Search

Objective To explore a method of constructing engineered sweat gland organoids in vitro by transdifferentiation of epidermal keratinocytes into sweat gland-like cells after lineage re-programming to achieve functional repair of sweat glands. Methods CRISPR/dCas9 system was used to up-regulate the expression of endogenous ectodermal dysplasia ectodysplasin (EDA) genes in human immortalized cuticle keratin HaCaTs, the positive HaCaTs were then screened and cultured. The cells were cultured according to HaCaT group (HaCaT cells cultured in sweat gland medium), HaCaT+Dox group (HaCaT cells cultured in sweat gland medium with 5 μg/ml Dox), HaCaT-E group (HaCaT-E cells cultured in sweat gland medium) and HaCaT-E+Dox group (HaCaT-E cells cultured in sweat gland medium with 5 μg/ml Dox), respectively. RT-PCR and Western blotting were used to detect the expression level of EDA in each group to verify plasmid transfection. In addition, the cells were divided as HaCaT-E group (cultured in sweat gland medium) and HaCaT-E+Dox group (cultured in sweat gland medium with 5 μg/ml Dox) to identify the expression levels of sweat gland-related markers using immunofluorescence staining to verify whether HaCaT were reprogrammed to sweat gland like cells. Furthermore, the sweat gland development microenvironment was imitatively reconstructed using Matrigel as the main extracellular scaffold, which induced sweat gland-like cells to assemble into sweat gland organoids, and the expression of sweat gland surface markers was detected by immunofluorescence staining. After transplantation of sweat gland organoids to mouse scalded paw pads, iodine-starch sweating experiment and histomorphology were performed to detect the involvement of engineered sweat gland organoids in sweat gland regeneration and skin wound repair. Results After transfection with CRISPR/dCas9 lentiviral expression system, positive HaCaT-E cells were obtained. RT-PCR showed the expression level of EDA mRNA in HaCaTE+ Dox group was up-regulated about (4.62±0.19) times than that in HaCaT group (P0.05). Western blotting showed the same trend as did by RT-PCR. After 2-7 days of inducted incubation with sweat gland culture medium containing doxycycline, the cells showed fusiform shape, lumenized networks, as well as up-regulated expression of sweat gland-related markers. After mixedly cultured in a 3-D culture system containing Matrigel for 7 days, the cells proliferated and volume increased to form sweat glands organoids with cystic cavity, and the sweat gland surface markers and functional markers cytokeratin 18 (CK18), α-smooth muscle actin (α-SMA) and aquaporin 5 (AQP5) were positively expressed. In vivo experiments showed that the iodine-starch sweating test was positive in mice, and tissue immunofluorescence staining showed that green fluorescent protein (GFP) positive cells were mainly distributed in the basal layer and subunit basal layer of the epidermis, and the tubular glandular structure was found in the deep and subcutaneous tissues of the dermis, and the GFP and sweat gland cell markers CK18 and α-SMA were observed. Conclusions Up-regulation of endogenous EDA gene by CRISPR/dCas9 system may successfully reprogram HaCaT into sweat gland-like cells. A three-dimensional culture environment constructed with Matrigel as the main extracellular scaffold can induced engineered sweat gland-like cells to self-assembly and develop into sweat gland organoids in vitro. The engineered organoids not only possessed the phenotype and structural characteristics of the sweat gland primordia, but also promoted the regeneration of sweat glands in vivo, thus achieving functional wound repair. DOI: 10.11855/j.issn.0577-7402.2020.04.07

Background Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands (SG), thus impairing the skin’s physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged skin. Methods The expression of the SG markers cytokeratin 5 (CK5), cytokeratin 10 (CK10), cytokeratin 18 (CK18), carcino-embryonic antigen (CEA), aquaporin 5 (AQP5) and α-smooth muscle actin (α-SMA) was assessed with quantitative PCR (qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells (iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of iSGCs. BALB/c nude mice were randomly divided into a normal group, SGM treatment group and iSGC transplantation group. Immunocytochemical analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of iSGCs. Results EDA overexpression drove HDF conversion into iSGCs in SG culture medium (SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18 and CEA in iSGCs, and flow cytometry data demonstrated (4.18 ± 0.04)% of iSGCs were CK5 positive and (4.36 ± 0.25)% of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealed significantly increased mRNA expression of CK5, CK18 and CEA in iSGCs, as well as activation of the duct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails, (23.05 ± 2.49)% of iSGCs expressed CK5+ and (55.79 ± 3.18)% of iSGCs expressed CK18+, respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79 ± 7.71)% vs. (70.59 ± 0.34)%, ns]. In vivo transplantation experiments showed approximately (5.2 ± 1.1)% of the mice were sweat test positive, and the histological analysis results indicated that regenerated SG structures were present in iSGCs-treated mice. Conclusion We developed a SG reprogramming strategy to generate functional iSGCs from HDFs by using the single factor EDA in combination with SGM and small molecules. The generation of iSGCs has important implications for future in situ skin regeneration with SG restoration.

