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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.

Inappropriate treatment at early stage of wound could result in the formation of pseudoepitheliomatous granuloma (PEG). The correlation of abnormal transdifferentiation of epithelial cells to immunologic cells and the occurrence of PEG lesion was investigated.Morphological change of epithelial tissue was observed with histopathology in 11 specimens of PEG lesions and 6 specimens of normal skins from PEG edge (PEG-N) from 11 patients with damaged skin. The expression characteristics and distribution of pan-cytokeratin (CKp), IV type collagen, laminin (LM), epithelial cadherin (E-Cad), beta-catenin (beta-Cat), focal adhesion kinase (FAK), stem cell factor (SCF) and its receptor-c-Kit, proliferating cell nuclear antigen(PCNA), and cluster of differentiation-14 (CD14), CD68 and mast cell tryptase (MCT) in PEG were detected with the immunohistochemical and the indirect immunofluorescent double-staining.In comparison with PEG-N, epithelial tissue take on squamous metaplasia, and stroma was infiltrated with intensive microvessels and inflammatory cells in the PEG lesion. Poor epithelial basal layer constitution, basal polarization, and migration of basal cells to stroma could be observed. In the ultrastructure, the loose intercellular junction of basal cells and the increased nucleus/cytoplasm ratio and intercellular space could be observed, neonatal monocytoid cells and macrophages and mast cells as a exuviate-like manner brooded from cytoplasm of original epithelial cells and basement membrane. protein expression of CKp and E-Cad by basal cells was significantly decreased, and the IV type collagen and LM protein could not be found in basement membrane of identical locus. By contrast, the immunoreactivity of beta-Cat and FAK was apparently increased. In addition, CD14(+) monocytes, CD68(+) macrophages, MCT(+) mast cells and CD68(+)/MCT(+) cells with various size, and these cells of stronger immuno-staining of SCF, c-Kit and PCNA antigen could be found in epithelial tissue and stroma.Epithelial cells in PEG related to wound are characteristized by transdifferentiation of epithelial cells to immunologic cells, wich may be associated with local infectious and inflammatory reaction, ultimately resulting in enhancement the ratio of beta-Cat/E-Cad signal and activation SCF-c-Kit signal pathway. The phenomena of transdifferentiation epithelial cells in the PEG lesion will help to recognize of the neoplatic immune and trauma repair mechanism.

To investigate gene expression of transforming growth factor-beta(1) (TGF-beta(1)) and its two upstream signalling factors (smad2 and smad3) in fetal skin at different gestational ages and postnatal skin and its potential biological significance.Fetal skin samples of human embryo were obtained from spontaneous abortions at different gestational ages ranging from 13 to 32 weeks, and also skin collected from patients undergoing plastic surgery. After morphological characteristics of skin at different developmental stages were examined histologically, gene expressions of TGF-beta(1), smad2 and smad3 in skin specimens at different developmental stages were examined with reverse transcription-polymerase chain reaction analysis (RT-PCR).Gene expression of TGF-beta(1), smad2 and smad3 could all be detected in fetal skin and skin after birth. In skin from early gestational fetus, gene expressions of TGF-beta(1) and smad2 were weak. Along with advance in gestational age, gene expression of these two genes in skin became progressively stronger. In skin from late gestational fetus and skin after birth, the transcription contents of these two genes were significantly increased compared with early gestation fetus (P0.05). On the contrary, gene expression of smad3 was apparently higher in younger fetal skin versus elder compared with that of late fetal skin (P0.05). In skins after birth, the levels of smad3 gene expression were elevated to the level similar to that in early gestational fetal skin.The signal pathway mediated by TGF-beta(1) might be involved in regulating development of the skin at embryonic stage and in designating cetaceous structure and function, and also in wound healing after birth. The relative lack in expression of TGF-beta(1) and smad2 genes in skins from younger fetuses might contribute to fetal scar-less healing, in which the role of smad3 needs to be further investigated.

To explore the change of gene expression of stress activated protein kinase (SAPK) and its upstream signal-regulated molecule--mitogen activated protein kinases(MAPKs) (MKK4 and MKK7) in hypertrophic scar and auto-control normal skin.The total RNA was isolated from 8 hypertrophic scars and 8 auto-control skin, and then mRNA was purified. The gene expressions of MKK4, MKK7 and SAPK were examined with reverse transcription-polymerase chain reaction(RT-PCR) method.In hypertrophic scar, both MKK7 and SAPK genes weakly expressed. In auto-control skin, the expression of these 2 genes was significantly elevated in comparison with hypertrophic scar (P0.01). The expression levels of these 2 genes were 1.5 times and 2.6 times as long as those of hypertrophic scar, respectively. Gene expression of MKK4 had no significant difference between auto-control skin and hypertrophic scar (P0.05).Decreased gene expression of MKK7 and SAPK which results in reducing cell apoptosis might be one of the mechanisms for controlling the formation of hypertrophic scar.

