| As we know,skin is the largest human organ.Its outer layer,which is called epidermis, has an ability of permanent self-renewal.This renewal composes of division,proliferation, and differentiation of a kind of self-renewal cell,which is called keratinocyte and in basal layer of epidermis.Skin keratinocyte mainly include epidermal stem cell(ESC),transit amplifying cell(TA cell),and postmiotic differentiating cell(PMD cell).Epidermal stem cell,located in basal layer of epidermis,proliferates and differentiates continuously to replace terminal differentiation cell,showing a key role in structural renovation and cell homeostasis,particularly in proceeding of wound healing.Based on trauma model of animals,Fu Xiao-bing et al found in proceeding of wound healing,as time passed, scatteredβ1-integrin and K19 positive cells appeared around wound edge,particularly in spinous or granular layer closer to the wound.These positive cells,regarded as epidermal stem cells,gradually became more and more in number,and were mostly concentrated on the border of wound edges till the wound healing.Up to now,it has not been clearly demonstrated the mechanism of epidermal stem cells appearing around wound.At the early stage of wound healing,there is a lot of secretion containing abundant cytokine,extracellular matrix,endotoxin and so on.These cytokines play a key role in wound healing,mainly including regulating various cell reactions,affecting on cell proliferation and migration,as well as on synthesis and releasing of extracellular matrix. Therefore,we speculated there might be some cytokines in secretion around the wound, cooperating with cell surface and intracellular receptors to affect on distribution and quantity of epidermal stem cells,which might be regarded as chemotaxis mediated by chemotatic factors and their receptors.To investigate the mechanism of distribution and quantity of epidermal stem cells around wound,our study initially established a rat model of skin wound,respectively applied immunohistochemistry in location of distribution of epidermal stem cells in different phase of wound healing,and ELISA in detection of chemotatic factor SDF-1 expression around the wound,also performed correlation analysis on distribution of epidermal stem cells and SDF-1 expression.Then a large amount of in vitro cultured epidermal stem cells were detected for two classic cell surface makers—β1-integrin and ceratin 19(K19),moreover,immunofluorescence chemical technique and flow cytometry were also applied to detect chemotatic factor receptor CXCR4 expression in epidermal stem cells cultured in vitro.Finally,we applied Transwell cultivation system to observe in vitro chemotaxis of wound secretion collected from burnt patients on epidermal stem cells,and made a preliminary investigation on its mechanism.Methods and results:1.Histological location of rat epidermal stem cells in different phase of wound healingWith application of immunohistochemistry,the results showed a positive staining ofβ1-integrin,epidermal stem cells surface markers K19 and BrdU stagnation agent in basal layer of epidermis and hair follicle outer root-sheath area of normal rat skin.In proceeding of wound healing,as time passed,scatteredβ1-integrin and K19 positive cells appeared around wound edge,particularly in spinous or granular layer closer to the wound.These positive cells,regarded as epidermal stem cells,gradually became more and more in number,and were mostly concentrated on the border of wound edges till the wound healing.2.Detection of SDF-1 expression around wound edgeEnzyme-linked immunosorbent assay(ELISA) was applied to detect SDF-1 expression in supernatant of adjacent tissue homogenate,and supernatant from normal tissue homogenate was regarded as control group.24 hours after injury,SDF-1 expression began to appear in adjacent tissues and increase with the proceeding of wound healing.It reached the maximum on the 7th day; however,in control group,as time passed,there was no significant difference of SDF-1 expression at all the phases.3.Isolation,cultivation and identification of human epidermal stem cellPostoperative foreskin tissues were obtained from young patients of 5-15 years old who had accepted circumcision.Two-step separation,including neutral protease and pancreatin,and rapid adhesion of epidermal stem cells to typeⅣcollogen were applied to screen cells.Selected stem cells grew well on typeⅣcollogen with uniform cell shape as "cobblestone".These cells,small and round with high proportion of caryoplasm ratio, showed characteristic of protocell form,with higher cloning efficiency and positive expression of K19 andβ1-integrin,suggesting that human stem cells in basal layer of epidermis could be successfully cultured following above steps.4.Detection of CXCR4 expression in human epidermal stem cellsImmunofluorescence chemistry was used to detect CXCR4 expression in epidermal stem cells cultured in vitro.The results showed:①there was positive FITC fluorescein staining on cell membrane and cytoplasm;②fluorescence intensity of CXCR4 expression in epidermal stem cell was about 43.5%,detected by flow cytometry.5.Collection of wound secretion and research on its in vitro chemotaxis in epidermal stem cellsWound secretion had been collected with sterile operation from burnt patients(n = 6) in our hospital.10 ml syringes were used to extract wound secretion or fluidify under scar. Supernatant was obtained after centrifugation,filtered by filter paper and micro filter,then conserved under -70℃.Based on Transwell cultivation system,the results of in vitro chemotaxis showed about 87±9.12 cells moved from upper to lower layer of microporous membrane in wound secretion group,obviously more than CXCR4 receptor group(21±2.71) and K-SFM control group(15±3.28)(P＜0.05).However,migrated cells in receptor group were slightly more than K-SMF control group,without significant differences(P＞0.05).Based on above results,we could conclude that:1.The nearer to wound,the more epidermal stem cells can be found;and the cells gradually increase with decrease of wound until complete healing.Ectopic distribution of epidermal stem cells may play a key role in wound re-epithelization.2.High SDF-1 expression appears in adjacent tissues and gradually increase with proceeding of wound healing.3.Chemotatic factors receptors CXCR4 expression can be found in epidermal stem cells.4.Wound secretion has significant chemotaxis on epidermal stem cells in vitro,with its effect mainly mediated by SDF-1/CXCR4,besides cooperation of other signal channels.
|
PDF Full Text Request