A tightly controlled activity of renin-angiotensin system (RAS) including renin, angiotensin-converting enzymes (ACEs), and angiotensin II (Ang II) receptors is critical not only for maintaining systemic hemodynamics and blood volume but also for controlling cell proliferation, differentiation, and tissue remodeling in target organs. ACE inhibitors or Ang II receptor type 1 (AT1R) blockers are widely used as first line drugs for the treatment of cardiovascular diseases that are caused by chronical activation of RAS. However, about 15% of patients using ACE inhibitors develop side effects in the skin and the underlying mechanisms have been poorly understood or even neglected. Herein we show an endogenous RAS in maintaining self-renewal and regeneration potential of epidermal stem cells (ESCs) thereby contributing to wound healing. Firstly, we found that ESCs may express ACE, and its members in wound edges were positively associated with wound healing in Captopril-treated rats. Secondly, we demonstrated that human ESCs had a functional RAS including ACE1, ACE2, Ang II, AT1R, and AT2R. ACE-Ang II axis maintains human ESC function via activation of both AT1R and AT2R, which are negatively regulated by each other. Ang II-induced activation of extracellular signal-regulated kinase (ERK) and signal transducers and activators of transcription (STAT)1 and STAT3 was mediated by the negative cross-talk between AT1R and AT2R in human ESCs. These results suggest that Ang II is a critical regulator of ESC function and ESC-mediated epidermal regeneration. Inappropriate interruption of Ang II-operated signaling may prejudice ESC function leading to impaired skin wound healing or even disease.

Objective To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on macrophage polarization under hypoxia. Methods hUC-MSCs were obtained by explants adherent culture and cultured under normoxia (21% O2) and hypoxia (5% O2). The multi-directional differentiation of hUC-MSCs was observed by osteogenic and adipogenic differentiation induction. Live/death staining was performed to detect the cell viability, and ELISA was executed to detect the protein content in supernatant of hUC-MSCs. Transwell chamber was employed to co-culture the hUC-MSCs cultured under normoxia and hypoxia and macrophage (THP-1) stimulated by lipopolysaccharide (IPS), then the polarization of THP-1 was detected by immunofluorescence, and the secretions of inflammatory factor and anti-inflammatory factor of THP-1 were detected by ELISA. Results hUC-MSCs cultured under hypoxia showed the ability of osteogenic and adipogenic multi-directional differentiation. Live/death staining showed the high cell viability of hUC-MSCs cultured under normoxia and hypoxia. The expression levels of prostaglandin E2 (PGE2) and indoleamine 2,3-dioxygenase (IDO) were significantly higher in the hUC-MSCs cultured under hypoxia than in those cultured under normoxia. hUCMSCs cultured under hypoxia promoted the polarization of THP-1 to M2, obviously reduced the expression of TNF-α and IL-1β, and increased the expression of IL-10 significantly. Conclusion hUC-MSCs cultured under hypoxia may promote the polarization of THP-1 to M2 and improve the viability of anti-inflammatory. DOI: 10.11855/j.issn.0577-7402.2016.07.01

Objective To study the therapeutic effect of exosome derived from lipopolysaccharides (LPS) priming mesenchymal stem cells (MSCs) for diabetic wound healing. Methods Human umbilical cord MSCs were treated with LPS (100ng/ml) for 2 days, the supernatant were then collected, and exosomes were harvested by density gradient centrifugation and identified. Diabetic cutaneous wounds were prepared and the animals were divided into the following three groups: control group, untreated MSCs derived exosome (un-exosome) treatment group and LPS primed MSCs derived exosome (LPS-exosome) treatment group. Exosomes (60μg) were injected dispersively into the wound edge daily for 10 days. After treatment, the therapeutic results were evaluated by gross observation of the wounds, the expression levels of inflammation related factors and macrophage subtype markers in the injured sites were detected by qRT-PCR at day 3, 7 and 14 after treatment. Results Compared with control group, the diabetic wound healing was obviously improved in LPS-exosome treatment group after treatment for 7 and 14 days, with faster wound close, depressed expression of pro-inflammatory factors IL -1, IL -12 and M1 macrophage surface marker iNOS, and up-regulation of anti-inflammatory factors IL-10, TGF-βand M2 macrophage surface marker CD163, the differences were significant (P

This paper introduces the proposal of the concept and effect of actual combat on Tactical Combat Casualty Care (TCCC), analyzes the principle and key technology of TCCC, and explores the system construction and military significance of TCCC, so as to provide reference and inspiration for development of our military TCCC. TCCC has played an important role in reducing the wound and death rate in modern war, and is of great significance for the progression of theory and practice on combat casualty care in our army. DOI: 10.11855/j.issn.0577-7402.2015.12.01

OBJECTIVE: To evaluate the effect of different microstructures prepared by three-dimensional (3D) bioprinting on proliferation and viability of the murine epithelial stem cells in vitro. METHODS: 3D cell-laden microstructures were constructed using 3 different printing nozzles with diameters of 210, 340, and 420 μm. Fluorescence microscopy and the live/dead assay kit were used to observe the proliferation and viability of the murine epithelial stem cells in the microstructures. RESULTS: All the 3D cell-laden micro-structures were capable of promoting the proliferation of murine epithelial stem cells. In the 3 groups of micro-structures, the cell viability decreased significantly with time until 7 days after printing (P < 0.01), but at 14 days after the printing, the cell viability increased significantly as compared with that at 7 days (P < 0.01). The viability of the cells was significantly higher in the microstructure printed using a 420 μm nozzle than in the microstructures printed with 210 μm and 340 μm nozzles (P < 0.01). CONCLUSION: The microstructure printed with a 420 μm nozzle can stably promote the proliferation of murine epithelial stem cells and maintain a high level of cell viability, suggesting the feasibility of constructing tissue-engineered epidermis and full-thickness skin graft using 3D bioprinting technique.