To observe the changes of matrix metalloproteinase-1,2/tissue inhibitor of metalloproteinase-1,2 (MMP-1,2 and TIMP-1,2) in granulation tissue after 30% TBSA deeper partial thickness scald, and explore the regulation mechanism of MMP-2/TIMP-2 during wound healing.150 male Wistar rats were randomly divided into 5 groups as follows: (1) normal control (n = 6); (2) injured control group (n = 36): which is subdivided into postburn 3 h, 6 h, 1 d, 3 d, 7 d and 14 d groups, respectively; (3) BDM group (n = 36): intravenous injected of 400 mg 2,3-butanedione monoxime in each rat was done after anesthesia; (4) H7 group (n = 36): Each rat was intravenous injected of 0.2 mg 1-5-isoquinolinyl-sulfony-2-methylpiperazine after anesthesia; (5) anti-c-fos group (n = 36): Each rat was intravenous injected of 5 microg c-fos monoclony antibody after anesthesia. The immunohistochemistry staining technique and the reverse transcription polymerase chain reaction (RT-PCR) were used for detecting.The expression of c-fos mRNA and protein was increased from 3 to 6 hours post-burn, and then decreased. The expression of MMP-1,2/TIMP-1,2 was delayed to 3 days post-burn compared with the expression of c-fos mRNA and protein. Treatment with BDM induced to raise c-fos mRNA and protein expression. The expression of MMP-1,2/TIMP-1,2 was also increased accordingly. However, following treatment with H7 inhibited the expression of c-fos mRNA and protein, MMP-1,2/TIMP-1,2 proteins expression decreased. Exogenous c-fos antibody could inhibit endogenous c-fos protein expression and the expression of MMP-1/TIMP-1,2 decreased, but MMP-2 has no notable changes.The expression of MMP-1,2 and TIMP-1,2 has closely relation protein kinases activated signaling pathways. The expression changes of MMP-1 and TIMP-1/TIMP-2 depend on c-fos expression. Oncogenes play an important role in the change process of wound matrix degradation and remodeling.

To explore the change in gene expression of angiogenesis-related factors vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), acid fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF), in fetal skin at different developmental stages and children skin and their potential biological significances.Fetal skin samples of human embryo were obtained from spontaneous abortion at different gestational ages ranging from 13 to 32 weeks, and children skin specimens were collected from child patients (4-12 years) undergoing plastic surgery. After morphological characteristics of skin at different developmental stages were defined histologically gene expressions of VEGF, Ang-1, aFGF and bFGF were examined with reverse transcription-polymerase chain reaction analysis (RT-PCR).The trend of changes in gene expression of VEGF, Ang-1, aFGF and bFGF was not same for different skin specimens at various developmental stages. In early gestational fetal skin, genes of VEGF and Ang-1 were strongly expressed, while in late gestational and childhood skins, gene expressions of VEGF and Ang-1 were apparently decreased. In skin of middle gestational stage, the level of aFGF gene expression was highest, and then it was progressively reduced. In childhood skin, this gene was weakly expressed. In marked contrast, the contents of transcripts of bFGF showed no substantial change in fetal skin at different developmental stages, whilst the mRNA content of bFGF was significantly decreased in childhood skin.VEGF, Ang-1, aFGF and bFGF might be involved in regulating angiogenesis in skin from fetuses of different gestational stages and children. The relative increase in gene transcription of VEGF and Ang-1 in younger fetal skin might be one of the reasons why cutaneous cells proliferate rapidly and the wounds heal without scar.

To investigate the change in hepatic and renal functions parameters after intestinal ischemia-reperfusion (I/R) injury, and to explore the effects of acidic fibroblast growth factor (aFGF) on hepatic and renal functions after intestinal I/R injury in rats.Seventy-eight Wistar rats were divided into four groups, which are sham-operated (C) group, ischemia (45 minutes) plus reperfusion (R), reconstructive human aFGF treatment (rhF), and wild type aFGF treatment (wtF) groups. The animals were sacrificed at 2, 6, 12 and 24 hours, respectively. Hepatic and renal functions were analyzed.In comparison with those in group C, the hepatic and renal functions were damaged in group R, rhF and wtF decreased. Treatment with rhF and wtF markedly abated the hepatic and renal dysfunction. The desquamation of intestine villi and infiltration of inflammation cells in the submucosa were observed in all groups.Hepatic and renal functions are damaged after intestinal I/R injury. Treatment with rhF and wtF could protect against multiple organ dysfunction associated with intestinal ischemia-reperfusiun injury.

