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@#Objective To optimize the extraction process of flavonoids from Broussonetia papyrifera leaves and explore the antioxidant effect of flavonoids on mouse epidermal stem cells.Methods The extraction process of flavonoids from Broussonetia papyrifera leaves was optimized by single factor experiment,including the liquid-solid ratio(15:1,20:1,25:1,30:1and 35:1),sodium hydroxide(NaOH) concentration(0.2%,0.4%,0.6%,0.8% and 1.0%),pH value(2.5,3.0,3.5,4.0and 4.5) and extraction temperature(60,65,70,75 and 80℃).Based on the results of single factor experiment,the optimal extraction process was determined by orthogonal test with the mass fraction of flavonoids as the evaluation index.CD49f~+/CD71~-mouse epidermal stem cells were isolated and cultivated by immunomagnetic bead method,and the effects of flavonoids on the cell relative viability and the contents of reduced glutathione(GSH) and malondialdehyde(MDA) were detected.Results The optimal extraction conditions of flavonoids were liquid-solid ratio of 30:1,0.6% NaOH,pH 4.5and extraction temperature of 75 ℃.Under these conditions,the average mass fraction of flavonoids extracted was 1.47%.Compared with the negative control group,when the flavonoids final concentration was 25 and 50 μg/mL,the cell relative viability increased significantly(F=1.427 and 13.747 respectively,each P <0.01);when the final concentration of flavonoids was 12.5,25 and 50 μg/mL,the content of GSH increased significantly(F=0.044,0.291 and 2.577 respectively,each P <0.05) and the content of MDA decreased significantly(F=3.568,4.909 and 1.400 respectively,each P <0.05).Conclusion The optimized extraction process of flavonoids from B.papyrifera leaves was stable and reliable,which is beneficial to the reuse of remaining stock solution after processing,and the extracted flavonoids can promote the proliferation of mouse epidermal stem cells and perform antioxidant activity.
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Stem cells are undifferentiated cells capable of generating, sustaining, and replacing terminally differentiated cells and tissues. They can be isolated from embryonic as well as almost all adult tissues including skin, but are also generated through genetic reprogramming of differentiated cells. Preclinical and clinical research has recently tremendously improved stem cell therapy, being a promising treatment option for various diseases in which current medical therapies fail to cure, prevent progression or relieve symptoms. With the main goal of regeneration or sustained genetic correction of damaged tissue, advanced tissue-engineering techniques are especially applicable for many dermatological diseases including wound healing, genodermatoses (like the severe blistering disorder epidermolysis bullosa) and chronic (auto-)inflammatory diseases. This review summarizes general aspects as well as current and future perspectives of stem cell therapy in dermatology.
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Adult , Humans , Blister , Dermatology , Epidermolysis Bullosa , Induced Pluripotent Stem Cells , Regeneration , Skin , Stem Cells , Wound HealingABSTRACT
Objective To study the molecule mechanism of ethanol extract of Loropetalum chinensis (EELC) in the treatment of diabetic skin ulcer. Methods In vitro epidermal stem cell (ESCs) culture technique and dual luciferase reporter gene detection system were used to study the mechanism of EELC in the treatment of diabetic skin ulcer. Results Totally 232.8 g powder was extracted from 4.5 kg Loropetalum chinensis by 75% ethanol, and the main ingredient was polyphenol. After treating for 48 h with different doses of EELC (50 and 100 μg/mL), the expression of WNT protein, β-catenin protein and C-myc protein was all up-regulated, and was positively correlated with the dose effect (P < 0.01). The target effect of EELC on the Wnt signaling pathway was further identified by the dual luciferase reporter gene assay system. Conclusion The results suggested that EELC could activate canonical Wnt signaling pathway, promote β-catenin protein accumulation in combination with transcription factor in the nucleus to activate the expression of specific target genes, promote ESCs proliferation and differentiation, and repair skin wounds of diabetic ulcers effectively as well.
