| In the past thirty years,diabetes has gradually become an epidemic throughout the world.There are more than 100 million diabetic patients in China,ranking the first over the world.Diabetic patients may experience skin damage during their lifetime,and most will turn to chronic.There is no special effective method for the treatment of diabetic wounds currently.Under the premise of controlling blood glucose,the main treatments for diabetic wounds include thoroughly debridement of the wound,protection from infection,promotion of wound healing and etiological treatment.Due to the existence of treatment for a long time,the frequency of recurrence rate is high,the clinical demand for a new and effective treatment greatly.Amniotic membrane is a layer with a thickness of about 8-12 um in the most inner layer of the placenta,without nerves and blood vessels.The amniotic membrane is composed of the epithelial layer and connective tissue outer layer.The epithelial layer is connected to the amniotic fluid,and connective tissue outer layer is connected to the chorionic membrane.The amniotic membrane was firstly used as a dressing for the treatment of wound surface in 1910.Since then,with the characteristics of cell migration and proliferation ability,low immunogenicity,relieve of wound pain by reducing inflammation and scar tissues,the containing of many growth factors and cytokines,it has been widely used in clinical.With the development of amniotic membrane preservation technology,amniotic membrane has also been widely applied in ophthalmic diseases,burn treatment,plastic repair,oral diseases and neurosurgery.The amniotic epithelial layer is composed of monolayer homogeneous cubic epithelial cells,which is called amniotic epithelial cells.Multiple studies have shown that human amniotic epithelial cells express stem cell markers and also have the ability to differentiate into all three embryonic germ layers,capable of secreting a variety of growth factors.Meanwhile,with the advantages of wide source,convenient separation,no immunogenic and tumorigenicity,amniotic epithelial cells have become a potentially useful and non-controversial cell source for transplantation and regenerative medicine.The application of amniotic epithelial cells in tissue repair has been widely reported.Nowadays,amniotic epithelial cells exhibit a certain advantage in the treatment of liver,nerve,lung,myocardium and even tumor.However,few researches have explored the role of amniotic epithelial cells in diabetic wounds.Therefore,we aim to extract and culture amniotic epithelial cells and apply them to diabetic wounds so as to observe its effect on wound healing,and to explore its mechanism.Part ?: Isolation and Identification of Human Amniotic Epithelial Cells in Vitro and Detection of Biological CharacteristicsObjective: Isolation and culture of human primary amniotic epithelial cells and identification.Methods: Amniotic membranes were collected from cesarean section placenta and amniotic epithelial cells were extracted by enzymatic digestion.The phenotypes of amniotic epithelial cells were detected by flow cytometry.Meanwhile,immunofluorescence and RT-PCR were used to detect the antigen of amniotic membrane epithelial cells.Finally,the differentiation potential of the amniotic epithelial cells was detected by three germ layer.Results: In vitro human amniotic epithelial cells microscopically observed cells into pebble-like growth,nuclear round,centered,cells were polygons,clear boundary,clear outline.Flow cytometry showed that cultured cells highly expressed CD29,CD44,CD73,CD90,CD105,but did not express CD45,CD34.Immunofluorescence showed that amnion epithelial cells highly expressed SSEA-3,SSEA-4 and OCT4.RT-PCR results showed that amnion epithelial cells expressed genes such as OCT4,Sox-2 and NANOG.Induction of differentiation experiments demonstrated that amniotic epithelial cells differentiate into three germ layers.Conclusion: Successful isolation and culture of amniotic epithelial cells,which can express a variety of stem cell markers,and can differentiate into the three germ layers,has great potential in tissue repair.Part II: Study on amniotic epithelial cells promote the healing of diabetic woundsObjective: To study the effect of amniotic epithelial cells on diabetic wound healing.Methods: The primary cultured amniotic epithelial cells were injected locally with diabetic wounds to observe wound healing.In order to evaluate the wound vascularization,macrophage polarization and degree of inflammatory response.The wound specimens were collected for pathology,immunohistochemistry and the detection of IL-1?,IL-6 and TNF-? and the promotion of healing factors(IGF-1,IGF-1,VEGF)condition by ELISA,Results:(1)Compared with PBS group,wound healing rate of amniotic epithelial cell group was significantly increased on the 10 th and 14 th postoperative days(P <0.01).