The Preliminary Study On The Migration And Trans-differentiation Of Bone Marrow Mesenchymal Stem Cells Into Corneal Epithelium Cells

Objective:1. To establish a persistent corneal epithelial defect model and investigate theinfluence of corneal epithelial defects of different phases on the proliferation capacity of bonemarrow Mesenchymal stem cells（BMSCs）.2. To imitate the model of transendothelial migration of BMSCs in vitro and explore the changesof BMSCs’ stransdifferentiation（adipogenic differentiation）capacity.3. To explore the possibility of post-penetrated BMSCs differentiating into corneal epithelialcells(CECs) under conditional medium.Methods:1.40healthy male wistar rats were selected and then randomly divided into8groups,and named as defect groups (A, B, C, D, E, F, G) and control group. The same area of cornealepithelium was removed daily on the left eye of all defect groups to construct a persistent cornealepithelial defect model. Rats in defect group A were sacrificed on the second day followingsuccessful modeling, then group B on the third day, and finally, group G on the eighth day.BMSCs were seperated and cultivated under aseptic conditions and the cell cycle was examinedby flow cytometry.2.The model of transendothelial migration of BMSCs was established in vitro, Then observe thecells with phase-contrast microscope,flow cytometry and quantitative analysis of adipogenicdifferentiation. Then the transdifferentiation capacity of BMSCs before and after migration wasevaluated.3. The post-penetrated BMSCs was cultured under conditional medium with corneal stromatissue and CECs supernatant for a week,then immunofluorescence was utilized to examine theexpression of cytokeratin12(CK12) in differentiated BMSCs.Results:1.The results of the cell cycle examination showed that the proportion of BMSCs atS-phase in group B and D was16.76%and17.95%, which was obviously increased (P<0.05)compared with other defect groups and control group, while the proportion of G1-phase cellswas72.26%and71.33%, significantly lower than others (P<0.05). But no conclusions can bedrawn that the proportion of S-phase and G1-phase cells between group B and D was statisticallydifferent(P>0.05), besides, no conclusions could be drawn that the proportion of S-phase andG1-phase cells among group A,C, E, F,G, and control group was statistically different (P> 0.05).2. Morphocytology observation of pre-and post-penetrated BMSCs revealed obviousdifference，and the quantitative analysis of adipogenic differentiation capacity showed that theOD value was0.0971and0.1016respectively, between which there was no statisticallysignificant difference (P>0.05).3. After co-cultured with conditional medium for7days, BMSCs’ morphology graduallychanged from long to polygon or triangle, but there was no CK12expressed in the cells byimmunofluorescence.Conclusion:1. In this experiment, the proliferative capacity of BMSCs significantly improved onday3and5during the consecutive process of corneal epithelial defect。2. Pre-and post-penetrated BMSCs have the capacity of adipogenic differentiation.3. BMSCs cultured under conditional medium for a week, presented some morphocytologychanges,but no CK12expression was discovered.