| Background:Failures of tissue repair after severe burn injury and other damages,which lead to many medical problems,even causes casualties and disabilities.Rapid re-epithelialization plays a key role in the formation of new epidermal tissue in the sealing of cutaneous wounds.The epidermal stem cells are the cellular basis of wound re-epithelialization.Maintaining its strong proliferation potential and high anti-apoptotic ability is necessary for the re-epithelialization and expansion of neonatal epidermal tissue.The number of epidermal stem cells and progenitor cells,which depends on the balance between the differentiation and proliferation of epidermal stem cells to precursor progenitor cells and the over-differentiation to terminal cells,is the key factor to determine the proliferation potential and anti-apoptotic ability of neonatal epidermal tissue.According to the literature,the process of proliferation and differentiation of epidermal stem cells can be roughly divided into four stages:self-renewal of epidermal stem cells,differentiation of epidermal stem cells into progenitor cells,proliferation of precursor progenitor cells,and differentiation of precursor progenitor cells into terminal cells.The process of self-renewal of epidermal stem cells and differentiation of epidermal stem cells into precursor progenitor cells is relatively slow,but the proliferation potential and anti-apoptotic ability of cells in these two stages are stronger;on the contrary,the process of differentiation of precursor progenitor cells into terminal cells is faster,but the proliferation potential and anti-apoptotic ability of cells in this stage are weaker.Therefore,the process of proliferation and differentiation of epidermal stem cells is the cytological basis of re-epithelialization.Previous studies have shown that DETCs,as an important component of the skin immune system,have a strong ability to promote re-epithelialization and play an important role in wound repair.After skin injury,the surrounding DETCs activate rapidly and migrate to the epidermis around wound area,secreting growth factors such as IGF-1 to promote the re-epithelialization process and improve wound healing.However,whether DETCs can promote wound healing by regulating the proliferation and differentiation of epidermal stem cells has not been reported yet.The purpose of this study is to clarify whether DETCs can promote skin wound healing by regulating the proliferation and differentiation of epidermal stem cells.Objective:To clarify that DTECs can accelerate the process of re-epithelialization and improve wound healing by regulating the proliferation and differentiation of epidermal stem cells.Methods:I.DETCs secrete IGF-1 to promote wound healing.Wound healing model was established on wild-type mice and TCR?-/-mice.The percentage of residual wound area at each time point was observed and the wound healing was compared between the two groups.Subsequently,TCR?-/-mice were subcutaneously injected with mice DETCs isolated and cultured in vitro.The model of TCR?-/-+DETCs was constructed to clarify the role of DETCs in the process of wound healing.The epidermis around wound area of wild type mice,TCR?-/-mice and TCR?-/-+DETCs mice were extracted respectively and the expression of IGF-1 in epidermis was detected by FACS and WB technique to clarify that DETCs participated in wound healing by secreting IGF-1.Finally,the recombinant mouse IGF-1 was subcutaneously injected into the wound of TCR?-/-mice,and the model of TCR?-/-+IGF-1 was constructed to clarify the role of IGF-1 in the process of wound healing.II.DETCs and IGF-1 significantly increased the number of epidermal stem cells in neonatal epidermal tissue around wound,and significantly reduced the number of epidermal terminal differentiation cells in neonatal epidermal tissue around wound.BrdU labeling animal model was constructed on wild-type mice and epidermal stem cells were labeled with BrdU.After the model was successfully constructed,neonatal epidermal tissues of wild-type mice,TCR?-/-mice,TCR?-/-+IGF-1 mice and TCR?