To evaluate the effect of fibroblast growth factor-10 (FGF-10) on the secretion of transforming growth factor-alpha (TGF-alpha), platelet-derived growth factor-AB (PDGF-AB) and vascular endothelial growth factor (VEGF) by human keratinocytes.Concentrations of FGF-10 used were 4, 16, 125 and 500 ng/ml. Serum-free keratinocyte growth medium without EGF or with EGF were as negative control and positive control, respectively. Cells were cultured at 2500, 37500 cells/cm 2 in dishes in serum-free medium and supernatants were collected at 24, 48 and 72 hours. The concentrations of TGF-alpha PDGF-AB and VEGF in cell culture supernatant were measured by using TGF-alpha, PDGF-AB and VEGF enzyme linked immunoadsorbent assay (ELISA) kits, respectively and cell numbers were counted by haemocytometer.For cells cultured at low density and cells were subconfluent, TGF-alpha in each group was low and there was no significant difference among them at 24 hours. At 48 hours, both in absolute concentrations and on a per-cell basis, FGF-10 enhanced the secretion of TGF-alpha by cells cultured at low density in a dose-dependent fashion. At 72 hours, the concentrations of TGF-alpha in all dose of FGF-10 were significantly higher than that in negative control (P0.05). The production of TGF-alpha in pg/10(6) cells in 500 ng/ml FGF-10 reached statistical significance compared with negative control (P0.05). The secretion of TGF-alpha in FGF-10 were lower than that in positive control at 48 and 72 hours. For cells cuttured at 37500 cells/cm(2), the secretion of TGF-alpha stimulated by FGF-10 was similar to that when cells cuttured at lower density. PDGF-AB in each group was undetectable. When cells cuttured at high density and reached 100 percent confluent, PDGF-AB could not be detected at 24 hours. At 48 hours, both in absolute concentrations and on a per-cell basis, PDGF-AB productions in 125 and 500 ng/ml of FGF-10 were markedly greater than those of negative control at 48 and 72 hours (P0.05), and the secretion of PDGF-AB stimulated by FGF-10 was in a dose-dependent fashion. For cells cultured at low density, although the concentrations of VEGF in 16-500 ng/ml of FGF-10 were significantly higher than that in negative control (P0.05) at 72 hours, on a per-cell basis, they were not greater than that of negative control. The secretion of VEGF in positive control was greater than that of FGF-10 at 24-72 hours.The effect of FGF-10 on increasing secretion of TGF-alpha and PDGF-AB might mediate the actions of FGF-10 on keratinocytes, fibroblasts and endothelial cells to promote re-epithelialization as well as granulation tissue deposition during wound healing.

To explore the effects of acute hypoxia and intermittent hypoxic acclimatization on gene expression of erythropoietin (EPO) and hypoxia-inducible factor-1alpha (HIF-1alpha) in rat hepatic and renal tissues.Twenty-four rats (weight was 180 to 220 g) were divided into three groups: normal control group (NC), intermittent hypoxic acclimatization (IH) and acute hypoxia groups (AH). Gene expression of EPO and HIF-1alpha were examined with Northern dot blot method.As compared with NC group, the levels of EPO gene expression in rat hepatic and renal tissues in AH group were both significantly elevated (P0.05 or P0.01). In IH group, the contents of EPO mRNA in hepatic and renal tissues were apparently decreased versus AH group (P0.01), whilst were not differently elevated in comparison with group NC (P0.05). In hepatic tissue from AH group, the content of HIF-1alpha mRNA were more than those in NC group and IH group, while the levels of gene expression of HIF-1alpha were no substantial change in renal tissues from different groups (all P0.05).Hypoxic acclimatization can inhibit the increment of EPO gene expression induced by acute hypoxia in rat hepatic and renal tissues, in which HIF-1alpha may play important roles.

To investigate the relationship between the formation of pseudoepitheliomatous hyperplasia (PEH) and the mechanism of loss of some components from the basement membrane in epithelial interstitial junction (EIJ) in the treatment of cutaneous wounds, when formation of PEH lesion was induced.Morphological change in epithelial tissue was observed with histopathologic method and electronic microscopy in 11 specimens of PEH lesions and 6 specimens of normal skin adjacent to PEH (PEH-N) from 11 patients with injured skin. The expression characteristics and distribution of pan-cytokeratin (p-CK), matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-9, proliferating cell nuclear antigen (PCNA), epithelial cadherin(E-Cad) and beta-catenin(beta-Cat) in EMJ were detected with immunohistochemical methods.Epithelial cells expressing P-CK presented squamous epithelialization and extended into deep layer of mesenchyma. In epithelium-mesenchyma junction, where IV type collagen and laminin were weakly expressed, the protein contents of p-CK, E-Cad, MMP-2, MMP-3 MMP-9 were decreased, whilst the immunochemical staining of beta-Cat and PCNA was apparently increased. In the junction, epithelial basal cells were observed to migrate and to depart from basal membrane; epithelial islands and isolated epithelial cells expressing p-CK in mesenchyma could be observed. Ultrastructural observation revealed deformation of epithelial basal cells, increment of nucleus/cytoplasm ratio, loosened intercellular junctions, decrement of electronic density of BM and derangement of BM structure could be observed.Reduction in the capability of epithelial basal cells adhesion, differentiation and formation of basement membrane and cytokeratin in PEH associated with wound may be the crucial cause which controls epithelial cells migration into mesenchyma. That the contents of ColIV and LN were decreased may not be associated with MMPs, but with enhancement of the ratio of beta-Cat/E-Cad signal might be the important mechanism of dedifferentiation of epithelial basal cells and the loss of ability of structure formation and cellular migration.

