Experimental Study Of Mechanism Of Bone Marrow Mesenchymal Stem Cell Transplantation In Cornea Limbal Alkali Burn Models:Engraftment And Involvement In Wound Healing

ObjectiveTo establish a methodology for isolation and expansion of MMSCs from bone marrow ex vivo easily;To investigate how MMSCs work,where they locate and the interact MMSCs with microenvironment after they transplant into the body;To study the mechanisms of MMSCs engraftment accelerated wound healing of Alkali-Burned Cornea limbal.Methods:The experimental technologies were used to find the best condition of isolation and expansion of MMSCs and its biological characteristic;The distributing of MMSCs implanted into the body was viewed under a confocal fluorescent microscope.The changes of the number of labeled-MMSCs in peripheral blood with time were evaluated by Flow Cytometry;The study will use the alkali-induce cornea injury rabbits as the experiment body.Gross,clinical,and histological criteria were used to compare healing of alkali-burned corneas in control group and MMSCs-transplanted group.Effects of MMSCs and allergenic reconstitution of alkali-induce injury rabbit with MMSCs on wound healing were evaluated.VEGF?VEGFR?IL-2 Gene expression patterns in control and MMSCs-transplanted corneas were surveyed with array-based quantitative RT-PCR.TIMP-2 protein expression patterns were surveyed with Western-blotting and concentration of VEGF and IL-2 in peripheral blood and aqueous in anterior chamber were surveyed with ELISA.CD45?MMP-2/TIMP-2?Vemitin?SMA Keratin-pan?PCNA?CD44 were stained with Immunofluorescent and express patterns were viewed under a confocal fluorescent microscope.The changes of the number of labeled-MMSCs in peripheral blood with time were evaluated by Flow Cytometry.Results:First portion1.The proliferation and differentiation potential were higher in culture medium contain 5-10%FBS,compared with other groups.There were difference significantly between the groups(P<0.05).but the morphology of MMSCs looked like spindle-shape and grow slowly.The MMSCs differentiated into adipocytes naturally.2.Immunostaining and FACS analysis showed that MMSCs from the standard inbred strains were negative for CD31.all the MMSCs tested were negative for the hematopoietic cell marker CD45 and HLA-??they varied in their expression of CD34~+ and CD90~+?CD29~+?CD44~+?CD106~+?HLA-I.3.The rate of cell migration were varied in different concentration of VEGF?IL-1 ex vivo.The rate of cell migration showed positive relationship with the numbers of labeled-MMSCs in peripheral at 3 days after implanted(r?0.561;P<0.05).Second portion:1.In peripheral blood,the number of labeled-MMSCs in alkali-induced injury group were higher in post-operation 3.21.60 days(respectively 2.56±0.11.2.98±0.09.1.97±0.07)and showed difference significantly,compared with them in control group in each time point,respectively 0.86±0.05;0.65±0.07;0.54±0.04.The initial concentration showed positive relationship with the numbers of labeled-MMSCs in peripheral at 3 days after implanted(r=0.597;P<0.05).2.There are little labeled-MMSCs distributed in normal rabbit eye tissue,such as cornea?conjunctiva?retinal?choroids?iris,but in alkali-induced injury rabbit tissue,a lot of labeled-MMSCs located in cornea limbal and decreased in cornea centre?conjunctiva?retinal?choroids?iris in turn.,moreover,the distribution of MMSCs in cornea limbal showed a special morphology of "Convergent".3.The research revealed that engraftment MMSCs in rabbit tissue of systemically occurs in important organ such as heart?liver?kidney?lung?spleen?muscle and brain.The numbers of MMSCs in lung at early stage are higher than that in other organs,and are lower in terminal stage.In contrast,the level of MMSCs in spleen are comparatively lower than that in other organs and was higher in terminal stage.The order,which numbers of MMSCs distributed in organ,decreased in lung?spleen?kidney?liver?heart?muscle and brain.In alkali-induced cornea limbal injury rabbit,the level of MMSCs have changed,in terminal stage of engraftment,number of MMSCs in spleen showed no different with them at early stage.Third portion1.In engraftment group,the area of Corneal neovascularization respectively(12.56±0.46;19.16±0.76;4.36±0.66)in different time after implanted MMSCs showed different significantly,compared with the control group((3.34±0.51;14.56±0.46;29.16±0.68));The density of the corneal opacity(2.8 ± 0.2)in third days post-operation showed no difference significantly between the control group(2.9±0.3),but showed the degree of cornea epithelial defects,and reepithelization were(3.1±.045;2.7±0.39;2.1±0.43)respectively,compared with the control groups showed Statistics significantly.2.The concentration VEGF(98.23±1.45)in