Objective: The present study was conducted to investigate the effects of helium-neon (He-Ne) laser irradiation on the proliferation, migration, and differentiation of cultured human epidermal stem cells (ESCs). Background data: A He-Ne laser with a wavelength of 632.8 nm is known to have photobiological effects, and is widely used for accelerating wound healing; however, the cellular mechanisms involved have not been completely understood. Methods: The ESCs were prepared from human foreskin, and irradiated by using He-Ne laser at 632.8 nm with 2 J/cm2. The ESC proliferation, migration, and differentiation were examined by using XTT assay, scratch assay, and flow cytometry technology, respectively. The phosphorylation of extracellular signal-regulated kinases (ERK) was analyzed by using Western blotting. Results: He-Ne laser irradiation markedly promoted cell proliferation and migration accompanied by an increase in the phosphorylation of ERK, but did not significantly influence cell differentiation. Conclusion: Our data indicated that photostimulation with a He-Ne laser resulted in a significant increase in human ESC proliferation and migration in vitro, which might contribute, at least partially, to accelerated wound re-epithelialization by low-level laser therapy.

Low intensity laser irradiation (LILI) was found to promote the regeneration of skeletal musclein vivobut the cellular mechanisms are not fully understood. Myoblasts, normally quiescent and inactivated in adult skeletal muscle, are a type of myogenic progenitor cells and considered as the major candidates responsible for muscle regeneration. The aim of the present study was to study the effect of LILI on the growth potential and cell-cycle progression of the cultured myoblasts. Primary myoblasts isolated from rat hind legs were cultured in nutrient-deficient medium for 36 hours and then irradiated by helium-neon laser at a certain energy density. Immunohistochemical and flow cytometric analysis revealed that laser irradiation could increase the expression of cellular proliferation marker and the amount of cell subpopulations in the proliferative phase as compared with the nonirradiated control group. Meanwhile, the expressions of cell-cycle regulatory proteins in the laser-treated myoblasts were markedly upregulated as compared to the unirradiated cells, indicating that LILI could promote the reentry of quiescent myoblasts into the cell division cycle. These results suggest that LILI at certain fluences could promote their proliferation, thus contributing to the skeletal muscle regeneration following trauma and myopathic diseases.

The new military reform has put forward multiple challenges to the defense and medical support abilities, so the concept of war wound rescue should be constantly updated. The present paper introduces the concept and development of tactical war wounds rescue, analyze the echelons for war wound care and the characteristics and duty of each echelon in tactical war wounds rescue, summarized the management and update of the "Tactical War Wounds Rescue Guideline". The article also discusses the present situation of war wounds rescue, and the significance in the development and construction the principles of war wounds rescue. DOI: 10.11855/j.issn.0577-7402.2015.11.02

Objective To explore the therapeutic effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) on bleomycin-induced skin scar in mice and its possible mechanism. Methods BM-MSCs were isolated and purified from C57BL/6 mice by direct adherence method. Thirty male C57BL/6 mice were randomly divided into control group, skin scar model group and BM-MSCs administration group (10 each). The mice in skin scar model group and BM-MSCs administration group were subcutaneously injected with 1ml bleomycin (1mg/ml), 3 hours later, the mice in BM-MSCs administration group were subcutaneously injected with 1×106 BM-MSCs suspended in 1ml phosphate-buffered saline (PBS) and in skin scar model group with 1ml PBS, while the mice in control group were subcutaneously injected with 1ml PBS at the aforementioned two time points respectively every day. After 4 weeks, the mice were sacrificed and fresh skin specimens were harvested for preparation of frozen section, and then the expression of transforming growth factor β1 (TGF-β1) was determined by immumofluorescence method. For the rest skin tissues, 4% paraformaldehyde-fixed and paraffin-embedded sections (5μm) were obtained, and the histopathological changes were observed with HE and Masson's Trichome staining, while the expression of α-smooth muscle actin (α-SMA) was observed by immunohistochemical method. Results The skin scar significantly alleviated, the collagen thickness of dermis significantly decreased, and the expression of TGF-β1 and α-SMA reduced in BM-MSCs administration group when compared with those in skin scar model group. Conclusion The transplantation of BM-MSCs could inhibit the bleomycin-induced skin scar formation in mice, and the mechanism may be related with the regulation of TGF-β1 expression.

