,with advance preparation special medium(Each hundred milliliters of culture solution containing 100 ul epidermal growth factor,100 ul transferrin,100 ul insulin,100 ul hydrocortisone,1ml non-essential amino acid,1ml glutamic acid,1ml streptomycin and penicillin,10 ml fetal bovine serum,and 86.6ml high-glucose dulbecco's modified eagle medium)of human corneal epithelium cells optimize cultivating cells,to ensure the normal growth and proliferation of cells.During the whole culturing and passaging period,morphologic characteristics of h CECs were observed by using the inverted microscope and HE staining.Maker of human corneal epithelium cell(Cytokeratin 3 and Cytokeratin 12)was detected by immunofluorescence.To established models of H2O2-induced human corneal epithelial cells oxidative damage.The morphological change and aging condition of h CECs before and after and oxidative damage were observed by using the inverted microscope.We have detected that the live and dead proportion of h CECs before and after H2O2-induced by using the viability and cytotoxicity assay.Apoptosis ratio,reactive oxygen species and mitochondrial membrane potential of H2O2-induced h CECs by before and after 24 h co-culture with hUCMSCs,which were detected through using the flow cytometry.The expression of growth factors(epidermal growth factor and basic fibroblast growth factor)before and after co-cuture by using the enzyme-linked immunosorbent assay.Results: We have successfully established the optimized culture system for h CECs by special medium.We have observed that h CECs did not appear signs of aging after several passages under the inverted microscope.On the contrary,h CECs can still maintain normal growth condition.The h CECs can express markers characteristics of corneal epithelium: CK3 and CK12.The CCK-8 assay showed that 1m M H2O2 for 24 hours did significantly decrease h CECs viability.Additionally,the morphology and quantity of h CECs were obviously changed.We have observed that blue agglutination particles of aging h CECs under the inverted microscope.The dead and broken proportion of h CECs was significantly higher than before H2O2-induced.After incubation with hUCMSCs for 24 hours,the apoptotic ratio and reactive oxygen species of h CECs was obviously decreased compared with that before co-culture,and the mitochondrial membrane potential was also up-regulated.The differences were statistically significant,respectively,for apoptosis detection(t=15.75,p=0.00),active oxygen detection(t=57.71,p=0.00),mitochondrial membrane potential detection(t=-3.258,p=0.031).We have observed that the h CECs and hUCMSCs co-cultured 24 h,the EGF and b FGF content of cell fluid was clearly increased compared with that before co-culture.The difference was also statistically significant,respectively,for EGF(t=-58.291,p=0.00),b FGF(t=-10.272,p=0.01).Conclusions: The hUCMSCs can exert protective and antiapoptotic effects on H2O2-induced h CECs oxidative damage by co-culture.We speculate that hUCMSCs play a role in the process,which by secreting certain trophic factors to promote damaged cells to complete repair and regeneration.Part ? Prothetic and Therapeutical Effect of Human Umbilical Cord-derived Mesenchymal Stem Cells against Corneal Alkali Injury by Subconjunctival InjectionObjective: To investigate the prothetic and therapeutic effect of human umbilical cord-derived mesenchymal stem cells against corneal alkali injury in rabbits by injecting hUCMSCs' suspension into the subconjunctival area of the operative eye after alkali burn.Method: To established models of rabbit acute corneal alkali burn.The corneal epithelial injury proportions were observed by fluorescence staining.Through HE-staining to determine the depth of injury.We have prepared that hUCMSCs' suspension and homogeneous negative control solution for injection therapy.The corneal epithelium healing area,transparency,corneal neovascularization(CNV),anterior chamber,conjunctival hyperemia,ciliary congestion and ocular secretions were dynamically observed by macro camera and slit lamp for a 2 weeks,which after the inject hUCMSCs into subconjunctival location.The healing condition of corneal epithelium and the expression of related proteins such as corneal epithelial marker protein(CK3 and CK12),corneal epithelial repair protein(?-SMA and Ki-67)were observed by histopathological staining.The expression of inflammatory factors(TNF-? and IL-1?)and growth factors(VEGF?EGF and KGF-2)in aqueous humor and corneal tissue by using the enzyme-linked immunosorbent assay.Results: We have successfully established the models of rabbit acute corneal alkali burn.Through the macro camera and slit lamp,observed that the ophthalmic indicators(corneal epithelial healing area,transparency,corneal neovascularization and anterior chamber sutitution)of hUCMSCs' suspension injection for treatment of alkali burn have significant improvement compared with the negative control group and the blank control group.HE staining showed that corneal epithelium of hUCMSCs group has been repaired completely,only leaving mild edema of the matrix.But in the control group,a large number of inflammatory cells infiltration and fibrous tissue hyperplasia in the corneal stroma.Immunohistochemical staining showed that the expression of CK3 and CK12 were positive in each group,and the expression of Ki-67 was positive in hUCMSCs group and negative in control group.The expression of ?-SMA was not observed in hUCMSCs group,while the expression of ?-SMA was positive in the control group,which indicated that the corneal stroma had fibrotic repair.Enzyme-linked immunosorbent assay shows that the expressions of TNF-? and IL-1? in hUCMSCs group were significantly lower than the control group(P<0.05),and the differences were statistically significant.The levels of VEGF in hUCMSCs group was significantly also lower than the control group(P<0.05).But the levels of EGF and KGF-2 in hUCMSCs were obviously higher than those control group,the differences were statistically significant,p= 0.00.Conclusions: For the first time by the way of hUCMSCs subconjunctival injection,can exert prothetic and therapeutic effects on corneal alkali injury.We speculated that hUCMSCs play many roles in the way,which through inhibiting the release of inflammatory mediators and the formation of VEGF,and through secreting certain nutritional factors(EGF and KGF-2)to promote the repair and remodeling of damaged tissue.