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Objective:To investigate the role of MicroRNA-9-1 in inducing epidermal stem cells(ESCs) differentiation into neurons.Methods:The lentiviral of MicroRNA-9-1 was constructed and transfected into rats epidermal stem cells.The experiment was divided into transfected group,non-transfected group and the negative control group.The β-mercaptoethanol was as an inducer for triggering the ESCs to differentiate into neurons.The GFP fluorescence expression of epidermal stem cells after transfection was observed under inverted fluorescence microscope.The protein and mRNA expression level of microtuble-associated protein 2 (MAP-2) was detected by immunocytochemical method and RT-PCR,respectively.Results:The result of Positive clone PCR confirmed successful construction of MicroRNA-9-1 in rats.Transfection after 48 h,the expressing of GFP fluorescence at peak in transfected group,and transfection efficiency reached (85.6+1.9)％.Most ESCs differentiated into neurons in transfected group after β-mercaptoethanol induction 7 h,and the effect was significantly better than non-transfected group and the negative control group.The protein ((87.3± 0.6)％) and mRNA (about twice over) expression levels of MAP-2 in transfected group was higher than those in non-transfected group and the negative control group (P＜0.05).Conclusion:The lentiviral of MicroRNA-9-1 has high transfection efficiency in rats ESCs,and could promoted ESCs differentiate into neurons under β-mercaptoethanol induced.
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Objective@#To explore the effects of hypoxia inducible factor-1α (HIF-1α) on P311 and its influence on the migration of murine epidermal stem cells (ESCs) under hypoxia in vitro.@*Methods@#Two kinds of murine ESCs were isolated and obtained from 15 neonatal wild-type C57BL/6J mice and 5 congeneric source P311 gene knock-out mice, respectively. The first passage of cells were used in the following experiments after morphologic observation and detection of expression of cell surface markers CD71 and CD49f with flow cytometer. (1) After cell scratch assay, according to the random number table (the same dividing method below), ESCs of P311 gene knock-out mice were divided into normoxia group (cells were cultured with complete medium in normoxic carbon dioxide incubator, and the subsequent normoxic treatments were the same) and hypoxia group (cells were cultured in hypoxic carbon dioxide incubator containing 1% oxygen, and the subsequent hypoxic treatments were the same), with 12 inserts in each group. ESCs of wild-type mice were divided into normoxia group, pure hypoxia group, dimethyl sulfoxide (DMSO) control group (2 μL DMSO solvent was added for 1 h of normoxia treatment before hypoxia treatment), HIF-1α inhibitor group (cells were treated with 11 μmol/L HIF-1 inhibitor of 2 μL under normoxia condition for 1 h before hypoxia treatment), HIF-1α stabilizer group (the cells were treated with 2 μmol/L FG-4592 of 2 μL under normoxia condition for 1 h before hypoxia treatment), with 12 inserts in each group. Three inserts of each time point in each group were adopted respectively to measure the residual width of scratch under inverted phase contrast microscope at post scratch hour (PSH) 0 (immediately), 12, 24, and 48. (2) After hypoxia treatment, the protein level of HIF-1α in ESCs of wild-type mice was detected by Western blotting at post hypoxia hour (PHH) 0, 12, 24, and 48. (3) ESCs of wild-type mice were divided into pure hypoxia group, DMSO control group, HIF-1α inhibitor group, and HIF-1α stabilizer group as that of experiment (1) with the same treatment. The mRNA expression of P311 and expression of P311 in ESCs were determined by real-time fluorescent quantitative reverse transcription polymerase chain reaction and immunocytochemical staining, respectively, at PHH 0 (immediately), 12, 24, and 48 (with sample numbers of 12). (4) The second passage of human embryonic kidney 293 (HEK-293) cells were divided into empty vector hypoxia group (cells were cultured under hypoxia condition after being transfected with empty vector plasmid), P311 normoxia group (cells were cultured under normoxia condition after being transfected with