Objective To investigate the expression of human telomerase reverse transcriptase(hTERT) in epidermal cells and the influence on basic fibroblasts growth factor(bFGF).Methods The precursor epithelial stem cells(ESCs) were derived by in vitro bFGF-induced dedifferentiation of human keratinocytes(HEK) cells.The phenotypic changes of derived ESCs were detected by immunocytochemical staining,and the hTERT expression was detected by flow cytometry,immunofluorescence staining and TRAP-silver staining.HEK without bFGF treatment were used as control,and ESCs simultaneously isolated from human epidermis were used as positive control.Results Immunohistochemical staining revealed that the expression of β1-integrin,CK19 and CK14 in derived ESCs was significantly up-regulated,while the CK10 expression was significantly down-regulated.Flow cytometry revealed that the percentage of hTERT+ cell subsets in experimental group(derived ESCs),control group and positive control group was 98.41%,0.77% and 99.76%,respectively.TRAP-silver staining analysis indicated that the telomerase activity was significantly up-regulated in experimental group compared with that in control group,while there was no significantly difference between experimental group and positive control group.Furthermore,immunofluorescence assay revealed that there was difference in subcellular localization of hTERT between experimental group(derived ESCs) and positive control group(ESCs).Conclusion Basic fibroblast growth factor may induce epidermal cells to dedifferentiate and regain the phenotype of ESCs,which leads to the changes in hTERT expression and activity,and induces its subcellular shift of the locus.

Objective To identify the potentiality of human umbilical cord Wharton’s jelly derived mesenchymal stem cells(hUCWJ-MSCs) differentiated into sweat gland-like cells under the cultivation of sweat gland-induction medium.Methods Sweat gland cells were harvested from normal skin by digesting with collagenase typeⅡ,heat-shocked and then the supernatants of medium were collected.The sweat gland-induction medium was prepared at 10% volume fraction.hUCWJ-MSCs were harvested by enzyme digestion,and the cell phenotypes were analyzed by flow cytometry(FCM).Alkaline phosphatase(ALP) and Oil red-O staining were then performed after culturing in osteogenic and adipogenic induction medium for 2-3 weeks respectively.The hUCWJ-MSCs were cultured in sweat gland-induction medium for 3 weeks,the changes of cell morphology were observed with inverted microscope;the cells cultured for 1,2 and 3 weeks were harvested,and the expression of sweat gland markers(CEA,CK14 and CK19) were determined by immunohistochemistry and FCM,the expression of sweat gland development gene(EDA) was determined by RT-PCR.Results The normal sweat gland cells exhibited clonal growth with a flagstone appearance,while the hUCWJ-MSCs showed spindle and myofibroblast-like phenotype,and the positive rate of CD44,CD105,CD34 and CEA detected by FCM was 97.37%,96.26%,0.56% and 0.52%,respectively.After cultured in osteogenic and adipogenic induction medium for 2-3 weeks,the hUCWJ-MSCs were induced into adipocytes of Oil red-O positive staining and osteocytes of ALP positive staining,respectively.After cultured in sweat gland-induction medium for 3 weeks,sweat gland-like structures were found,and sweat gland markers CEA,CK14 and CK19 were positive in differentiated hUCWJ-MSCs when detected by immunohistochemistry,the positive rate detected by FCM was 54.37%,60.25% and 62.13%,respectively.RT-PCR analysis revealed a high level expression of gene EDA in differentiated hUCWJ-MSCs.Conclusion The hUCWJ-MSCs has a potentiality of differentiation into sweat gland-like cells.

The segmentation lesion of peripheral nerve will seriously impair the motion and sensation of the patients, and the satisfactory recovery of segmented peripheral nerve by autograft or allograft is still a great challenge posing to the neurosurgery. Apart from autograft for nerve repair, different allograft has been studying. In this study, a scaffold fabricated with polylactic acid-co-glycolic acid (PLGA) copolymer and gelatin was evaluated to be a potential artificial nerve scaffold in vitro. The effect of different mass ratio between PLGA and gelatin upon the characteristics of PLGA-gelatin scaffolds such as microstructure, mechanical property, degradation behavior in PBS, cell adhesion property were investigated. The results showed the homogeneity and mechanical property of the scaffolds became poor with the increase of gelatin, and the rate of max water-uptake and the mass loss of scaffolds increases with the increase of gelatin, and the cells could adhere to the scaffolds. Those indicated the scaffolds fabricated by the PLGA-gelatin complex had excellent biocompatibility, suitable mechanical property and sustained-release characteristics, which would meet the requirements for artificial nerve scaffold.

There has been no large-scale naval combat in the last 30 years. With the rapid development of battleships, weapons manufacturing and electronic technology, naval combat will present some new characteristics. Additionally, naval combat is facing unprecedented challenges. In this paper, we discuss the topic of medical rescue at sea: what challenges we face and what we could do. The contents discussed in this paper contain battlefield self-aid buddy care, clinical skills, organized health services, medical training and future medical research programs. We also discuss the characteristics of modern naval combat, medical rescue challenges, medical treatment highlights and future developments of medical rescue at sea.

