| Part One:The effects of DETC on wound healing.BackgroundSkin is the body's most superficial barrier which plays an important role in protecting against external damage.In clinical,trauma,burn and chronic skin ulcers are common injuries to the skin.It is important for these patients to close the wound effectively and timely.Skin wound healing is a complex process mediated by various kinds of cells and influenced by many factors.Dendritic epidermal T lymphocytes(DETC),as a kind of??T cells,are the main kind of lymphocytes in mouse epidermis.During wound healing,DETC migrate to wound edge to promote wound healing by secreting various kinds of soluble cytokines,recruitmenting of leukocytes,secreting inflammatory factors,etc.However,excessive inflammatory responses on wound have adverse effects on wound healing process.It is necessary to confirm the effects of DETC on wound healing process.ObjectiveTo investigate the effects of DETC on wound healing.MethodsTo explore the impact of DETC on wound healing.Wound healing model was constructed in WT mice and Tcr?gene knock-out mice.Comparison of the percentage of unhealed wound area was conducted between two group of mice over time.Comparison of the length of new epithelium between two groups of mice was conducted by HE staining.Comparison of PCNA expression in new epithelium between two groups of mice was conducted by western blotting.Further,wound healing model was constructed in Tcr?gene knock-out mice as a control group.DETC isolated and cultured in vitro was added to the wound margin of Tcr?gene knock-out mice as experimental group.Comparison of the percentage of unhealed wound area was conducted between two group of mice over time.Comparison of the length of new epithelium between two groups of mice was conducted by HE staining.Comparison of PCNA expression in new epithelium between two groups of mice was conducted by western blotting.Results1.DETC deficiency inhibited wound healing process and proliferation of epidermal cells.Compared with WT mice,the wound healing process of Tcr?gene knock-out mice was slower.3 days after wound,the length of new epithelium of Tcr?gene knock-out mice was359±15(?m),lesser than 462±26(?m)of WT mice(P<0.001).PCNA/GAPDH of Tcr?gene knock-out mice was 1.25±0.04,lower than 2.01±0.09 of WT mice(P<0.01).2.DETC have a positive impact on wound healing and proliferation of epidermal cells.Compared with Tcr?gene knock-out mice,the wound healing process of Tcr?gene knock-out mice+DETC was faster.3 days after wound,the length of new epithelium of Tcr?gene knock-out mice+DETC was 465±31(?m),longer than 375±21(?m)of Tcr?gene knock-out mice(P<0.05).PCNA/GAPDH of Tcr?gene knock-out mice+DETC was 1.62±0.08,higher than 1.05±0.14 of WT mice(P<0.05).ConclusionDETC promote wound healing partly by accelerating the proliferation of epiderma cells.Part Two:The effects of DETC on ESC in wounded epidermis.BackgroundESC are immature cells in a relatively resting state in the basal layer of normal epidermis.However,in wounded skin,ESC can proliferate quickly and promote wound healing.ESC in wound edge play a significant role in wound healing.Recently,many studies have shown that DETC play an important role in wound healing process and the proliferation of ESC is the basic process of wound coverage.However,there were no studies about the relationship between DETC and ESC.Our recent findings showed DETC promote the stemness maintenance and proliferation of ESC by producing IGF-1.However,it still lacked experimental evidence in vivo.It is necessary to investigate the effects of DETC on ESC in wounded epidermis.ObjectiveTo investigate the effects of DETC on ESC in wounded epidermis.Methods1.To explore the impact of DETC on ESC in normal skin.Epidermal cells were isolated from in WT mice and Tcr?gene knock-out mice.Comparison of the proportion of CD49~+CD71~-ESC and K15~+ESC on normal skin was conducted by flow cytometry methods between two groups of mice.Comparison of the number of K15~+ESC in normal skin was conducted by immunofluorescence methods between two groups of mice.2.To explore the impact of DETC on ESC in wounded epidermis.Full sickness wounds were constructed in WT mice and Tcr?gene knock-out mice.Three days after that,the proportion of CD49~+CD71~-ESC and K15~+ESC in wound margin was conducted by flow cytometry methods.The number of K15~+ESC in wound margin was conducted by immunofluorescence methods.3.To further explore the impact of DETC addition on the number of ESC in wound margin.Full sickness wounds was constructed in Tcr?gene knock-out mice as a control group.DETC isolated and cultured in vitro was added to the wound margin of Tcr?gene knock-out mice as experimental group.Three days after that,comparison of the proportion of CD49~+CD71~-ESC and K15~+ESC in wound margin was conducted by flow cytometry methods between two groups of mice.Comparison of the number of K15~+ESC in wound margin was also conducted by immunofluorescence methods between two groups of mice.Results1.Deficiency of DETC do not influence the number of ESC in normal epidermis.The results of flow cytometry showed that the deficiency of DETC had no impact on the proportion of CD49~+CD71~-ESC and K15~+ESC in normal skin(P>0.05,P>0.05).The results of immunofluorescence showed that the deficiency of DETC didn't influence the number of K15~+ESC in normal skin(P>0.05).2.Deficiency of DETC diminish the number of of ESC in wounded epidermis.The results of flow cytometry showed that the deficiency of DETC reduced the proportion of CD49~+CD71~-ESC and K15~+ESC in wound margin of Tcr?gene knock-out mice(P<0.001,P<0.001).The results of immunofluorescence showed that the deficiency of DETC reduced the number of K15~+ESC in wound margin