P311 reporter gene plasmid), P311 hypoxia group (cells were cultured under hypoxia condition after being transfected with P311 reporter gene plasmid), P311 hypoxia+ HIF-1α inhibitor group (cells which were incubated with HIF-1α inhibitor were cultured under hypoxia condition after being transfected with P311 reporter gene plasmid). The luciferase activity was detected at post culture hour (PCH) 0 and 12, respectively, and then the P311 transcriptional regulatory binding site of HIF-1α and the promoter sequence of P311 were predicted and searched by bioinformatics methods. Data were processed with factorial design variance analysis, one-way analysis of variance, LSD test and Bonferroni correction.@*Results@#(1) The results of ESCs. The cells showed cobblestone-like pattern and different clonal morphology due to the different cell proliferation potential. The proportion of CD71-CD49f+ cells accounted for about 85%. The identification results indicated that the cells showed strong stem cell properties and high purity. Compared with those in cells of normoxia group of P311 gene knock-out mice, the residual widths of scratch of cells in pure hypoxia group were smaller at PSH 12 and 24 (with P values below 0.05), and those in hypoxia group, normoxia group of wild-type mice, DMSO control group, HIF-1α inhibitor group, and HIF-1α stabilizer group were smaller at PSH 12 (with P values below 0.05). Compared with those in cells of normoxia group of wild-type mice, the residual widths of scratch of cells in hypoxia group, pure hypoxia group, and DMSO control group were smaller at PSH 12 and 24 (with P values below 0.05), and the residual width of scratch of cells in HIF-1α stabilizer group was smaller at PSH 12 (P<0.05). Compared with those of cells in pure hypoxia group, the residual widths of scratch of cells in hypoxia group were wider at PSH 12 and 24 (with P values below 0.05), and the residual width of scratch of cells in HIF-1α inhibitor group was wider at PSH 12 (P<0.05), and those of cells in HIF-1α stabilizer group were smaller at PSH 12 and 24 (with P values below 0.05). There was no obvious difference in the width of scratch in cells among the 7 groups (F=19.02, P>0.05). The protein levels of HIF-1α in ESCs of wild-type mice at PHH 0, 12, 24, and 48 were respectively 1.02±0.05, 2.56±0.09, 1.60±0.17, and 1.17±0.03. Compared with that at PHH 0, the protein level of HIF-1α at PHH 12 was significantly enhanced (P<0.01). It began to decline at PHH 24 but was still higher than that at PHH 0 (P<0.05), and the protein level of HIF-1α at PHH 48 was close to the normoxia level (P>0.05). Compared with those of cells in pure hypoxia group, the mRNA expressions of P311 of cells in HIF-1α inhibitor group were significantly decreased at each time point (with P values below 0.05), and those in HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). Compared with those of cells in HIF-1α inhibitor group, the mRNA expressions of P311 of cells in DMSO control group and HIF-1α stabilizer group were significantly increased at PHH 0, 12, and 24 (with P values below 0.05). Compared with those of cells in pure hypoxia group, the expressions of P311 of cells in HIF-1α inhibitor group were significantly decreased at each time point (with P values below 0.05), while those in HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). Compared with those of cells in HIF-1α inhibitor group, the expressions of P311 of cells in DMSO control group and HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). (2) The results of HEK-293 cells. At PCH 0, there was no significant difference in the luciferase activity among cells of empty vector hypoxia group, P311 normoxia group, P311 hypoxia group, and P311 hypoxia+ HIF-1α inhibitor group (F=13.33, P>0.05). At PCH 12, the luciferase activity of cells in P311 hypoxia group was higher than that in empty vector hypoxia group (P<0.01). The luciferase activity of cells in hypoxia group was higher than that in P311 normoxia group (P<0.05), while that of cells in P311 hypoxia+ HIF-1α inhibitor group was lower than that in P311 hypoxia group (P<0.01).@*Conclusions@#HIF-1α may increase the migration of murine ESCs through inducing the expression of P311 at the early stage of hypoxia.