-/-+DETCs mice were extracted and BrdU-labeled epidermal stem cells were examined by immunohistochemical technique to determine the effect of DETCs and IGF-1 on the number of epidermal stem cells in neonatal epithelial tissues.In addition,K15-positive epidermal stem cells were detected by immunofluorescence technique,and the effect of DETCs and IGF-1 on the number of epidermal stem cells were further clarified.K10-positive epidermal terminal differentiation cells were detected by immunofluorescence technique,and the effect of DETCs and IGF-1 on the number of epidermal terminal differentiation cells in neonatal epithelial tissues were determined.III.DETCs and IGF-1 can enhance the proliferation and self-renewal of epidermal stem cells,and inhibit the differentiation of epidermal stem cells into epidermal terminal differentiation cells in vitro.Isolation and culture of mouse epidermal stem cells and mouse DETCs in vitro,and co-culture of both cells was conducted to construct an epidermal stem cells-DETCs co-culture model.Mouse epidermal stem cells were isolated and cultured in vitro and stimulated by recombinant mouse IGF-1 to construct an epidermal stem cells-IGF-1stimulation model.By FACS,the epidermal stem cells in the epidermal stem cells-DETCs co-culture model and epidermal stem cells-IGF-1 stimulation model were stained with EdU and CD49f-CD71 to clarify the effects of DETCs and IGF-1 on the proliferation and self-renewal of epidermal stem cells.By FACS,K14-positive cells and K10-positive epidermal terminal differentiation cells in the epidermal stem cells-DETCs co-culture model and epidermal stem cells-IGF-1 stimulation model were detected to clarify the effect of DETCs and IGF-1 on the differentiation of epidermal stem cells into epidermal terminal differentiation cells.Results:I.DETCs secrete IGF-1 to promote wound healing1.Lack of DETCs delayed wound healing in mice.There were significant differences in the percentage of residual wound area between wild-type mice and TCR?-/-mice on the 4th,6th and 8th day after wound excision.The healing rate of TCR?-/-mice was significantly delayed compared with wild-type mice.2.Adoptive importation of DETCs could promote wound healing in TCR?-/-mice.There were significant differences in the percentage of residual wound area between TCR?-/-mice and TCR?-/-+DETCs mice on the 4th,6th and 8th day after wound excision.The adoptive importation of DETCs into TCR?-/-mice significantly accelerated their wound healing.3.IGF-1 is a key effector molecule of DETCs in promoting wound healing.?1?The expression of IGF-1 protein in epidermis around wound of TCR?-/-mice was significantly lower than that of wild-type mice,while the expression of IGF-1 protein in epidermis around wound of TCR?-/-mice was significantly increased after the adoptive importation of DETCs.?2?The percentage of DETCs expressing IGF-1 in epidermis around wound of TCR?-/-mice was significantly lower than that of wild-type mice,while the percentage of DETCs expressing IGF-1 in epidermis around wound of TCR?-/-mice was significantly increased after the adoptive importation of DETCs,which indicated that DETCs were the most important cell secreting IGF-1 in epidermis around wound.?3?There were significant differences in the percentage of residual wound area between TCR?-/-mice and TCR?-/-+IGF-1 mice on the 4th,6th and 8th day after wound excision.The adoptive importation of IGF-1 into TCR?-/-mice significantly accelerated their wound healing.II.DETCs and IGF-1 significantly increase the number of epidermal stem cells in neonatal epidermal tissue around wound1.The number of BrdU-labeled epidermal stem cells in neonatal epidermal tissues of TCR?-/-group was 5.00±3.00/view field,which was significantly lower than that of wild-type group?48.67±3.06/view field??P<0.0001?;the number of BrdU-labeled epidermal stem cells in neonatal epidermal tissues of TCR?-/-+DETCs group was 31.00±3.61/view field,and the number of BrdU-labeled epidermal stem cells in neonatal epidermal tissues of TCR?-/-+IGF-1 group was 24.67±2.08/view field,which were both significantly higher than that of TCR?-/-group?5.00±3.00/view field??P<0.0001,P<0.001?.2.The number of K15-positive epidermal stem cells in the neonatal epidermis of TCR?-/-group was 11.67±4.04/view field,which was significantly lower than that of wild-type group?163.30±14.05/view field??P<0.0001?;the number of K15-positive epidermal stem cells in the neonatal epidermis of TCR?-/-+DETCs group was 103.30±12.66/view field,and the number of K15-positive epidermal stem cells in the neonatal epidermis of TCR?