peripheral blood and in anterior chamber aqueous(145.12±2.01)were higher in engraftment group than in control group at 3 days after implanted(P<0.05).The concentration of VEGF in peripheral blood(56.12±1.56;50.36±1.25)and aqueous(98.23±1.26;56.12±1.98)showed difference significantly between the engraftment group and the control group at 21?60 days post-operation(P<0.05).The concentration of IL-2 in peripheral blood and aqueous in post-operation 3?21?60 days were lower than them the control groups.Significant differences were observed between the groups.3.Colocalization of immunofluorescence indicates TIMP-2,the inhibitor of MMP-2 was highly expressed in the rabbit cornea in experimental group,we detected the level of TIMP-2 protein by Western-blot and the result reveal that they increased in the rabbit cornea transplanted with MMSCs systemically.Colocalization of immunofluorescence indicates that CD31 had observed as early as 3 days after transplantation significantly,but the level express of CD31 decreased significantly in 60 days.The result proved the MMSCs could melted into endothelium of inflammation-related angiogenesis.4.Compared to rabbits that did not receive MSC transplantation,the MSC-implanted rabbits showed strong expression of PCNA.Cell proliferate activity was demonstrated by PCNA.PCNA was expressed not only in the limbal basal Layer,but it also extended from the periphery of the corneal epithelium to the central regenerated corneal epithelium,PCNA expression was much more pronounced on day 21,and it maintained a high level of expression at 60 month.The higher levels of the expression of rabbit keratin-pan,a marker for epithelial cells in implanted-MMSCs group have observed in diffenert time after implanted.5.Expression of CD45 was significantly depressed in rabbit eyes transplanted with MMSCs in comparison with control group.The MSC-transplanted rabbits possessed high levels of Vimentin in the stroma on day 3?21 and at 60 days,whereas the corneal stroma of rabbits without MSC transplantation demonstrated a very low level of Vimentin expression.Colocalization of immunofluorescence indicates that a significant portion of the surviving engrafted cells had begun to express smooth muscle-specific a-actin.Muscle-specific protein expression was observed as early as 3 days after transplantation Group and persisted at subsequent examination time points.6.Used a real-time PCR assay that specifically targets VEGF VEGFR IL-2 to quantify levels of MMSCs in the cornea of transplant recipients.Our results reveal that VEGF VEGFR in the cornea of alkali injury at early stage at high levels,but IL-2 is at low levels significantly in response to the MSC-implanted rabbits all different time.Interleukin 2 was significantly depressed in rabbit eyes transplanted with MMSCs in comparison with control group.Conclusion1.Morphology?yields?the proliferation,and differentiation potential of MMSCs are adapted to be an effective cell therapy when they are cultured in appropriate conditions.They can be purified in vitro by 5%?10%serum and select cells to culture,which adhered at 24?48 hours when they plated in dishes.MMSCs have a property of nature differentiation potential.VEGF?IL-1 can enhance bone marrow stromal cell migration in interface culture.2.Lung tissue is a dominating location where MMSCs are intercepted,when they engraft into the body.The MMSCs engraftment into organisms can home the injury-induced cornea and function by systemically.3.The MMSCs could merge into endothelium of angiogenesis and reduce the corneal inflammation-related angiogenesis and develop a microenvironment.where the cornea can be re-epithelium and transparency.MMSCs can reduce the neovascularization and accelerate migration of corneal epithelium to promote the wound healing by modulated express the metal protein enzyme inhibition-2.All these data suggested that inhibition of both inflammation and inflammation-related angiogenesis may partially account for the recovery of the damaged rabbit corneal limbal upon transplantation of MMSCs.We also show that administration of MMSCs immediately after TIMP-2 challenge protects cornea tissue from alkali induced cornea limbal injury,as evidenced by a significant reduction in inflammation,collagen deposition,and MMP activation within cornea tissue.Collectively,these findings indicate that systemic administration of MMSCs may be beneficial in the treatment of cornea disease even if engraftment levels of the administered cells are comparatively low.Therapeutic effect may be a result of inhibition of revascularization and inflammation.4.This result revealed that MMSCs can be an immune suppressive drug,which will be several novel methods using to promote wound heading in cornea injury.