| Background:With the development of the technique of uterine operation and the increase of the uterine operation,the basal layer of endometrium is injured and the thickness of functional layer is difficult to recover to the threshold thickness.This has become one of the main causes of refractory infertility in the field of reproduction.As heterogeneous adult stem cells,mesenchymal stem cells(MSCs)has strong ability of self-renewal and multi-directional differentiation,and can secrete many cytokines.It is a new treatment method for endometrial injury.Hepatocyte growth factor(HGF),as one of the cytokines secreted by MSCs,plays a significant role in vascular repair and mesenchymal to epithelial transformation(MET).Therefore,we speculate that MSCs transfected with HGF gene can produce more HGF which is beneficial to the repair of endometrial injury while retaining the characteristics of mesenchymal stem cells.It is expected that HGF gene transfected MSCs can obtain better therapeutic effect,and verify its role through HGF and its downstream pathway.Objective:To investigate the effect and mechanism of HGF gene transfected MSCs in mouse model of endometrial damage.Methods:Umbilical cord was collected and cultured to obtain MSCs,than the plasmid expressing HGF was transfected into MSCs by electroporation.The proliferation and activity of the HGF gene transfected MSCs were detected,and the surface specific antibody was detected by flow cytometry.After identifying the cell viability and characteristics of HGF gene transfected MSCs,the ability of HGF gene transfected MSCs to secrete HGF was measured by enzyme-linked immunosorbent assay.The ideal mouse model of endometrial injury was established by injecting 95% ethanol into the uterine cavity of female mice of childbearing age.After average grouping,saline,normal MSCs and HGF gene transfected MSCs were injected into the mouse model of endometrial injury through tail vein.The therapeutic effect was quantified by measuring the thickness of endometrial epithelium and the number of endometrial glands.The proliferative,apoptotic,mesenchymal and epithelial markers of endometrial cells were observed by immunofluorescence.Real time fluorescent quantitative polymerase chain reaction was used to assess the expression of angiogenesis related factors.The activation of HGF receptor c-Met and downstream signal AKT protein was compared by western blot.Results:After the plasmid of HGF was transfected into MSCs,there was no difference in the morphology and cell proliferation between HGF gene transfected MSCs and ordinary MSCs under the same culture conditions.The surface markers of HGF gene transfected MSCs were determined,the expression of CD73,CD90 and CD105 was consistent with that of ordinary MSCs.In addition,HGF gene transfected MSCs can secrete more HGF than ordinary MSCs.Observing the therapeutic effect of HGF gene transfected MSCs on mouse model of endometrial injury,it was found that HGF gene transfected MSCs had better effect in promoting the repair of damaged endometrial epithelium than ordinary MSCs.Morphologically,the damaged uterus recovered obviously.Through the observation and statistics of HE staining sections of the uterus,it was found that the thickness of the epithelium and the number of the damaged endometrium increased significantly(P<0.05).From the molecular view,MSCs transfected with HGF gene increased the number of proliferating cells and decreased the degree of apoptosis(P<0.05).To verify whether HGF gene transfected MSCs can reverse the mechanism of endometrial injury: epithelial mesenchymal transition(EMT)and microvascular injury,we counted the expression levels of the corresponding markers.The results showed that HGF gene transfected MSCs could promote the process of MET and angiogenesis(P<0.05).Finally,Western blotting confirmed that these repair effects were related to the activation of HGF receptor c-Met and downstream AKT signaling pathway.Conclusions:Compared with normal MSCs,HGF gene transfected MSCs had more significant repair effect on damaged endometrial epithelium.The mechanism is to promote the process of MET and angiogenesis,and the mechanism is achieved by activating the receptor c-Met and downstream AKT pathway. |
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