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Objective@#To investigate the effects of PRX-2 gene on phenotype changes in epidermal stem cells differentiating into sweat gland cells.@*Methods@#Epidermal stem cells and sweat gland cells separated and cultured from healthy foreskin and adult full-thick skin respectively, were identified by immunofluorescence staining. Lentiviral vector-mediated overexpression and knockdown of PRX-2 gene in epidermal stem cells were performed respectively, with empty vector-mediated epidermal stem cells as a control group. Overexpression、blank control and knowdown group′s PRX-2 expressions in gene and protein levels were detected using RT-PCR and Western blot technology. The ESCs of each group were co-cultured with sweat gland cells through transwell plate, and the expressions of CEA and β1 integrin in epidermal stem cells were determined by flow cytometry before and after co-culturing.@*Results@#Epidermal stem cells and sweat gland cells were in line with their respective specific antigens. Before co-cultured, epidermal stem cells highly expressed β1 integrin (98.69±0.67)%, hardly expressed CEA (6.20±3.15)%. After co-cultured, β1 integrin expression levels were showed as knockdown group (19.30±0.53)%<blank control group (65.77±2.32)% <overexpress group (92.63±10.97)%, and CEA expression levels as knockdown (95.43±2.36)%> blank control group (51.20±0.79)%> overexpress group (45.91±0.93)%. There had significant differences between those of each two groups.@*Conclusions@#PRX-2 gene can inhibit the phenotypic change of Epidermal Stem Cells differentiating into Sweat Gland Cells and improve the ability to maintain their own specific antigens.
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Objective To observe the effects of low intensity electromagnetic fields (LIEMFs) in promoting the reconstruction of full skin loss wounds grafted with human epidermal stem cells (ESCs). Methods Fifty nude mice aged 7 to 8 weeks with full skin loss wounds were equally divided into 3 experimental subgroups ( 1 Hz, 10Hz and 50Hz) and two control groups (a cell suspension control group and a blank control group) , with ten mice each. In the 3 experimental subgroups and the cell suspension control group, ESCs separated from human foreskin and cultured in vitro were grafted to the wounds using collagen sponge scaffolds. The experimental subgroups were then stimulated with an LIEMF (magnetic field intensity 5mT) at the appropriate frequency for 30min/day for 15 days. The blank control group was put under the same conditions without the cell suspension and LIEMF. The healing rates of the wounds were observed, and tissue slices were stained and observed under a light microscope. The inner structure of the regenerating skin was observed using transmission electron microscopy. Results The ESCs were successfully grafted. A few human integrin β1 positive stained cells appeared in the regenerating skin. The average healing rates in the experimental subgroups were significantly superior to those of the control groups. Well differentiated epidermis and dermis could be seen in the regenerating skin in all of the experimental groups. The epidermis had more cell layers and was thicker than in the control groups. More desmosome, hemidesmosome and keratin filaments were seen among the epidemic cells of the experimental groups. Conclusions LIEMF promotes the healing of full skin loss wounds grafted with ESCs in nude mice, and can promote complete repair of skin defects and the regeneration of skin function.
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The bulge of hair follicles contains a mixed population of stem cells,including epidermal stem cells,neural crest stem cells,as well as another type of stem cells which can be induced into melanocytes.Bulge stem cells have the ability to divide,proliferate and are pluripotent as other adult stem cells.The neural crest stem cells can differentiate in vitro into neurons,glia and melanocytes under proper induction.Meanwhile,it was proved that implanting these cells into the gap region of damaged nerves could greatly enhance the regeneration rate of nerve and restoration of nerve function.In this review,distribution of the stated stem cells in hair follicle bulge and their characteristics of differentiating into neurons were discussed,as well as their possible application in regenerative medicine.