AIM:To investigate the effects of EGF on the characteristics of phosphrylated p44/42 MAPK expression and its biological significance in EGF-induced gut repair after ischemia-reperfusion (I/R) injury. METHODS:A total of 80 Wistar rats were randomly divided into four groups, namely EGF treated group (E), normal saline control (R), ischemia group (I) and sham operated control (C). In group E and R, the rats were treated with intravenous EGF 100 µg/kg/rat or normal saline respectively after 45 minutes of superior mesenteric artery occlusion. Blood samples were collected at 2, 6, 12 and 24 hours after reperfusion and plasma D-lactate were determined. Tissue samples from intestine were also taken for histological analysis and immunohistochemical analysis of phospho-p44/42 MAPK. RESULTS:The changes of histological structure and D-lactate indicated that the intestinal barrier was damaged after intestinal I/R injury, while EGF treatment significantly improved the outcome. In group C and I positive signals of phospho-p44/ 42 MAPK were mainly located in the cytoplasm of the intestinal villi and crypts, while in group I positive cells increased significantly (P

The repair and regeneration of tissue is a well-discussed topic. Over the past 20 years, with the development of genetics, auxology, stem cell biology, and tissue engineering, tissue repair and regeneration have rapidly developed as emerging "Regenerative Medicine". Regenerative medicine has significant market demand in China. Based on national statistics, injury and poisoning patients rank third in afflictions in city hospitals (accounting for 9.13%) and rank second in afflictions in county hospitals (accounting for 14.07%). Totally, approximately one hundred million patients suffered from traumatic, genetic and metabolic diseases in China and demand reparative and regenerative medical treatment each year. The Chinese government and its related departments have always attached great importance and support to the development of regenerative medicine, and the Chinese academic circle is involved in a very wide range of diseases and injuries including regenerative medical theory and technology. Stem cell biology, organ engineering and duplication, tissue engineering research and production have developed rapidly, and great portion of these studies have started to appear in applications, which have aroused extensive concerns in international professional circle. In the next 10 years, the Chinese regenerative medical system will be further improved, in both statute and rules, clinical translation will be further accelerated. Breakthroughs are expected in induced differentiation of stem cells and synchronous repair and regeneration of multiple organs, construction of major organs by tissue engineering, large-scale applications of tissue engineering products, and other aspects.

Patients with severe full-thickness burn injury suffer from their inability to maintain body temperature through perspiration because the complete destructed sweat glands can not be regenerated. Bone marrow-derived mesenchymal stem cells (BM-MSCs) represent an ideal stem-cell source for cell therapy because of their easy purification and multipotency. In this study, we attempted to induce human BM-MSCs to differentiate into sweat gland cells for sweat gland regeneration through ectodysplasin (EDA) gene transfection.The dynamic expression of EDA and EDA receptor (EDAR) were firstly observed in the sweat gland formation during embryological development. After transfection with EDA expression vector, human BM-MSCs were transplanted into the injured areas of burn animal models. The regeneration of sweat glands was identified by perspiration test and immunohistochemical analysis.Endogenous expression of EDA and EDAR correlated with sweat gland development in human fetal skin. After EDA transfection, BM-MSC acquired a sweat-gland-cell phenotype, evidenced by their expression of sweat gland markers by flow cytometry analysis. Immunohistochemical staining revealed a markedly contribution of EDA-transfected BM-MSCs to the regeneration of sweat glands in the scalded paws. Positive rate for perspiration test for the paws treated with EDA-transfected BM-MSCs was significantly higher than those treated with BM-MSCs or EDA expression vector (P0.05).Our results confirmed the important role of EDA in the development of sweat gland. BM-MSCs transfected with EDA significantly improved the sweat-gland regeneration. This study suggests the potential application of EDA-modified MSCs for the repair and regeneration of injured skin and its appendages.

To investigate the effect of ecdysterone on the proliferation of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro.hUCMSCs isolated by enzyme digestion from human umbilical cord tissues were cultured and identified for the surface antigens using fluorescence-activated cell sorting (FACS). The cells were treated with ecdysterone at the concentrations of 0, 25, 50, 100, 150, and 200 µg/ml, and the changes in the cell proliferation were detected using MTT assay.The third-passage hUCMSCs were positive for CD29 and CD105 and negative for CD34 and CD45 as shown by flow cytometry. Treatment with ecdysterone resulted in significantly increased cell proliferation as compared to the control cells (P0.05), but no significant differences were found in cells treated with 100, 150, and 200 µg/ml ecdysterone (P0.05). The growth curves of the cells also demonstrated the definite effect of ecdysterone in promoting the proliferation of hUCMSCs.Ecdysterone can promote the proliferation of hUCMSCs in vitro with the optimal concentration of 100 µg/ml, suggesting its potential value in the enrichment of mesenchymal stem cells.