(2)Pathological examination showed that the thickness of nascent granulation tissue in amniotic epithelial cell group on day 10 was significantly increased compared with PBS group(P <0.01).CD31 immunohistochemistry showed that the density of nascent capillaries in the wound surface of amniotic epithelial cell group was significantly higher than that in PBS group(P <0.01).The density of M1 macrophages in amniotic epithelial cell group was significantly lower than that in PBS group(P <0.01).The density of M2 macrophages was higher(P <0.05)and the ratio of M1 / M2 was also significantly lower(P <0.01).The expression of VEGF,TGF-?1 and IGF-1 in amniotic epithelial cell group was significantly higher than that in PBS group,while the expression of IL-1?,IL-6 and TNF-? was significantly lower than those in PBS.(3)Amniotic epithelial cells were implanted into wounds on day 5,and a large number of amniotic epithelial cells could be observed on the wound surface.On the 10 th day of wounding,a small amount of amniotic epithelial cells could be observed in the wound surface.Conclusion: Amniotic epithelial cells promote healing of diabetic wounds by regulating macrophage polarization,reducing wound inflammatory response,and promoting wound angiogenesis;Amniotic epithelial cells remain on the wound for a short period of time,and their paracrine effects may play a major role in promoting wound healing in diabetic patients.Part III: Study on the mechanism of amniotic epithelial cells to promote the healing of diabetic woundObjective: A preliminary study on the mechanism of amniotic epithelial cells promoting wound healing in diabetic patientsMethods:(1)The macrophages were isolated and cultured in vitro.The effects of conditioned medium of amniotic epithelial cells on the polarization of macrophages were observed by stimulation with IFN-? and TNF-?.Cells were collected for RT-PCR detection of the gene expression.We designed three groups: 10% fetal bovine serum DMEM medium+IFN-?and TNF-? positive control group,amniotic epithelial cell conditioned medium +IFN-?and TNF-? group,without IFN-?and TNF-? blank control group.(2)Human umbilical vein endothelial cells were cultured in vitro.The effects of amniotic epithelial cell culture on the proliferation,migration and tube formation of human umbilical vein endothelial cells were observed by CCK-8 kit,Transwell chamber and tube formation test.We designed three groups: 10% fetal bovine serum DMEM medium positive control group,amniotic epithelial cell conditioned medium group,DMEM without IFN-?and TNF-? as the blank control group.Results:(1)Application of amniotic epithelial cells cultured for TNF-? and IFN-? stimulated macrophages can reduce fine dendritic protrusions around cells.Macrophages in amniotic epithelial cell culture conditions group showed that the expression of CD206(M2 group macrophages marker)was significantly higher than that in the control group(P<0.01)and positive control group(P<0.01);and the expression of iNOS(M1 group macrophage marker was significantly lower than that in the control group(P<0.05)and positive control group(P<0.01).(2)CCK-8 kit detection of human umbilical vein endothelial cell proliferation activity,the application of amniotic epithelial conditioned medium treatment group can significantly promote the proliferation of human umbilical vein endothelial cells than the negative control group(P <0.01).The results of Transwell assay showed that the number of cells in the conditioned medium of amniotic epithelial cells increased significantly compared with the negative control group(P <0.01).The results of tube formation assay showed that the number of tubes in conditioned medium of amniotic epithelial cells increased significantly compared with that in the negative control group(P <0.01).Conclusion: Amniotic epithelial cells can significantly increase the proliferation,migration and tube formation of human umbilical vein endothelial cells.It can also reverse the conversion of macrophages to M1 and promote the transformation of M1 macrophages into M2,and thus promote the diabetic wounds healing. |
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