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Objective To observe the effects of low frequency electromagnetic fields (LFEMFs) on the proliferation of human epidermal stem cells (hESCs) cultured in a three dimensional environment so as to provide an experimental basis for applying LFEMF in skin tissue engineering.Methods hESCs from human prepuces were isolated and purified by the method of rapid adherence to collagen type ⅣV. They were grafted into a type-I collagen sponge or chitosan scaffold in vitro, and then stimulated with different frequencies of LFEMF ( 1 Hz, 10 Hz or 50 Hz) at a magnetic field intensity of 5 mT for 30 min/d. The cells' growth and proliferation were tracked using hematoxylin and eosin (HE) and diamine pheny1 indole (DAPI) staining and observed under the scanning electron microscope at different time points ( on 2nd, 7th, 10th and 14th days of LFEMF intervention). The amounts of cell proliferation at every time point were analyzed and compared.Results LFEMFs of different frequencies showed significantly different efficacy in promoting hESC proliferation. The two scaffolds also showed significantly different effects.By the 10th day, hESCs had grown significantly better on collagen sponge scaffolds than on the chitosan ones. All LFEMF frequencies could promote proliferation of hESCs, but the differences in their effects were statistically significant.Conclusion Collagen sponge may be a preferable scaffold for hESCs cultured in vitro. Rapid proliferation of ESCs in three-dimensional settings can be promoted by LFEMF intervention. LFEMF has relatively great potential in skin tissue engineering.
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Objective To investigate the changes of human epidermal stem cells after transfected with human telomerase reverse transcriptase(hTERT)gene.Methods The plasmid pIRES2-EGFP and plasmid pIRES2-EGFP-hTERT encoding hTERT were transfected into in vitro cultured human fetal epidermal stem cells by liposome-mediated transfection.Then,the positive cells were selected with G418.The mRNA and protein expressions of hTERT were detected by reserve transcriptase-polymerase chain reaction(RT-PCR)and Western blot.The telomerase activity and the proliferation and cycle of human epidermal stem cells were detected by telomeric repeat amplification protocol(TRAP)-ELISA and flow cytometry respectively.Results RT-PCR and Western blot techniques detected weak mRNA and protein expressions of hTERT gene in untransfected and vacant vector transfected cells but high level of mRNA and protein expressions of hTERT gene in pIRES2-EGFP-hTERT transfected cells.Compared with untransfected and vacant vector transfected cells,the pIRES2-EGFP-hTERT transfected cells had higher telomerase activity,with lower proportion of cells at G_0/G_1 phase,higher proportion of cells at S and G_2/M phases and enhanced proliferation ability.Conclusion Transfection with hTERT gene can markedly enhance mRNA and protein expressions,telomerase activity and proliferation ability of hTERT gene of human epidermal stem cells euhured in vitro.
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Objective: To study whether normal adult stem cells can have malignant transformation (have tumor characteristics) when exposed to tumor microenvironment. Methods: We established an in vitro model of melanoma cell-induced malignant transformation of epidermal stem cells in a cell co-culture system. The morphological changes of epidermal stem cells were observed before and after induction by phase-contrast microscope; the expression of E-cadherin and P53 mutant protein were examined by immunofluorescence method. Soft agar test was used to examine the colony forming ability of epidermal stem cells after co-culture. Results: Seven days after co-culture with A-375 cells, epidermal stem cells began to form visible colonies. The expression of E-cadherin protein was decreased and P53 mutant protein was observed in some cells. Soft agar test showed that 0. 55% epidermal stem cells formed colony in the soft agar. Conclusion: This study shows that epidermal stem cells can obtain the characteristics of tumor cells when exposed to inducement of A-375 cells, suggesting that tumor microenvironment can cause disturbance in self renewing and differentiation of stem cells.
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Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.