TTo explore the method for inducing the dedifferentiation of epidermal cells into their progenitor stem cells in vitro without external gene intervention.HEK cells obtained from Casacade were induced to reverse their differentiated process and produce immature stem-like cells, namely the dedifferentiation derived epidermal stem cells (dESCs), by induction with basic fibroblasts growth factors (bFGF) in vitro. Immunochemical staining, flow FACS analysis, RT-PCR and immunofluorescent staining were used to detect the phenotypic and functional changes of the differentiated epidermal cells, using human epidermal stem cells (ESCs) as the positive control.Immunohistochemical staining revealed that the expressions of β₁-integrin, CK19 and CK14 were up-regulated, while CK10 expression was down-regulated significantly after bFGF treatment. Two-color flow cytometric analysis of α₆-integrin and CD71 showed that the percentages of α₆(+)CD71(-), α₆(+)CD71(+) and CD71(+) expressing populations reached 13.24%, 58.26% and 23.12% of the total isolated cells, as compared with those of the control (0.12%, 3.06%, 51.50%) and positive control cells (37.49%, 45.13%, 5.86%). RT-PCR analysis indicated that the relative gene expressions of β₁-integrin, CK19 and CK14 increased in bFGF treatment group, whereas the expression of CK10 was significantly suppressed. Although there was no significant difference in the expression levels of β₁ integrin, CK19 and CK10 between the bFGF-treated and the positive controls, the expression of CK14 in bFGF-treated cells showed a 1.4-fold increase as compared with that in ESCs (P0.05). Immunofluorescent staining showed that a regional difference in the subcellular localization of telomerase between dESCs and ESCs.bFGF can induce the epidermal cells to convert into epidermal precursor cells. Although they are more likely to be transient amplifying cells, the method for reprogramming somatic epidermal cells into their progenitors by bFGF induction other than genetic manipulation offers a new approach to generate residual healthy stem cells for wound repair and regeneration.

To investigate the effect of ecdysterone (EDS) on the proliferation of human bone marrow mesenchymal stem cells (hMSCs) in vitro.hMSCs were isolated from human bone marrow cell suspension by density gradient centrifugation. The expression of integrins CD44, CD105, CD34 and CD29 were examined by immunocytochemical method. EDS at 10, 25, 50 or 100 microg/ml were added in hMSC culture system, using the routine culture medium for hMSCs as control. The cell viability were analyzed by MTT assay and the cell cycle changes were examined by flow cytometry.The optical density (OD) differed significant between the EDS treatment groups and the control group (P0.01), and 25 microg/ml EDS group showed the highest OD value (P0.01) without significant differences among 10, 50 and 100 microg/ml EDS groups (P0.05). Flow cytometry showed that treatment of the cells with 25 microg/ml EDS significantly increased the cell percentages in S and G(2)M phases and the proliferation index (PI) of the cells as compared with the control group.Within a given concentration range, EDS can promote the proliferation of hMSCs in vitro, and this effect can be the most obvious at the concentration of 25 microg/ml. The effect of EDS in promoting the proliferation of hMSCs does not positively correlate to EDS concentration administered.

Stem cells are defined by their capacity of self-renewal and multilineage differentiation, which make them uniquely situated to treat a broad spectrum of human diseases. Based on a series of remarkable studies in several fields of regenerative medicine, their application is not too far from the clinical practice. Full-thickness burns and severe traumas can injure skin and its appendages, which protect animals from water loss, temperature change, radiation, trauma and infection. In adults, the normal outcome of repair of massive full-thickness burns is fibrosis and scarring without any appendages, such as hair follicles, sweat and sebaceous glands. Perfect skin regeneration has been considered impossible due to the limited regenerative capacity of epider-mal keratinocytes, which are generally thought to be the key source of the epidermis and skin appendages. Currently, researches on stem cells, such as epidermal stem cells, der-mal stem cells, mesenchymal stem cells from bone marrow, and embryonic stem cells, bring promise to functional repair of skin after severe burn injury, namely, complete regeneration of skin and its appendages. In this study, we present an overview of the most recent advances in skin repair and regeneration by using stem cells.

To investigate the role of the signal routes P38, ERK, and Rho in the differentiation of bone marrow mesenchymal stem cells (MSCs) into epidermoid cells.(1) MSCs were separated from the bone marrow of Wistar rats by Ficoll-Pague lymphocyte separating medium and proliferated in culture medium. Then the MSCs were immunocytochemically stained to detect the expression of surface antigens. (2) The MSCs were randomly divided into 3 groups: control group; pure induction induced group, cultured with epithelial growth factor (EGF) added into the culture fluid, and Rho inhibition group, cultured with EGF and HA1077, a ROK inhibitor, added into the culture fluid. One, 3, 5, and 7 days later FC was used to detect the levels of phosphorylated P38 and ERK. (3) MSCs were randomly divided into 4 groups: control group, cultured with low-sugar DMEM complete culture fluid; pure induction group, cultured with supernatant of rat fibroblasts and EGF added into the culture fluid, p38 blocking group, with SB203580, inhibitor of P38 added into the culture fluid; and ERK blocking group, with PD98059, inhibitor of ERK added into the culture fluid. Seven days later, SP method was used to detect the expression of CK5/8 and CK19 induced by MSCs. (4) MSCs were randomly divided into 4 groups: control group; pure induction group, with supernatant of rat fibroblasts and EGF added into the culture fluid; and RHO blocking group, with HA1007 added into the culture fluid. Seven days later, FC was used to detect the expression of CK5/8 and CK19.(1) Both FC and immunocytochemistry showed that the MSCs were uniformly positive in CD29 and CD44, but did not express CD34 and CD45. (2) The phosphorylated P38 rate remained 0.01% in the control group. The phosphorylated P38 rate was 0.04%, significantly higher than that of the control group (0.01%, P0.05) at day 5, and then lowered to 0.01% at day 5 in the pure induction group; and became 6.17%, 4.13%, 3.97%, and 0.41% respectively at day 1, 3, 5, and 7, all significantly higher than those of the control group (all P0.05), in the Rho inhibition group. The phosphorylated ERK level was 4.23% in the control group; became 0.39% and 0.40% at day 3 and day 5 (both P0.05), and then returned to 5.10% at day 7 in the pure induction group; and was not significantly changed at days 1, 3, and 5, and then became 0.41%, significantly lower than that of the control group (P0.05), in the Rho blocking group, (3) The control group was CK5/8 and CK19 negative. The CK5/8 and CK19 rates at day 7 of the pure induction group were 3.01% and 6.47% respectively, both significantly higher than those of the p38 inhibition group (1.43% and 5.41% respectively, both P0.05). The CK5/8 and CK19 expression rates of the ERK inhibition group were 5.54% and 7.56% respectively, both significantly higher than those of the pure induction group (both P0.05), (4) The CK5/8 and CK19 expression rates of the HA1077 group were 21.65% and 39.41% pure, both significantly higher than those of the pure induction group (1.81% and 10.19% respectively, both P0.05).p38 route may play an active role in the differentiation of MSCs into epidermoid cells. Blocking of the upstream signal Rho may enhance the activation of p38 route and then promote the differentiation of MSCs into epidermoid cells.

