| Part I The Establishment and Evaluation of Alkali Burn Related Total Limbal Stem Cell Deficiency Mouse ModelObjectiveTo establish the mouse model of alkali burn related total limbal stem cell deficiency (LSCD), and to provide a simple and effective model for further research work.MethodFirst, put an annular filter paper infiltrating with0.5mol/L sodium hydroxide with3mm inner diameter and5mm external diameter on the corneal limbus of a6-8weeks old mouse for30seconds, and then remove. Complete ocular examinations were performed, including slit-lamp examination, fluorescein staining and impression cytology after1d,3d,5d,7d,10d,14d,21d and30d. In addition, hematoxylin-eosin staining was made after the eyeballs were removed and embed in the paraffin.Results①Slit-lamp examination:The limbus became ischemic, a gray ring, the size of which was consistent with the diameter of the annular filter paper, was observed on Day1. The cornea became edematous and opacified on Day3-5. The corneal opacification and edematous were relieved on Day7-10. The circumlimbal hyperemia occurred with hyperplastic vessels, and extended up to the corneal stroma on Day14. Much more hyperplastic vessels were observed around the corneal limbus on Day21. The cornea became conjunctivalization and neovascularization on Day30.②Hematoxylin-eosin staining:Large number of inflammatory cells were infiltrated in corneal epithelium and stroma on Day1-5. The edematous of corneal stroma was relieved on Day7-10.2-3layers of corneal epithelium was observed on Day14. On Day21, goblet cells were observed on corneal epithelium. Hyperplastic vessels with mature red blood cells inside were observed on corneal stroma on Day30.③Impression cytology:A lot of goblet cells with positive staining of PAS were observed on corneal epitheliu.ConclusionThe method of annular filter paper infiltrating with sodium hydroxide is effective in establishing total limbal stem cell deficiency mouse model, which can provide a simple and stable model for further research work. Part II The Observation of Mouse Bone Marrow Mesenchymal Stem Cells Homing to cornea after ocular alkali burnsObjectiveTo observe the timing and location of mouse bone marrow mesenchymal stem cells homing to cornea after ocular alkali burns.Method80mouse models were randomly divided into three group:group of normal bone marrow function, mobilized bone marrow function with G-CSF and subconjunctival injection with GFP transfected mouse bone marrow mesenchymal stem cells. The operated and contralateral control eyeballs were removed at1d,3d,5d,7d,10d,14d,21d and30d. The immunofluorescence staining of CD29, CD44and CD45and immunohistochemical staining of SDF-1were performed. Laser scanning confocal microscope was used to detect GFP transfected mouse bone marrow mesenchymal stem cells. Real-time quantitative polymerase chain reaction was performed to analyse the expression of CD44, CD45, CD90, CD105and SDF-1.Results①Immunofluorescence staining:No positive staining of CD29, CD44and CD45was found on Day1-10. A few positive staining of CD29and CD44in corneal limbus were detected on Day14-30.②Laser scanning confocal microscope:No GFP transfected mouse bone marrow mesenchymal stem cells was detected in cornea and subconjunctiva.③Real-time quantitative polymerase chain reaction:No expression of CD29, CD44, CD45, CD90and CD105was detected on Day1-10, a low level expression was detected on Day14-30.④Immunohistochemical staining:The positive staining of SDF-1was observed on Day7, and became less gradually. No positive staining was observed on Day21.Conclusion①A few mouse bone marrow mesenchymal stem cells were detected homing to corneal limbus after14-30days of ocular alkali burns.②The expression of SDF-1, which was consistent with myocardial tissue, was not the key factor affecting the homing of mouse bone marrow mesenchymal stem cells. Part III The influence of inflammatory cytokine and hyperosmosis after ocular alkali burns on mouse bone marrow mesenchymal stem cellsObjectiveTo observe the influence of inflammatory cytokine (IL-1β) and hyperosmosis (406Osm) after ocular alkali burns on mouse bone marrow mesenchymal stem cells.MethodMouse bone marrow mesenchymal stem cells were isolated and incubated in vitro, and then divided into five groups:group of control,5ng/ml IL-1β, lOng/ml IL-1β,20ng/ml IL-1β and hyperosmosis. MTT assay, cell clone forming test and scratch assay were performed.Results①Cell morphology:The original generation of cultured mouse bone marrow mesenchymal stem cells were fusiform with abundant cytoplasm and circular large nuclei. The group of5ng/ml IL-1β:There was no difference in cell morphology between the control and5ng/ml IL-1β group. With the increase of concentration of IL-1β in medium, more floating cells appeared. The group of hyperosmosis:The cultured cells present as irregular shape with shrinking and reflective cell membrane.