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Objective:To explore a method for isolation and culture of human epidermal stem cells. Methods:The Epidermal stem cell were isolated by adhering to collagen typeⅣafter obtained by digesting human foreskin with DispaseⅡand Tryp and culture in vitro in K-SFM. The expressions of?1-integrin and keratin 19 (K19) in epidermal stem cell were detected with immunocytochemical methods,and the colony forming efficiency was also studied. Keratinocytes were served also detected. Results:It was revealed by histologieal observation that colonieswere formed 24 hours after inoculation. The isolated and cultured cell cloning efficiency was higher than that of the control group. Positive expression of?1-integrin and K19 of cultured cells was detected by immunocytochemistry. Conclusion:Adult epidermal stem cells could be successfully isolated and cultured by adhension with typeⅣcollagen and culture with K-SFM
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Objective To observe the effect of Yuhonggao on proliferation and differentiation of epidermal stem cells and investigate the mechanism of promoting wound healing. Methods The whole layer skin defection rat was used and randomly divided into 3 groups:Yuhonggao group,Jingwanhong group,model group,with normal rats as control. The condition of skin wound surface and healing time were observed and recorded,and then draw the wound skin. The p63 and?1 integrin expression,the marker of epidermal stem cells,were measured and compared by immunohistochemistry staining. Results Average wound healing time hadn’t significant difference between the Yuhonggao group (13.5? 0.9)d and Jingwanhong group (12.6?0.9)d,which were better than model group (P
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Objective To explore the differentiation potency of ES cell-derived epidermal stem cells compounded by dermal analogs reconstructed with bone MSCs in hypodermis.Methods The dermal analogs were reconstructed with rat bone MSCs and compound gel-gelatin sponge.E14-ES cells,labeled with Hoechst 33342,were cocultured with human amnion.Four days later,epidermal stem cell clones were formed.The skin analogs,reconstructed with ES cell-derived epidermal stem cells and dermal analogs,were transplanted into 129 mice hypodermis.The differentiation tissue of skin analogs was sampled at 2,4,6,8 weeks.The sections were observed with HE staining,immunohistochemical and di-labeled immunofluorescence methods to test the expression of ?1 integrin,CK15,CK19,CEA,CK18.Results The sections were showed tubular or follicular like structures formed with simple or stratified epithelium at 2 and 4 weeks.Keratinized stratified squamous epithelium,sweat glands-like,sebaceous glands-like and hair follicles-like structures were observed at 6 week and 8 week after transplantation.The cells labeled by Hoechst 33342,formed tubular or follicular like structures,expressed?1 integrin,CK15,CK19,CEA and CK18positive respectively at 2 and 4 weeks.The sweat glands-like structure expressed CEA and CK18 positive respectively at 6 and 8 weeks.There were more sebaceous glands-like structures.Conclusion ES cell-derived epidermal stem cells compounded by dermal analogs reconstructed with bone MSCs can differentiate into keratinized stratified squamous epithelium,sweat glands-like,sebaceous glands-like and hair follicles-like structures in hypodermis.
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Objective:To explore side population(SP) cells in human epithelial cells and to observe the expression of the universal stem cell marker ABCG2,epidermal stem cell markers ?6 integrin and ?1 integrin on SP cells.Methods:Epithelial cells were obtained by digesting human skins with Dispase II and Trypsin and stained with Hoechst33342 and PI.The SP cells were analyzed and sorted by the fluorescence-activated cell sorter.Then the expression of ABCG2,?6 integrin and ?1 integrin was analyzed by flow cytometry.Results:SP cells accounted for 0.2%-0.3% of total human epithelial cells.Only a small part of cells expressed ABCG2,integrin ?6 and integrin ?1 of both SP cells and total human epithelial cells detected by flow cytometry.The difference of positive rates between SP cells and total human epithelial cells was not significant.Conclusion:SP cells accounted for 0.2%-0.3% of total human epithelial cells.The difference of positive rates of stem cells marker ABCG2,integrin ?6 and integrin ?1 between SP cells and total human epithelial cells was not significant.