-/-+IGF-1 group was 41.00±5.29/view field,which were both significantly higher than that of TCR?-/-group?11.67±4.04view field??P<0.0001,P<0.05?.III.DETCs and IGF-1 significantly decrease the number of epidermal terminal differentiation cells in neonatal epidermal tissue around woundThe number of K10-positive epidermal terminal differentiation cells in the neonatal epidermis of TCR?-/-group was 69.00±9.54/view field,which was significantly higher than that of wild-type group?4.333±1.53/view field??P<0.0001?.The number of K10-positive epidermal terminal differentiation cells in the neonatal epidermis of TCR?-/-+DETCs group was 12.67±3.79/view field?P<0.0001?,and the number of K10 positive epidermal terminal differentiation cells in the neonatal epidermis of TCR?-/-+IGF-1 group was 43.33±8.51/view field,which were both significantly lower than that of TCR?-/-group?69.00±9.54/view field??P<0.0001,P<0.01?.IV.DETCs and IGF-1 enhance the proliferation and self-renewal of epidermal stem cells in vitro1.Epidermal stem cells were cultured in vitro,and the epidermal stem cells-DETCs co-culture model and epidermal stem cells-IGF-1 stimulation model were constructed.FACS showed that the percentage of EdU-positive proliferating cells in the control group was 32.30±1.27?%?,which was significantly lower than that in the DETCs co-culture group?43.53±0.62?%???P<0.01?;the percentage of EdU-positive proliferating cells in the control group was 32.43±0.69?%?,which was significantly lower than that in the IGF-1stimulation group?42.13±0.92?%???P<0.01?.These results suggest that DETCs and IGF-1promote the proliferation of epidermal stem cells.2.Epidermal stem cells were cultured in vitro,and the epidermal stem cells-DETCs co-culture model and epidermal stem cells-IGF-1 stimulation model were constructed.FACS showed that the percentage of CD49f+-CD71-cells in the control group was56.40±0.32?%?,which was significantly lower than that in the DETCs co-culture group?66.53±0.50?%???P<0.0001?;the percentage of CD49f+-CD71-cells in the control group was 74.90±0.73?%?,which was significantly lower than that in the IGF-1 stimulation group?81.13±1.27?%???P<0.05?.These results indicate that DETCs and IGF-1 promote the proliferation and self-renewal of epidermal stem cells.V.DETCs and IGF-1 inhibit the differentiation of epidermal stem cells into epidermal terminal differentiation cells in vitro1.Epidermal stem cells were cultured in vitro,and the epidermal stem cells-DETCs co-culture model and epidermal stem cells-IGF-1 stimulation model were constructed.FACS showed that the percentage of K14-positive cells in control group was 54.90±1.32?%?,which was significantly higher than that in DETCs co-culture group?69.27±1.70?%???P<0.001?;the percentage of K14-positive cells in control group was 52.00±1.80?%?,significantly higher than that in IGF-1 stimulation group?66.83±0.97?%???P<0.01?.These results suggest that DETCs and IGF-1 inhibit the differentiation of epidermal stem cells.2.Epidermal stem cells were cultured in vitro,and the epidermal stem cells-DETCs co-culture model and epidermal stem cells-IGF-1 stimulation model were constructed.FACS showed that the percentage of K10-positive epidermal terminal differentiation cells in the control group was 67.13±1.19?%?,which was significantly higher than that in the DETCs co-culture group?55.67±0.70?%???P<0.01?;the percentage of K10-positive epidermal terminal differentiation cells in the control group was 58.17±0.30?%?,which was significantly higher than that in the IGF-1 stimulation group of?53.53±1.12?%???P<0.05?.It further indicates that DETCs and IGF-1 inhibit the differentiation of epidermal stem cells into epidermal terminal differentiation cells.Conclusion:DETCs can promote the proliferation of epidermal stem cells and inhibit their differentiation into epidermal terminal differentiation cells by secreting IGF-1,so as to increase the proportion of epidermal stem cells and reduce the proportion of epidermal terminal differentiation cells in neonatal epidermis around wounds,raise the proliferation and anti-apoptotic potential of neonatal epidermis,promote the process of re-epithelialization and thus improve wound healing. |
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