The Role Of Schwann Cell-like Cells Derived From Human Amniotic Membrane Mesenchymal Stem Cells Transplantation In Flap Nerve Regeneration

Objective: Isolation,culture and identification of human amniotic membrane mesenchymal stem cells in vitro,and induced differentiation into Schwann cell-like cells in vitro.To observe the effect of transplantation of two kinds of cells on nerve regeneration of the rat flaps.Methods: 1)Amniotic was taken from the full-term cesarean delivery,human amniotic membrane mesenchymal stem cells were isolated from placenta via trypsin and collagenase digestion,and the supernatant was discarded to remove human amniotic epithelial cells after 48 h,and according to the same method 1~2generations,purification h AMSCs through via adherent method.The Flow cytometry and IF identification h AMSCs.2)The P3 generation of h AMSCs grown on day 6 was induced to differentiate into i SCs,after induced differentiation after,the expression of S-100?P75 and GFAP of i SCs and P3 generation of h AMSCs were detected by immunofluorescence staining;The expression of S-100?P75 and GFAP protein of i SCs and P3 generation of h AMSCs were detected by Western blot analysis;The expression of S-100?P75 and GFAP gene of i SCs and P3 generation of h AMSCs were detected by fluorescent quantitative real-time PCR;The concentration of BDNF and NGF in the supernatant of h AMSCs culture group at 6days and 1d?4d?7d?10d?13d?16d?19d after h AMSCs induction.3)Animal models were divided into three groups: h AMSCs transplantation group(A),i SCs transplantation group(B)and perforator flap model control group(C),25 rats in each group.The proliferation period of P3 h AMSCs and i SCs after induction,respectively,to mark each rat flap at the skin more injection of 1×106corresponding cells,C group injected PBS.IF was used to detect the nerve regeneration.Results: 1)A large number of stable proliferation of h AMSCs can be obtained via trypsinand collagenase digestion,purification h AMSCs through adherent method.Primary culture h AMSCs were seen growing adherently after 48 h,the cells density can reach 80% to 90%after 5 to 6d.flow cytometry results: CD90(99.61%)?CD44(99.64%)?CD73(99.82%)?CD105(91.84%),CD34?CD11b?CD19?CD45?HLA-DR(0.12%).Immunofluorescence staining showed that h AMSCs were highly expressed in vimentin and did not express CK19.2)Immunofluorescence staining showed that the expression of i SCs group S-100?P75 and GFAP was significantly higher than that of h AMSCs group(P<0.05);Western blot analysis showed that the expression of i SCs group S-100?P75 and GFAP protein was significantly higher than that of h AMSCs group(P<0.05);Fluorescent quantitative real-time PCR showed that the expression of i SCs group S-100 ? P75 and GFAP gene was significantly higher than that of h AMSCs group(P<0.05);The result of the BDNF and NGF in the supernatant of h AMSCs cultured for 6 days and 1 day,4d,7d,10 d,13d,16 d,19d after induction of h AMSCs were:The concentration of the BDNF and NGF were expressed in cultured h AMSCs for 6days,the concentration of the BDNF and NGF were decreased after joining induced liquid 1,with the induced liquid 2 and induced liquid 3 in turn add,the concentration of the BDNF and NGF were gradually increased,and significantly higher than the h AMSCs cultured for 6 days,indicating that the secret a large number of BDNF and NGF during the induction of differentiation,providing a potential trophic supports for nerve regeneration.3)On the 2d,5d,7d,9d and 14 d post-cell transplantation,the flap tissue immunofluorescence staining respectively,the number and the diameter of the regenerated nerve fibers in flaps in the i SCs group was significantly higher than the h AMSCs.Conclusion: 1)A large number of h AMSCs can be obtained via trypsin and collagenase digestion,purification h AMSCs through trypsin digestion and adherent method;2)Through the Immunofluorescence?Western blot?q PCR and ELISA method were used to identification respectively,which would proved that h AMSCs can be inducted into Schwann cell-like cells with the proper chemical factor regulation in vitro.In addition,a large number of promoting nerve growth factor were released during theprocess of differentiation.3)h AMSCs and i SCs were transplanted into the denervated flap model,and the regeneration of nerve was observed respectively.The number and the diameter of the regenerated nerve in flaps in the i SCs group was higher than the h AMSCs group,which through a large number of BDNF and NGF were released.