of Tcr?gene knock-out mice(P<0.001).3.DETC increase the number of ESC in wound margin of Tcr?gene knock-out mice.The results of flow cytometry showed that the addition of DETC increased the proportion of CD49~+CD71~-ESC and K15~+ESC in wound margin of Tcr?gene knock-out mice(P<0.001,P<0.01).The results of immunofluorescence showed that the addition of DETC increased the number of K15~+ESC in wound margin of Tcr?gene knock-out mice(P<0.01).ConclusionDETC increase the number of ESC in wounded epidermis.Part Three:DETC promote the stemness maintenance and proliferation of ESC by producing exosome.BackgroundESC are the stem cells impelling wound re-epithelialization.The number of ESC in wound edge influence the speed of wound healing.The stemness maintenance and proliferation capabilities of ESC determine the number of ESC in wound edge.However,during wound healing process,ESC proliferate quickly accompanied by the reduction of stemness maintenance.Our recent resourch have shown that DETC promote the stemness maintenance and proliferation of ESC by secreting IGF-1.However,the effects of IGF-1 were too weak to fully explain the effects of DETC on stemness maintenance and proliferation of ESC.Exosome is an efficient medium of signal communication between cells which has more activity than free cytokines.Many immunocytes can secrete exosome.However,there is no research on exosome of DETC.It is essential that we invesitage whether DETC affect wound healing process through regulating the function of ESC by secreting exosome.ObjectiveTo investigate the effects of DETC on stemness maintaness and proliferation of ESC and related mechanism.Methods1.To explore whether DETC produce exosome.Exosome of DETC was isolated from its culture medium.Typical protein of exosome was detected by western blotting.The structure of DETC exosome was detected by transmission electron microscopy.The diameter of exosome was detected by nanoparticle size analyzer.2.To detect whether DETC affect the stemness maintenance and proliferation of ESC.ESC was isolated and culture in vitro as a control group.DETC and DETC treated by exosomal inhibitor was used to coculture with ESC as experimental groups.Comparison of the proportion of ESC was conducted by flow cytometry methods between the three groups.After that,ESC was labeled with CFSE and treated by the same methods mentioned above.Comparison of the proliferation of ESC was conducted by flow cytometry methods between the three groups.3.To detect whether DETC-derived exosome influence the stemness maintenance and proliferation of ESC.ESC was isolated and culture in vitro as a control group.Exosome of DETC was used to treat ESC.Comparison of the proportion of ESC was conducted by flow cytometry methods between these groups.After that,ESC was labeled by CFSE and treated by same methods mentioned above.Comparison of the proliferation of ESC was conducted by flow cytometry methods between these groups.Results1.DETC produce exosome.The results of western blotting showed that the typical proteins of DETC exosome included CD63 and Tumor Susceptibility Gene 101(TSG101)and excluded Canexin.The result of transmission electron microscopy showed that DETC exosome had an elliptical vesicle-like shape.DETC exosome also had an outer complete membrane and a low-density center.The diameter of DETC exosome was 50-140nm.2.DETC promote the stemness maintenance of ESC by producing exosome.(1)When ESC was cultured in a high seeding density,the proportion of CD49~+CD71~-ESC of DETC+ESC culture group was 74.02±2.12(%),higher than that of control group(54.66±1.34(%)(P<0.001))and DETC(treated by exosomal inhibitor)+ESC group(62.88±1.70(%)(P<0.01)).The proportion of K15~+ESC of DETC+ESC culture group was 73.38±2.14(%),higher than that of control group(58.70±0.90(%)(P<0.001))and DETC(treated by exosomal inhibitor)+ESC group(62.58±2.71(%)(P<0.05))(2)When ESC was cultured in a low seeding density,the proportion of CD49~+CD71~-ESC of DETC+ESC group was 53.46±1.41(%)higher than that of control group(16.78±0.93(%)(P<0.001))and DETC(treated by exosomal inhibitor)+ESC group(28.02±1.42(%)(P<0.001)).The proportion of K15~+ESC of DETC+ESC culture group was 48.74±1.92(%)higher than that of control group(16.78±0.93(%)(P<0.001))and DETC(treated by exosomal inhibitor)+ESC group(38.46±0.96(%)(P<0.01)).3.DETC exosome promote the stemness maintenance of ESC.The proportion of CD49~+CD71~-ESC of control group,5?g/ml exosome group,15?g/ml exosome group and 45?g/ml exosome group were 15.62±1.17(%),33.18±1.92(%),46.62±1.50(%),and 48.46±2.79(%)respectively.The results of three experimental groups were all higher than control group(P<0.001,P<0.001,P<0.001).The proportion of K15~+ESC ofcontrol group,5?g/ml exosome group,15?g/ml exosome group and 45?g/ml exosome groupwere 21.24±0.98(%),29.82±1.56(%),42.20±1.93(%),and 46.58±2.30(%)respectively.The results of three experimental groups were all higher than that of control group(P<0.01,P<0.001,P<0.001).4.DETC promote the proliferation of ESC by producing exosome.The percentage of proliferative ESC(CFSE~-ESC)of DETC+ESC group was 58.58±1.65(%),higher than that of control group(10.08±0.75(%)(P<0.001))and DETC(treated by exosomal inhibitor)+ESC group(24.74±0.96(%)(P<0.001)).5.DETC exosome promote the proliferation of ESC.The percentage of CFSE~-ESC of control group,5?g/ml exosome group,15?g/ml exosome group and 45?g/ml exosome group were 14.66±1.13(%),31.88±0.75(%),43.70±1.70(%),and 48.62±2.13(%)respectively.The results of three experimental groups are all higher than control group(P<0.001,P<0.001,P<0.001).ConclusionDETC promote the stemness maintenance and proliferation of ESC by producing exosome. |
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