To study the differences of gene expression between earlier gestational skin and later gestational skin of rats with the aids of single primer amplification (SPA) and high-density oligonucleotide DNA array to understand the molecular mechanism of scarless healing.Total RNAs were isolated from fetal rat skin of the scarless (E15) and scar-forming (E18) periods of gestation (term = 21.5 days). The RNAs from earlier gestational skin (EGS) and later gestational skin (LGS) were both reversely transcribed to cDNAs, then labeled with the incorporation of fluorescent dCTP for preparing the hybridization probes by SPA method. The mixed probes were then hybridized to the oligonucleotide DNA arrays which contained 5,705 probes representing 5,705 rat genes. After highly stringent washing, these DNA arrays were scanned for fluorescent signals to display the differentially expressed genes between the 2 groups of skin.Among 5,705 rat genes, there were 53 genes (0.93 percent) with differentially expressed levels between EGS and LGS groups, 27 genes, including fibroblast growth factor 2 (FGF2) and follistatin were up-regulated (0.47%) and 26 genes were down-regulated (0.46%) in fetal skin during scarless period versus scar-forming period. Higher expressions of FGF2 and follistatin in EGS than those in LGS were also revealed by RT-PCR method.High-density oligonucleotide DNA array provided a powerful tool for investigating differential gene expression in earlier and later gestational fetal skins. This technology validates that the mechanism of fetal scarless healing is very complicate and the change of many gene expressions is associated with fetal scarless healing.

To investigate the expression characteristics of basic fibroblast growth factor (bFGF) and its receptors, flg (FGFR1) and bek (FGFR2), in fetal skin at different gestational ages underlying the relevance of these 3 proteins to skin development and the mechanisms underlying the phenotypic transition from scarless to scar-forming healing.Eighteen specimens of fetal skin biopsies of human embryo were obtained from spontaneous abortions at different gestational ages of 13-32 weeks. Gene expression of bFGF, bek and flg was examined with reverse transcription-polymerase chain reaction (RT-PCR). The dynamic expression and distribution of these 3 proteins were detected with streptavidin peroxidase (SP) immunohistochemical staining method.In the early gestational fetal skin, genes of bFGF and flg were strongly expressed and more protein contents of these 2 proteins were found as compared with the genes at late gestation fetal skin (2.446+/-0.116 and 2.066+/-0.152 versus 2.157+/-0.101 and 1.818+/-0.086, respectively, P0.05). On the contrary, the levels of gene expression and protein content of bek were not differently expressed in the early gestational fetal skin versus the late ones. Protein particles of bFGF were mainly distributed in the epidermal cells and some fibroblasts. Bek was mainly located in the cell membrane and cytoplasm of epidermal cells while flg protein was principally located in the epidermal cells, endothelial cells and some fibroblasts.The endogenous bFGF and their receptors might be involved in the cutaneous development at fetal stage. The differently expressing levels of bFGF and flg during gestation may be related to scarless or scar-forming repair during gestation.

To investigate the cellular phenotype conversion during human mesenchymal stem cells (hMSCs) cocultured with injured human sweat gland cells (hSGCs) in vitro.HMSCs and hSGCs were isolated and cultured and expanded respectively. The antigens expression of hMSCs and hSGCs were detected by two-steps immunocytochemistry. HMSCs were labeled with BrdU. The hSGCs were heat-shocked at 47 degrees C for 40 min when they reached 70% confluency, then cooled for 1-2 h at 37 degrees C and (1 - 2) x 10(5) BrdU-labeled hMSCs were added before incubation for up to 2 weeks. The cocultures were observed by phase contrast microscopy and detected by double-staining immunocytochemistry using CEA and BrdU as primary antibodies.The cultured hMSCs and hSGCs were clonogenic growth. HMSCs were positive for anti-CD44 and anti-CD105 staining and negative for anti-CD34 and anti-CEA staining. HSGCs express CK7, CK18, CK19 and CEA. The positive rate of BrdU labeled-hMSCs was 90%. The majority of hSGCs lost cell-cell contact after heat-shock. 2 weeks after cocultured, some cocultured cells were positive for both anti-CEA and anti-BrdU staining and some cocultures had more than two nuclei which stained with two different colors by double-staining immunocytochemistry. Statistic results showed 1%-5% of the hMSCs added to the coculture system were recovered as double-staining cells expressing BrdU and CEA while only 0.01%-0.05% cells stained with two different colors in nuclei. The multi-nucleated cells were wide and flatten.HMSCs could differentiate into hSGCs in vitro under injured microenvironment. The mechanisms of which may be that hMSCs differentiate into hSGCs directly or by cell fusion, even nucleus fusion.

