| Background: Human epidermal cornified cells continually shed from the surface of the epidermis.Replenishment of the differentiation compartment depends on proliferation of keratinocyte stem cells in the basal layer. Stem cells have an unlimited capacity for self-renewal and the ability to generate daughter cells that undergo terminal differentiation . They not only play a central role in homeostasis and wound repair,but also represent a major target of tumor initiation and gene therapy.However, we've known so little about the properties of keratinocyte stem cells(KSC) since they are minority keratinocyte with few accurate molecular markers.It need further studies about localization,isolation,cultivation and characterization of KSC in vivo as well as in vitro.Objective: to observe the distribution and amount of KSC in normal adult skins,abortive fetal skins and scar skins and to speculate the role of KSC in skin maturity,wound healing and scar forming.Another aim is to isolate KSC accurately,culture it with proper conditions and characterize its proliferation and differentiation in vitro. Methods: (1). Skin specimens were harvested from the corresponding sites from healthy adult donors,abortive fetal skins and from scar of patients 3 years after abdomen operations.The expression of integrinβ1,a molecular marker for KSC and involucrin,a marker for terminally differentiation cells were determined in each specimens by immunofluorescence technique. (2).Primary human keratinocyte were isolated from young foreskins and cultured with clonal density. Then clone formed by KSC was isolated, cultured and passaged consecutively with the presence of a feeder-layer of 3T3 cells,fetal or child's fibroblasts.The colony forming efficiency(CFE) was determined before each subculturing.Meanwhile the expression of integrinβ1 and involucrin in daughter clones of KSC were detected by immunofluorescence technique.Results: 1. The distribution of KSC in different skins:(1). the integrinβ1-bright cells were found clustering in stem cell niche adjacent to the tip of the dermal papillae in normal adult abdomen skins and at the tips of the deep rete ridges in palms,whereas they were found in the bulge region of the hair follicle.(2).Most basal cells in fetal skin were integrinβ1-positive cells which suggest there are more abundant KSC in fetal skin than in adult skin.(3).KSC was found to distribute in a mono-layer style with no stem cell niche existing in scar.The average fluorescence intensity is lower than it is in normal adult skin.(4).the expression of involucrin was found in the keratinocyte just above basal layer in scar,which indicate the abnormal proliferation and differentiation of KSC in scar skins. 2. The prompt proliferation and differentiation of the primary keratinocyte could be observed. 3. The CFE was 8%-10% for keratinocyte isolated from children younger than ten-year old,while CFE was 5%-8% for keratinocyte isolated from adults in their twenties. 4. CFE of keratinocyte that adhere most rapidly to type IV collagen was fluctuated from 16.2% to 19.5% compare to CFE of less than 10% for unfractioned keratinocyte,which indicate it's a available but inaccurate way to isolate KSC by adhesion to type IV collagen. 5. CFE of cells in a KSC clone was 96.3% ,while average CFE of all their daughter cells was 48.2%,37.2,33.4%,28.6%,27.4% successively during consecutive subculturing for five times. The decreasing of CFE from more than 95% to a stable level of about 25% can be observed.Whereas CFE could be kept for more than 95% if the mega-clone was isolated alone from other KSC daughter cells and disaggregated,replated to form further clones. 6. Three type of clones include holoclone,meroclone and paraclone co-exist in each generation of KSC daughter cells. Holoclone was formed by KSC and characterized by the expression of integrinβ1while mereclone and paraclone was formed by Transit Amplifying cell and characterized by the expression of involucrin partly or totally. 7. The KSC clone can be isolated,cultured and p... |
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