②MTT assay:The value of OD in control group was0.498±0.043(n=5) and0.499±0.038(n=5) in5ng/ml IL-1group. There was no statistical significance between the two groups. But The difference was of significance in statistics among the control and10ng/ml IL-1β,20ng/ml IL-1P and hyperosmosis group.③Cell clone forming test:The cell clone forming rate in control group was18.200±1.923%(n=3), and decreased to17.400±2.074%(n=3)in5ng/ml IL-1group. But there was no statistical significance between the two groups. The cell clone forming rate was further decreased in groups of10ng/ml IL-1β,20ng/ml IL-1β and hyperosmosis, the difference was of significance in statistics among them and the control.④Seratch assay:The cell migration ability was not affected in5ng/ml IL-1group, but apparently decreased in groups of10ng/ml IL-1β,20ng/ml IL-1β and hyperosmosis in24hours,48hours and72hours. The difference was of significance in statistics among them and the control.Conclusion①The hyperosmosis in ocular surface apparently affected clone forming rate and migration ability of mouse bone marrow mesenchymal stem cells.②Low concentration of IL-1β was proved to be non-cytotoxicity, and had no influence on cell clone forming rate and migration ability.③With the increase of concentration of IL-1β in medium, cell toxicity gradually appeared, and clone forming rate and migration ability was inevitably affected. Part IV The Observation of Mouse Bone Marrow Mesenchymal Stem Cells Homing to cornea in the stable stage after ocular alkali burnsObjectiveTo observe the timing and location of mouse bone marrow mesenchymal stem cells homing to cornea in the stable stage after ocular alkali burns.Method80mouse models in the stable stage of ocular alkali burns were randomly divided into three group:normal bone marrow function group, mobilized bone marrow function group and subconjunctival injection group. The operated and contralateral control eyeballs were removed at Id,3d,5d,7d,10d,14d,21d and30d. The immunofluorescence staining of CD29, CD44and CD45were performed. Laser scanning confocal microscope was used to detect GFP transfected mouse bone marrow mesenchymal stem cells. Real-time quantitative polymerase chain reaction was performed to analyse the expression of CD44, CD45, CD90, CD105, ABCG2, P63, CK3/12and CK19.Results②Slit-lamp examination:The corneal clarity was partly restored, and corneal neovascularization decreased in mobilized bone marrow function group and subconjunctival injection group. There was no obvious change in normal bone marrow function group.②Immunofluorescence staining:A few positive staining of CD29and CD44in corneal limbus were detected in normal bone marrow function group. Similarly, a few positive staining of CD29and CD44in corneal limbus were detected on Day1-3, and sharply increased on Day5-10, gradually decreased on Day14in mobilized bone marrow function group. No positive staining was found on Day21-30.③Laser scanning confocal microscope:A lot of GFP positive cells in corneal limbus were detected on Day1-3, but gradually decreased as time passed. No positive staining was found on Day10.④Real-time quantitative polymerase chain reaction:A low level expression of CD29, CD44, CD90and CD105was detected in normal bone marrow function group. In mobilized bone marrow function group, the expression of CD29, CD44, CD90and CD105obviously increased on Day5, maintained a high level on Day7-10, and gradually decreased after Day14. A higher level expression of CD29, CD44, CD90and CD105was detected in subconjunctival injection group, but gradually decreased as time passed. No positive staining was found on Day10.When compared to normal bone marrow function group, a higher level expression of ABCG2, P63and CK3/12, and lower level of CK19were found in mobilized bone marrow function group. The difference was of significance in statistics between the two group. When compared to mobilized bone marrow function group, a higher level expression of ABCG2, P63and CK3/12, and lower level of CK19were found in subconjunctival injection group. But there was no statistical significance between the two groups.Conclusion①Much more mouse bone marrow mesenchymal stem cells were detected homing to corneal limbus in the stable stage after ocular alkali burns. Mobilizing bone marrow function with G-CSF benefited in improving homing efficience.②The effect of ocular surface reconstruction in subconjunctival injection group was a litter better than that in mobilized bone marrow function group.③The homing mouse bone marrow mesenchymal stem cells were located in the corneal limbus. |
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