To investigate the situation of neurofilament protein (NFP) and epidermal growth factor receptor (EGFR) staining in scars and normal human skin, and attempt to explain the etiology of scarring formation.The samples of scar were obtained from post-burn (11-26 months) patients undergoing plastic operation in our burn unit recently, and the samples of control came from skin donor site of the same patient correspondingly. Immunohistochemistry staining technique was employed to examine the protein expression in scars and skin. The expression of neurofilament protein (NFP-200) and EGFR in hyperplastic scars (n=12) and normal human (n=12) were investigated with light microscope.The expression of NFP-200 and EGFR in hyperplastic scars were much higher than that in normal human skin,and NFP-200 immuno-reaction nerve fibers were progressively stronger with scarring formation, then were gradually reduced with scarring maturation. The change of EGFR expression was similar to NFP-200 expression.These studies are undertaken to elucidate the relationship between scars formation and nerve reinnervation in cutaneous cells. The activity of EGFR might be associated with the nerve reinnervation. The expression of EGFR influence on wound healing. Therefore, neuroregulation might play a role in the pathogenesis of scars.

To study the relationship between the changes in serum hyaluronic acid (HA) concentrations and injury of hepatocytes in severe burn patients.Radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) were used to detect HA and tumor necrosis factor-alpha(TNF-alpha) concentrations in serum, and biochemical indexes for hepatic function and renal function in burn patients that were divided into different groups: systemic inflammatory response syndrome(SIRS), multiple organ dysfunction syndrome(MODS). Multiple organ failure(MOF) and non-surviving patient groups.In two weeks after burns, the serum HA concentration attained a high level. With the increase of severity of disease, the values of the serum HA concentration were elevated progressively (P0.001). At the onset of MOF till death, the serum HA content was rapidly decreased. Lineal correlation analysis indicated that the change in the serum HA concentration was closely correlated with SIRS, burn sepsis, MODS and death (P0.01). It was positively correlated with TNF-alpha, aspartate aminotransferase (AST) and total bilirubin (P0.05 or P0.01), and negatively correlated with plasma albumin and albumin/globulin(both P0.05).The increment in serum HA levels is relate with the impairment of the hepatic endothelial cells due to TNF-alpha etc. The HA content in serum can be used as a sensitive indicator for judging injury of liver, seriousness of the injury, and prognosis of the patient.

To observe the protein expression of phosphorylated form of P38 mitogen-activated protein kinase(P38MAPK) and c-Jun in hypertrophic scar skin and to explore their influences on the formation and maturation of hypertrophic scar.The expression intensity and distribution of phosphorylated form of P38MAPK and c-Jun were examined with immunohistochemistry and pathological methods in 16 cases of hypertrophic scar skin and 8 cases of normal skin.In normal skin, the positive signals of phosphorylated form of P38MAPK mostly distributed in basal lamina cells of epidermis, while c-Jun was mainly located in epidermal cells and endothelial cells. The positive cellular rates of two proteins were 21.3% +/- 3.6% and 33.4% +/- 3.5% respectively. In proliferative hypertrophic scar skin, the particles of phosphorylated P38MAPK and c-Jun were mainly located in epidermal cells and some fibroblasts. The positive cellular rates of two proteins were significantly elevated to 69.5% +/- 3.3% and 59.6% +/- 4.3% respectively (P0.01). In mature hypertrophic scar, the expression of these proteins decreased but was still higher than that of normal skin.The formation and maturation of hypertrophic scar might be associated with the alteration of phosphorylated P38MAPK and c-Jun protein expression in hypertrophic scar.

To establish a method for the isolation of porcine mesenchymal stem cells (MSCs) from bone marrow and to demonstrate their differentiation ex vivo into various mesenchymal tissue cells.MSCs were isolated from bone marrow and purified by centrifuge and in vitro. The proliferation and growth characteristics were observed in primary and passage culture. Cell cycle was analyzed by measuring DNA content with FAC-Scan flow cytometer and cell multipotent was identified with specific staining.The adherent, fibroblast-like cells were confluent in single layer after plating for 12-14 days. The cultured MSCs in vitro differentiated into osteoblasts. The cell cycle analysis showed that 80% of MSCs were in G0/G1 phase.Porcine MSCs can be isolated from postnatal bone marrow through their adherent ability. Porcine MSCs may be introduced as a valuable model system to study the mesenchymal lineages for basic research and tissue engineering.

