| Objective This research aimed to explore wheather the human umbilical cord mesenchymal stem cells could be the optimal feeding layer to replace the rat source of 3T3 fibroblasts as an ideal breeding layer to amplify the limbal stem cells and to maintain its stem cell characteristics.Method In vitro cultivation of HUCMSCs then induce the cells to differentiate into fat,osteogenesis cells,and to detect the expression of cell surface markers of CD31、CD45、CD90 by flow cytometry.Using the mitomycin C concentration of 0 ug/ml,1ug/ml,2 ug/ml,4 ug/ml and 6 ug/ml,8 ug/ml to preprocess the umbilical cord mesenchymal stem cells,the enzymes standard instrument selection to seclect the lowest concentration to maintain the umbilical cord mesenchymal stem cell activity stably.Co-culturing the original rabbits’ corneal limbus stem cells with the HUCMSCs and NIH-3T3 cell after mitomycin C preprocessing.Observed the cloning forming,calculated and compared the clone formation rate(CFE)of each group.Using immunofluorescence staining method to detect and compare the expression of the limbal stem cells marker of ABCB5,IPO13 and CK3/12 of each group.Then we can evaluate the two aspects which of maintaining stem cells and proliferation to determiner wheather the human umbilical cord mesenchymal stem cells can be used as a breeding layer to replace the 3T3 fibroblasts.Results The homogeneity of human umbilical cord mesenchymal stem cells cultured in vitro is good,after adipogenic and osteogenic induction,the cells have the potential to differentiate into fat,and bone cells;limbal stem cells can express CD90 cell adhesion molecules at the positive rate of 88.2 %.Cultured umbilical cord mesenchymal stem cells did not express CD45 markers of hematopoietic cells,the positive rate was 0.2%,nor the endothelial cell markers CD31,the positive rate was 0.4%;The cell activity of HUCMSCs pretreated with different concentrations of mitomycin C was measured by microplate reader,and the minimum mitomycin C concentration was maintained at 4ug / ml for the stability of umbilical cord mesenchymal stem cells.After co-culture with rabbit limbal stem cells for 8 days,the colony of corneal limbal stem cells from the two feeder layer cells were observed under microscope.The corneal limbal stem cells from the 3T3 feeder layer group were more dense and tightened,the colony forming efficiency of HUMSCs、NIH-3T3 feeder layer group and LSCs without feeder layer group is(4.10±0.56)%,(4.67±0.76)% and(0.83±0.35)% respectively,and the difference between the three groups was statistically significant(F= 37.898,P < 0.01).There was no significant difference in colony forming efficiency between umbilical cord mesenchymal stem cells and NIH-3T3(t=1.19,P>0.05).But there was significant difference between umbilical cord mesenchymal stem cells,NIH-3T3 and non-feeder layer group(T=6.87,8.06,all P<0.01).The marker ABCB5 and IPO13 of the colony which from the two feeder layer measured by immunofluorescence technique were at high expression but CK3/12 was at low expression,and the expression of CK3/12 was higher than that of umbilical cord mesenchymal stem cells in 3T3 feeder layer group,and the expression of the others stem cell markers was not significantly different in the two feeding groups.Conclusion Umbilical cord mesenchymal stem cells can breeder limbal stem cells which from rabbits and maintain their characteristics.There is no difference in the colony forming efficiency and maintain the state of undifferentiated of the corneal limbal stem cell compared with 3T3 feeder layer group,but the ability to promote the proliferation of limbal stem cells is weaker and the ability to maintain limbal stem cell characteristics is slightly stronger.Therefore,umbilical cord mesenchymal stem cells can be used as an ideal feeder layer to replace mouse 3T3 fibroblasts in vitro to culture limbal stem cells. |
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