In hypertrophic scar tissue, no sweet gland and hair follicle exist usually because of the dermal and epidermal damage in extensive thermal skin injury, thus imparing regulation of body temperature. This study was designed to reveal the morphological and distributional characteristics of the sweat glands in normal skin and hypertrophic scar obtained from children and adults, and to study the possible interfering effects of the scar on regeneration of the sweat gland after burn injury.Biopsies of hypertrophic scar were taken from four children (4 - 10 years) and four adults (35 - 51 years). Normal, uninjured full-thickness skin adjacent to the scar of each patient was used as control. Keratin 19 (K19) was used as the marker for epidermal stem cells and secretory portion of the sweat glands, and keratin 14 (K14) for the tube portion, respectively. Immunohistochemical and histological evaluations were performed.Histological and immunohistochemical staining of skin tissue sections from both the children and adults showed K19 positive cells in the basement membrane of epidermis of normal skin. These cells were seen only single layer and arranged regularly. The secretory or duct portion of the eccrine sweat glands was situated in the dermis and epidermal layer. However, in the scar tissue, K19 positive cells were scant in the basal layer, and the anatomic location of the secretory portion of sweat glands changed. They were located between the border of the scar and reticular layer of the dermis. These secretory portions of sweat glands were expanded and were organized irregularly. But a few K14 positive cells were scattered in the scar tissues in cyclic form.There are some residual sweat glands in scar tissues, in which the regeneration process of active sweat glands is present. Possibly the sweat glands could regenerate from adult epidermal stem cells or residual sweat glands in the wound bed after burn injury.

To explore the effects of bone marrow mesenchymal stem cells (MSCs) on the quality of healing of porcine skin wounds after burn injury so as to provide a new method for clinical skin repair in the future.Seventy-two deep-partial thickness burn wounds were produced on the back of 6 minipigs and then the pigs were randomly divided into 6 groups: saline control, MSCs treatment, MSCs plus basic fibroblast growth factor (bFGF) treatment, MSCs plus epidermal growth factor (EGF) treatment, bFGF treatment or EGF treatment only. MSCs were isolated from porcine marrow and cultured in vitro. After labeling with BrdU, MSCs were autografted onto the skin wounds. 7, 14, 21 and 42 days after the injury, the area of the wounds were measured and the histological examination was performed to evaluate the velocity and quality of wound healing.1, 2 and 4 weeks after transplantation, immunohistochemical examinations were carried out to detect the positive staining of BrdU, cytokeratin and S-100 to evaluate the wound healing quality.The area of wounds was decreased at day 7 and most of these wounds were healed on day 21 after injury. Although there was no significant statistical difference in the contraction rate among these six groups, however, the reduced wound areas in the MSCs plus bFGF treatment group was 15% to 20% more than that in the other 5 groups. Histological examination and semi-quantitative analysis demonstrated that the number of vessels and the expression density of S-100 in the MSCs plus bFGF treatment wounds were significantly enhanced than that in the other groups.MSCs autografting may benefit to enhance the wound healing quality in porcine skin, which may open a new way to reach a "perfect repair" after skin injury.

Keloid is an intricate lesion that is probably regulated by many genes. In this study, the authors used the technique of complementary DNA (cDNA) microarray to analyse abnormal gene expression in keloids and normal control skins.The polymerase chain reaction (PCR) products of 8400 genes were spotted in an array on chemical-material-coated-glass plates. The DNAs were fixed on the glass plates. The total RNAs were isolated from freshly excised human keloid and normal control skins, and the mRNAs were then purified. The mRNA from both keloid and normal control skins were reversely transcribed to cDNAs, with the incorporation of fluorescent dUTP, for preparing the hybridisation probes. The mixed probes were then hybridised to the cDNA microarray. After thorough washing, the cDNA microarray was scanned for differing fluorescent signals from two types of tissues. Gene expression of tissue growth factor-beta1 (TGF-beta1) and of c-myc was detected with both RT-PCR and Northern blot hybridisation to confirm the effectiveness of cDNA microarray.Among the 8400 human genes, 402 were detected with different expression levels between keloid and normal control skins. Two hundred and fifty genes, including TGF-beta1 and c-myc, were up-regulated and 152 genes were down-regulated. Higher expressions of TGF-beta1 and c-myc in keloid were also revealed using RT-PCR and Northern blot methods.cDNA microarray analysis provides a powerful tool for investigating differential gene expression in keloid and normal control skins. Keloid is a complicated lesion with many genes involved.