To investigate the effect of serum from normal and burned rats on the biological behaviors of bone marrow stromal cells (BMSCs).Bone marrow was extracted from sacrificed Wistar rats and BMSCs were then incubated in F-12 medium in the presence of fetal calf serum (FCS, group F), normal rat serum (group N), serum harvested from burned rats at 3 days (group B1) and 14 days (group B2) after burn injury. The growth curve, cell cycle, apoptosis rate and contents of DNA of BMSCs were investigated.The growth curves of cells incubated in different rat serum were similar, but were significantly different from that of cells incubated in FCS. The doubling time of cells incubated in rat serum from group N, B1 and B2 were 23.7, 18.6 and 20.2 hours, respectively, which were significantly shortened compared with that in group F. Compared with group F, the numbers of cells at platform phase in group N, B1 and B2 were greater. The ratios of cells at phase G0/G1 in group B1 and B2 were significantly lower than those in other groups (all P0.01), but the ratios of cells at phase S were adverse (P0.01). Contents of DNA in cells incubated in rat serum were greater than that in cells incubated in FCS (P0.05 or P0.01). Contents of DNA in cells incubated in serum from burned rats were greater than that in cells in normal rat serum (P0.01). The rate of cell apoptosis in group B1 was higher than those in other groups (P0.01).Rat serum is more effective to stimulate proliferation of BMSCs from rat in vitro than FCS. There are probably many factors affecting the growth of BMSCs, the contents of which changed with the time after burn injury. The effects of serum from burned rats on the growth of BMSCs are complex.

To investigate the expression and location of epidermal growth factor (EGF) and its receptor (EGFR) in dermal chronic ulcers and normal skin in order to explore their influence on ulcer formation.The expression intensity and distribution of EGF and EGFR were detected with pathological method and immunohistochemistry method in 8 cases of dermal ulcers, 8 cases of edge of ulcer and 8 cases of normal skin.The Positive signals of EGF could be found in epidermal cells, endothelial cells and some fibroblasts; EGFR was principally located in the cytoplasm and cellular membrane of these cells mentioned above in normal skin. From normal skin, edge of ulcer to ulcerative tissues, the protein contents of EGF and EGFR were decreased progressively. In ulcerative tissues, EGF was mostly distributed in monocytes and macrophages while EGFR was chiefly sited in monocytes. When compared with normal skins, the protein expression of EGF and EGFR was notably reduced in ulcerative tissues (both P0.01). The positive cellular ratios of two proteins were reduced to (7.1+/-5.2) % and (8.8+/-5.5) % of those in normal skin respectively (all P0.01).The formation of dermal chronic ulcers is closely associated with the reduction of EGF and EGFR protein expression which may lead to binding obstruction between EGF and its receptor.

To investigate the effect of recombinant human platelet-derived growth factor (rhPDGF-BB) on cutaneous incisal wound healing in diabetic rats and this factor does-effect relationship.Thirty Wistar diabetic rats were used in this study. Four full-thickness skin wounds of 2.54 cm(2) were incised in the back of each rat. The wounds were divided into five groups: three groups were treated with different dosage of rhPDGF-BB gel (14.0 microg/cm(2) wound, 7.0 microg/cm(2) wound, 3.5 microg/cm(2) wound), other two groups were treated with vehicle or untreated, respectively. rhPDGF-BB gel was topically applied to the wounds from 1 to 14 days. The wound healing effect was assessed by the measurements of the wound area, subcutaneous chamber and histological changes of each group.On 14 days, the wounds of the three groups treated with rhPDGF-BB contracted to (0.22+/-0.30)cm(2), (0.05+/-0.06)cm(2), (0.32+/-0.32)cm(2), respectively. The area of the middle dosage groups was significantly smaller than those treated with vehicle 0.23+/-0.22 cm(2) (P0.05) and untreated (0.22+/-0.25) cm(2) (P0.05). On 7 days, the subcutaneous chambers of the three treated groups contracted to (0.04+/-0.03)ml, (0.02+/-0.02)ml, (0.06+/-0.03)ml, and there was a statistic significance when comparing middle dosage group with vehicle group (0.06+/-0.03) ml (P0.05) and untreated group (0.07+/-0.05 ml (P0.05). As for histological examination, much more granulation formation was shown in the wound bed in middle dosage group than that in other four groups on 7 days, and some of the wounds treated with middle dosage were healing on 14 days.It suggested that topical application of rhPDGF-BB might improve wound healing in diabetic rats. The effective dosage of rhPDGF-BB is 7.0 microg/cm(2) wound.