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Objective To explore the plasticity of transdifferentiation of epidermal stem cells (ESCs) into corneal epithelial cells. Methods Epidermal stem cells enriched by adhering to type Ⅳ collage were induced in vitro by coculture with corneal stromal fibroblasts. The method of HCC were used to show the expression of K12 which is expressed specially in corneal epithelial cells.Results ESCs with greater proliferation potential strongly expressed K19 and ?_ 1 - integrin. After two-week induction, the K12 positive cells could be detected. Conclusion The data suggested that the epidermal stem cells have the potential to transdifferentiate into corneal epithelial cells in vitro.
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Objective To study the effect of neuropeptide on migration of epidermal stem cells in wound repair. Methods 90 new born rats (3～4d old) were randomly divided into three groups: SP group, capsaicin group, and normal group. BrdU labeling, combined with specific protein markers of epidermal stem cells, K19 and ?1 integrin, were used to identify epidermal stem cells. The migration of epidermal stem cells was observed in full thickness skin wound on back on 21 days after injury. Substance P was applied to the skin wound after injury in SP group. The status of migration of epidermal stem cells in SP group was compared with that in capsaicin group, in which capsaicin was injected to destroy sensory neuron before skin injury, and with normal group, in which the skin wound was not treated with any medication. Results Wounds of rats in SP group were healed 18 days after injury. It was shortened by three days compared with normal group. Only 25.54% of wound area was healed in capsaicin group on day 18. There were many epidermal stem cells in the edge of the wound and granulation tissue in Group SP. Only a small number of epidermal stem cells were seen in capsaicin group, where SP release was blocked by chemical destruction of sensory neurons, in the wound edge, but not in granulation tissue. The amount of epidermal stem cells as seen in the wound edge, but not in granulation tissue, in normal group was less than in the SP group but more than in capsaicin group. Conclusions SP obviously promotes wound healing and shortens the healing period. SP can induce epidermal stem cells to migrate into the skin wound edge and granulation tissue.
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Epidermal stem cells (ESCs) play a critical role in homeostasis and wound repair of skin tissue. Since ESCs are rare (fewer than 10% in total basal cells population) and lack specific markers,it is difficult to isolate and identify them from keratinocytes. Currently ,isolation of ESCs was achieved mainly by fast adhesion of ESCs to extracelluar matrix or flow cytometry. Several specific markers have been found in recent years for the identification of ESCs.
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AIM: To investigate the mechanism of proliferation and differentiation of human epidermal stem cells cultured in vitro under the influence of compressive stress.METHODS: Epidermal stem cells were isolated by adhering to type IV collagen and were cultured with conditioned medium,then were detected by Powervision~(TM) two-step immunohistochemical method with keratin 19 and cell cycle analysis.The cultured epidermal stem cells transplanted on silica gel membranes,which were put in a new apparatus,was designed to offer cell culture and intermittent compressive stress(4 kPa,6 kPa,8 kPa,10 kPa,12 kPa) for 2 h,3 times a day simultaneously.A week later,cells on silica gel membranes were identified with keratin 19 and 10 by Powervision~(TM) two-step immunohistochemical method.RESULTS: The new apparatus offered cell culture and intermittent compressive stress simultaneously.The isolated and cultured epidermal stem cells were identified with keratin 19 positive and 84.80 percent of them were showed in G_1 period with cell cycle analysis.Cells on silica gel membranes had been subjected intermittent compressive stress above 8 kPa for a week.The number of the cells was increased,which was more than that in control group.However,some cells identified by immunohistochemical staining with keratin 10 positive were detected among the disposed epidermal stem cells.CONCLUSION: The intermittent compressive stress above 8 kPa induces and promotes epidermal stem cells to proliferate and differentiate,indicating that epidermal stem cells respond to mechanical stress,probably is one of their major biological features.