| Purpose The mesenchymal stem cells from femur and tibia of SD rats were isolated, purified and identified.The purpose of present study was to study the survival of marrow mesenchymal stem cells(MMSCs)and the immune status of recipient after allogenetic transplantation of MMSCs,and to investigate whether the local allogenetic transplantation of MMSCs and its reconstruction on alkali burned rat corneal surface are feasible.Method1.The MMSCs from femur and tibia of SD rats were isolated,passaged, amplified and puritied by adheresion-screening method.Cell morphology were observed by microscope.MMSCs are induced toward differentiation of Adipose-like cells and bone-like cells.The flow cytometry was utilized to detect the expression of CD29,CD44,CD90,CD31,CD34,CD45 molecule.2.Twenty Adult Sprague-Dawley(SD)rats were randomly devided into transplantation group(10 rats)and control group(10 rats).MMSCs were labeled by DiI fluorescent.The cell suspension containing 5×10~3 cells/μL MMSCs was injected On the nasal and temporal subconjunctica in the right eyes of 10 SD rats,and the culture medium was used in a same way in other 10 SD rats as control.At 4 weeks after transplantation,the survivals of DiI-labled MMSCs were invastigated under the confocal microscope,and recipient growth and ocular manifestations were clinically observed.The expression of MHC-ⅡmRNA between two groups were compared.3.MMSCs(5×10~3 cells/μL)allotransplantation for moderate alkali Burned 40 Rats Corneal Surface(DiI-MMSCs allotransplantation for 10 rats),and culture medium for moderate alkali Burned 30 Rats Corneal Surface(control group)were performed.After allogenetic MMSCs transplantation,rat cornea clarity and neovascularization in transplantation group and control group were observed.At the 4th week after DiI-labled MMSCs transplantation,expression and distribution of corneal epithelial cell specific marker CK12,Cx43 were observed by confocal microscopy.Expression of EGF and IL-1RA were compared by RT-PCR,and E-cadherin by Western blotting between transplantation group and control group at 3d, 2w and 4w.MMSCs(5×10~3 cells/μL)allotransplantation for moderate alkali Burned 28 Rats Corneal Surface(DiI-MMSCs allotransplantation for 10 rats),and culture medium for moderate alkali Burned 1 Rats Corneal Surface(control group)were performed.After allogenetic MMSCs transplantation,rat cornea clarity and neovacularization in transplantation group and control group were observed.At the 4th week after DiI-labled MMSCs transplantation,expression and distribution of comeal epithelial cell specific marker CK12,Cx43 were observed,by confocal microscopy.Expression of IL-1RA were compared by RT-PCR between transplantation group and control group at 3d,2w and 4w.Result1.Cutured adherent cells present spindle fibroblast-like growth and swirl-like arrangement.Red adipose vacuole in endochylema was showed in adipose-like cell and black calcium salts deposition was found in bone-like cells differentiated by MMSCs.Cultured adherent cells presented positive expression for CD29(99.19%), CD44(98.34%),CD90(97.94%)and absent expression of CD31(0.15%),CD34(0.69 %),CD45(1.21%).2.A lots of MMSCs labled by DiI were seen under the limbal and subconjunctiva in the 4th week after transplantation.No obvious abnormality was found in the eyes during the clinical observation.MHC-ⅡmRNA was expressed in cornea of allograft group compared with the control group.3.In MMSCs allotransplantation for moderate alkali burned rat corneal surface,compared with the control group,corneas in transplantation group at the 4th week presented reduction in vascularity(P＜0.05)and obvious improvement in clarity(P＜0.05).HE staining showed three to four layer cells in epithelial lamina and less neovascularization and inflammatory cells in stroma.A lot of DiI-labled MMSCs appeared in cornea limbus,which express CK12 and Cx43,and them in near central region which no express CK12 and Cx43,and not in central region.By RT-PCR, corneas in transplantation group expressed more EGF and IL-1RA than in control group at the 2w and 4w.,and most discrepancy at the 2w.By Western blotting, corneas in transplantation group expressed small amounts E-cadherin at the 4w. In MMSCs allotransplantation for severe alkali burned rat corneal surface, compared with the control group,corneas in transplantation group at the 4th week presented reduction in vascularity(P＜0.05)and no obvious improvement in clarity.HE staining showed less basal cells in epithelial lamina and less NV and Inflammatory cells in stroma.A lot of DiI-labled MMSCs appeared in cornea limbus,which expressed no CK12 and Cx43 and not in central region or near central region of cornea by confocal microscopy.By RT-PCR,corneas in transplantation group expressed more IL-1RA than in control group at the 2w and 4w.,and most discrepancy at the 2w.Conclusion1.Adherent cells from femur and tibia of SD rats are identificated as MMSCs based on cell morphology,differentiation and cell surface immune molecule. adheresion-screening and passage are good method for MMSCs purification.2.local allogenetic transplantation of MMSCs is safe and feasible.It is a new,no immunologic rejection and less injury way on ocular surface diseases treatment.3.Allogenetic MMSCs after early being subconjunctivally transplanted into moderate alkali Burned Rat Cornea,can reconstruct rat corneal surface.It is effective on inhibition angiogenesis of severe alkali burned cat Cornea.MMSCs migration and differentiation rely on microenvironment.MMSCs repair cornea surface by regulating cytokines and promoting residuary cells proliferation,not differentiating into injuryed cells.
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