To explore the expressive characteristics of epidermal growth factor (EGF) and its receptor (EGFR) in tissues of fetal and adult intestines.The expression intensity and distribution of EGF and EGFR were detected with pathological and immunohistochemical methods in 6 specimens of adult (16-54 years) intestines and 18 specimens of fetal intestines with different gestational ages (13-31 weeks).Positive protein particles of EGF and EGFR could be detected in tissues of fetal and adult intestines. The protein expressions of EGF and EGFR were elevated progressively with the gestational age. EGF was mainly located in the cytoplasm and extracellular matrix of intestinal villus cells, endothelial cells and tunica serosa epithelial cells, while EGFR chiefly distributed in the cellular membrane of these cells.The endogenous EGF and EGFR might be involved in the intestinal development at embryonic stage, in the structural and functional maintenance at adult stage, and in the wound healing after injury.

To construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs.The BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1. At the same time, mesenchymal stem cells (MSCs) were isolated and cultured in vitro. The plasmid carrying the BMP7 gene was transfected into MSCs.PCR and digesting demonstrated that the eucaryotic expression plasmid -pcDNA-BMP7 was obtained. RT-PCR and immunohistochemical methods showed that the BMP7 gene was expressed in MSCs.Construction of an eucaryotic expression plasmid carrying BMP7 gene and expression in MSCs provide a sound basis for gene therapy using the BMP7 gene and the ideal seeds for tissue engineering.

To observe the expression characteristics of EGFR and FGFR-2 in normal skin from fetal and adult, and attempted to probe the molecule mechanism of fetal scarless healing.The skin samples of fetal and adult were taken from abortive fetus of obstetrics unit and donor site of plastic operation patients in our burn unit, respectively. EGFR and FGFR-2 were used as the biochemical markers for reparative cells. Immunohistochemistry staining technique was employed to determine the expressive levels of different epithelial cells markers.There were EGFR and FGFR-2 antibody positive cells in normal skin from fetal and adult, but the expressive levels of EGFR and FGFR-2 protein had apparent difference, with the time of fetation increasing, the EGFR and FGFR-2 positive expression rate became stronger gradually. The number of FGFR-2 antibody positive cells found in adult skin was much more than that in fetal skin.There were the inherent differences of EGFR and FGFR-2 antibody immunohistochemistry staining in cells of adult and fetal skin. which may be an essential facet of fetal scarless healing.

To investigate the expression characteristics of protooncogene c-jun and its related gene p38 in different developmental intestinal epithelial cells in rats, and to explore their biological roles in the intestinal wound healing after injury.Immunohistochemical techniques were used to detect the expressions of c-jun, p38 and proliferating cell nuclear antigen (PCNA).The positive expression of c-jun in intestinal epithelial cells at the early development stage (from E14 d to P28 d) was much stronger and more extensive than that in mature rats, and migrated from bottom to top of villus with epithelial cellular development. The positive expression of PCNA was similar with c-jun during the same time, but the distinction between them was location of their expression. The expression of c-jun in mature rat only lay in the top of villus, while the expression of PCNA was limited to intestinal crypts. The expression of p38 during stages of development and mature, mainly was in mitogenic cells.The results indicate that the strong expression of tumor gene c-jun at the early development and mature stages of intestinal epithelial cells at the special region is related to cellular differentiation, and p38 is probable correlate with cellular mitogenesis.

To explore the expression changes of proliferating cell nuclear antigen (PCNA) in oral normal mucosa and recurrent aphthous ulcers(RAU).The expression intensity and distribution of PCNA were examined with immunohistochemistry method in 15 cases of recurrent oral aphthous and normal oral mucosa.In RAU, there was large amount of inflamed cells. In normal oral mucosa, the expression intensity of PCNA was significant higher than that in oral aphthous (P0.05).The inhibition of epidermal cells proliferation in RAU may be one of the mechanisms which one involved in RAU formation.

To study the redistribution of epidermal stem cells in regenerating wound tissues, and to elucidate the role of epidermal stem cells during wound repair.80 circular full-thickness wounds were produced on both sides of the back in 20 male Wistar rats (4 wounds in each animal). Then the 80 wounds were randomly divided into 2 groups: group A (treated with sulfadiazine silver- sulfadiazine zinc cream, n = 40) and group B (without any treatment, n = 40). The infection and healing process of the wounds were observed with naked eyes. Five mice were killed one time 3, 7, 14, and 21 days after the wounding. Two wounds with surrounding normal skin tissues were collected from each mouse. Routine histological examination was conducted with HE staining. Beta(1) integrin and keratin19 (K19), markers of epidermal stem cells, were employed to determine the distribution of epidermal stem cells with streptavidin-peroxidase (SP) immunohistochemical method.The healing rate of wounds was 80% (32/40) in group A, and 60% (24/40) in group B. No beta(1) integrin and K19 positive cells was found in the granulation tissue of all wounds in these 2 groups at any time point during the healing process. However, a few beta1 integrin and K19 positive cells, bearing no anatomic relation with the epidermal stem cells in the basal layer, were found scattering in the stratum spinosum and stratum granulosum of the epidermis on the wound edges. The closer to the wound the more concentrated the beta(1) integrin and keratin19 positive cells. Their numbers increased gradually along with the shrinking of the wound surface until the wound completely healed. The numbers of the beta(1) integrin and keratin19 positive cells decreased gradually after epithelialization. The numbers of the beta(1) integrin and keratin19 positive cells were obviously lower in the infected wounds than in the uninfected wounds.Epidermal stem cells participated in the healing of wound. The function of redistribution of epidermal stem cells in wound edges seems to promote the re-epithelialization of granulating wounds.

