Effect on enhancing wound healing of rat bone marrow mesenchymal stem cells by silencing Tcf3 / 中国医师杂志

ABSTRACT

Objective:To investigate the role and mechanism of rat bone marrow mesenchymal stem cells (BMSC) in wound healing.Methods:Isolated BMSCs were cultured in vitro and induced to differentiate into epithelioid cells to promote wound healing by epidermal growth factor (EGF) and fibroblast growth factor (FGF). The expression levels of Tcf3, cytokeratin-18 (CK-18), CK-19 and p63 were evaluated by Western blot and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Cell activity was detected by cell counting kit-8 (CCK-8) and clone formation test. The level of Tcf3 protein and the degree of skin repair were evaluated by immunohistochemical staining and HE staining. Then, small interfering RNA (siRNA) targeting rat Tcf3 gene was used to silence Tcf3 , and a rat wound healing model was established to detect the effect of Tcf3 on wound healing. Results:The results of Western blot and qRT-PCR showed that EGF and FGF could promote the expression of CK-18, CK-19 and p63 in BMSC and reduce the expression of Tcf3 ( P<0.05). Immunohistochemical staining and HE staining showed that after silencing Tcf3 in BMSC, CK-18, CK-19 and p63 mRNA and protein were highly expressed and the proliferation ability was enhanced ( P<0.05). Luciferase and Western blot results showed that after silencing Tcf3 in BMSC, the Wnt activity and nuclear expression of β- Catenin was enhanced, while after adding XAV-393 to BMSC medium, the Wnt activity and the nuclear expression of β- catenin decreased ( P<0.05). Compared with si-Tcf3 group, the clone formation and cell growth ability of si-Tcf3 + XAV-393 group decreased after adding XAV-393 to BMSC medium on the basis of silencing Tcf3 ( P<0.05). Double luciferase assay showed that after silencing Tcf3 in BMSC, the activation level of Notch 1 pathway increased, but after adding Notch 1 signal inhibitor DAPT, its activity and expression level decreased ( P<0.05). Compared with si-Tcf3 group, the protein expression of CK-18, CK-19 and p63 in siRNA-Tcf3 + DAPT group decreased ( P<0.05). In the rat trauma model, the expression levels of Tcf3 protein and mRNA in skin increased significantly on the 4th, 8th and 12th day after injury (especially on the 12th day) ( P<0.05). The expression level of Tcf3 in si-Tcf3 BMSC group was lower than that in normal BMSC group ( P<0.05). Compared with the normal BMSC group, the wound healing effect of si-Tcf3 BMSC group was more significant thicker dermis, more hair follicles and greater proliferation space. Conclusions:Silencing Tcf3 can enhance the wound healing effect of BMSC in rats, which will provide a new way to accelerate skin regeneration.