Induction And Application Research Of Dedifferention Epithelial Stem Cells

Objective:1.To compare some biological characteristics between epithelial stem cells and those derived from dedifferentiation through three mothods including heating,H₂O₂ and bFGF in order to get the most efficient approach of dedifferention.2.To investigate repairing effectiveness after transplanting the the defferention epithelial stem cells into the skin wounds of BALB mice.Methords:1.Epithelial stem cells were isolated from the fresh circumcised foreskins.Epithelial cells were achieved by the culture and passage of epithelial stem cells.The biological features of epithelial stem cells and epithelial cells were tested:â‘ using inverted phase contrast microscope to observe the cells morphology,â‘¡testing the cells immunochemical staining,â‘¢testing the cells cycle and proliferation index by flow cytometry.2.The epithelial stem cells isolated from the same foreskin were divided equally and randomly 5 groups,1 of which were selected to be frozen and reserved as original epithelial stem cells at -70℃,4 of which were cultured and transferred to mature epithelial cells.3 dedifferentiation-inducing groups were established through heating,H₂O₂,and bFGF.The 3 dedifferention groups underwent some tests on 3d,7d,14d after treatment with the ESCs and ECs:â‘ cells morphology change,â‘¡phenotypic change ofβ1,CK10,CK19,â‘¢the number of positive cells ofβ1 was analysed by statistical method.3.The model of BALB/c mice full-thickness skin loss wounds were made in aseptic operation condition.Two circular 6mm-diameter skin loss wounds were cut on every BALB/c mice's back.24 wounds of 12 BALB/c mice were divided equally and randomly into 4 groups,DESCs,ESCs,ECs and NS group.Then the cell suspensions of DESCs,ESCs and ECs,which labelled with DAPI,were injected respectively into the wound edges with concentration of 2×10⁶/ml.And the dose was 0.2ml per treatment wound.The same dose normal saline was injected in the other wound.On the postoperative 0d,3d,7d,all of the wound areas were measured and analyzed with statistical methods.On the postoperative 7d,the sections were made,which were examined by fluorescence microscope and HE staining.Result:1.Morphologically,the epithelial stem cells were small,round,and had a high karyoplasmic ratio.However,the epithelial cells were flat,irregular, and had a low karyoplasmic ratio.Immuncytochemically,β1,CK19 expressions were positive and CK10 were negative in the epithelial stem cells.The results were adverse in the epithelial cells.The cell cycles analysis showed the number of G2/M cells were higher in the epithelial stem cells than in the epithelial cells.2. Statistically,the number ofβ1 positive cells was higher in the 3 treatment groups than in the ECs group on the 3d,7d,14d after the procedure(p＜0.01).There were no significant difference among the 3 treatment groups and the original epithelial stem cells group on the 14d after the procedure(p＞0.05).3.The wound areas of DESCs and ESCs were smaller than the other 2 control wound areas on postoperative 3d and 7d(p＜0.01).There were no significant difference between DESCs and ESCs groups.The fluorescence microscope revealed the dedifferentiation epthelial stem cells and original epthelial stem cells labelled with DAPI were disposed in the skin and subcutaneous tissue of the repaired wound, where no ECs labelled with DAPI were found.HE staining sections showed the repairing quality of treatment wounds was higher than control wounds,with regenerating glands and less fibrous connective tissues.Conclusion:The comparison investigation showed heating,H₂O₂,bFGF could induce the epithelial cells to dedifferentiate.The efficiency of bFGF is higher than another 2 appoaches on the 3d and 7d after the treantment.Both of the epithelial stem cells and dedifferentiation epithelial stem cells could promote the wound repair,and the their effect were on the same level.