To investigate the different location and the expression characteristics of epidermal stem cells in normal adult skin and scar tissue.Skin tissue specimens were harvested from the corresponding sites from 6 healthy volunteers and from scar tissue of 6 patients 1 year after major deep burn. beta1 integrin and keratin 19 (K19) were employed as the biochemical markers for stem cells and transit amplifying cells identification and keratin 14 (K14) and keratin 10 (K10) as markers for post-mitotic cells and terminally differentiated cells respectively. Integrin and keratin were determined by Elivision two-step immunohistochemistry.The expression of beta1 integrin and the K19 positive cell count in the epithelial basal layers of scar tissue were evidently decreased and weakened than those in normal adult healthy skin. Furthermore, the positive cells expressing K14 in epidermis of scar tissue were only located in 2 - 3 layers of basal epidermis, and their number was much less than that in normal adult skin. Whereas the cells positively expressing K10 were distributed wider in area than that in normal healthy skin. The epidermal stem cells and transit amplifying cells in scar epidermis were much less in number than that in normal skin. The differentiation process of scar epidermal stem cells was different from that of normal skin. And the proportions of post-mitotic cells and terminally differentiated cells were abnormal.The results indicated that the self-renewal ability of the scar epidermis was decreased, and the differentiation process of it was in disorder, which may be a reason for the abnormality of structure and function of the epidermis in scar, and a reason for the decreased ability of wound healing of scar tissue.

To investigate the feasibility of differentiation of bone marrow mesenchymal stem cells (MSCs) into vascular endothelial cells and the mechanism of its involvement in wound healing.Porcine MSCs were harvested from porcine marrow, and they were isolated and purified by density gradient centrifugation. After being cultured and amplified in vitro, the MSCs were labelled with BrdU (5-bromodeoxy-uridine). Full skin loss wound was created on the back of the mini-swine whose bone marrow was obtained. The labelled MSCs with fibrin glue as the vector were regrafted back to the donor animal wound. The wound tissue specimens were harvested at 2, 4, 6, 8 and 12 post-operation weeks and were immunohistochemically stained by BrdU and factor VIII (FVIII) for comparative study.Most BrdU positive cells aggregated around small blood vessels in the granulation tissue of the wounds. Only individual vascular endothelial cells were BrdU positive. There was FVIII expression in the cytoplasm of BrdU positive cells.MSCs were closely correlated with the formation of small blood vessels in granulation tissue during wound healing process. The porcine MSCs possessed the potential to differentiate into vascular entoehelial cells and to participate in wound healing under the micro-enviroment of the wound.

To observe the differences in protein contents of three transforming growth factor-beta (TGF-beta) isoforms, beta 1, beta 2, beta 3 and their receptor(I) in hypertrophic scar and normal skin and to explore their influence on scar formation.Eight cases of hypertrophic scar and their corresponding normal skin were detected to compare the expression and distribution of TGF-beta 1, beta 2, beta 3 and receptor(I) with immunohistochemistry and common pathological methods.Positive signals of TGF-beta 1, beta 2, and beta 3 could all be detected in normal skin, mainly in the cytoplasm and extracellular matrix of epidermal cells; in addition, those factors could also be found in interfollicular keratinocytes and sweat gland cells; and the positive particles of TGF-beta R(I) were mostly located in the membrane of keratinocytes and some fibroblasts. In hypertrophic scar, TGF-beta 1 and beta 3 could be detected in epidermal basal cells; TGF-beta 2 chiefly distributed in epidermal cells and some fibroblast cells; the protein contents of TGF-beta 1 and beta 3 were significantly lower than that of normal skin, while the change of TGF-beta 2 content was undistinguished when compared with normal skin. In two kinds of tissues, the distribution and the content of TGF-beta R(I) had no obviously difference.The different expression and distribution of TGF-beta 1, beta 2 and beta 3 between hypertrophic scar and normal skin may be associated with the mechanism controlling scar formation, in which the role of the TGF-beta R (I) and downstream signal factors need to be further studied.

To explore the possible mechanisms of skin regeneration through the epidermal stem cells stimulated by epidermal growth factor (EGF).At 8 and 14 days after treatment with EGF, the tissue specimens from 8 skin ulcered patients who were treated with EGF were used to evaluate the distribution and differentiation of epidermal stem cells. The expression of beta 1 integrin, keratin 19 (K19), keratin 14(K14) and keratin 10 (K10) in skin was detected with SP immunohistochemical methods. Hematoxylin and eosin staining method were used to observe the tissue structure. Another 7 biopsies from ulcered patients without EGF management were used as the control.The results from the hematoxylin and eosin staining showed that the epidermis in EGF treated wounds was thick and the epidermal ridges were enlarged both in 8 and 14 days compared with those in control skin. Immunohistochemical staining from beta 1 integrin and K 19 showed that all tissues treated with EGF were rich in epidermal stem cells both in 8 and 14 days. These stem cells were bigger in size and larger in number and localized at the base of the epidermis. In contrast, the positive expression cells of beta 1 integrin and K 19 in control group in the same time were scanty. It was found that there were some stem cell islands in epidermis treated with EGF in day 14 and absent from the control group. The expression of K14 and K10 could be observed in those terminally differentiating epidermal cells in both groups.The results indicate that the possible mechanisms of skin regeneration stimulated by EGF comes from the mitogenic effects and differentiation of skin stem cells.

Dr Hare and colleagues1 investigated the efficacy and safety of allogeneic mesenchymal stem cells (MSCs) administered by transendocardial stem cell injection compared with autologous MSCs in patients with chronic left ventricular dysfunction secondary to myocardial infarction. The results are encouraging; however, we have some concerns about the study.

Robert Kirsner and colleagues (Sept 15, p 977) describe a spray formulation of allogeneic neonatal keratinocytes and fi broblasts for the treatment of chronic venous leg ulcers. Such therapies are novel and of major interest. However, although this technique certainly has potential, we have some concerns about the interpretation of the results of Kirsner and colleagues’ study. First, it is not clear whether Kirsner and co-workers tested the viability of the fibroblasts and keratinocytes. Viable cells are crucial in the treatment of ulcers. Second, why are there no representative fi gures to show that wound healing was greater with 0·5×106 cells/mL every 14 days than with vehicle alone? Third, the follow-up (12 weeks) was too short to show the eff ect of spray-applied cell therapy with human allogeneic fi broblasts and keratinocytes on longterm outcomes. Were there any such follow-up fi ndings with regard to this technology? Finally, this spray-applied cell therapy contains compound solutions, including cells and matrix. It would be interesting to know which part is responsible for the clinical eff ects, and whether the mechanisms of action are direct (diff erentiation into myofi broblast or vascular endothelial cells), indirect (cell–cell contact or paracrine eff ects), or both.

Serious trauma and accidental injury are the leading causes of death among people younger than 45 years old in China. Thus, the prevention and control of serious trauma and accidental injury are important for reducing these deaths. The concept is timely but difficult. Here, we review the current state of serious trauma and accidents in China and other countries, focusing on road accidents, and provide our personal perspectives and suggestions on how to prevent and control these serious injuries in China.

Mitogenic and non-mitogenic activities of fibroblast growth factor (FGF) are coupled to a range of biological functions, from cell proliferation and differentiation to the onset of many diseases. Recent reports have shown that acidic fibroblast growth factor (aFGF) has a powerful anti-apoptosis function, which may have potentially therapeutical effect on gut ischemia and reperfusion injuries. However, whether this function depends on its mitogenic or non-mitogenic activity remains unclear. In this study, we identified the source of its anti-apoptosis function with a mutant, aFGF28-154 and observed its effect on reducing gut ischemia and reperfusion injury.aFGF28-154 was generated by amplification of appropriate DNA fragments followed by subcloning the products into pET-3c vectors, then they were expressed in BL21 (DE3) cells and purified on an M2 agarose affinity column. This mutant aFGF28-154 maintained its non-mitogenic activity and lost its mitogenic activity. With a dexamethasone (DEX)-induced mouse thymocyte apoptosis model in vitro and in vivo, we studied the anti-apoptotic function of aFGF28-154. Also, in vivo study was performed to further confirm whether aFGF28-154 could significantly reduce apoptosis in gut epithelium after gut ischemia-reperfusion injury in rats. Based on these studies, the possible signal transduction pathways involved were studied.With a dexamethasone (DEX)-induced mouse thymocyte apoptosis model in vitro and in vivo, we found that the anti-apoptotic function of aFGF28-154 was significantly enhanced when compared with the wild type aFGF. In vivo study further confirmed that aFGF28-154 significantly reduced apoptosis in gut epithelium after gut ischemia-reperfusion injury in rats. The mechanisms of anti-apoptosis function of aFGF28-154 did not depend on its mitogenic activity and were mainly associated with its non-mitogenic activities, including the intracellular calcium ion balance protection, ERK1/2 activation sustaining and cell cycle balance.These findings emphasize the importance of non-mitogenic effects of aFGF, and have implications for its therapeutic use in preventing apoptosis and other injuries in tissues and internal organs triggered by ischemia-reperfusion injury.

Contributing reviewers The editors of Military Medical Research would like to thank all our reviewers who have contributed to the journal in volume 1 (2014) and 2 (2015).