To identify the stem cell islands in regenerated epidermis, the same biopsies used in our previous experiments were used in this study. CD87 immunohistochemicy and PAS staining were used. The scant CD87 positive cells could be found in basal membrane. Also, the positive staining of PAS could be seen in the basal membrane in both normal and regenerated epidermis. No CD87 positive cells were found in the spinous and granular layers. The results indicate that the error in metrology could be excluded from both CD87 immunohistochemicy and PAS staining, and that the stem cell islands may come from the cell reversion in regenerated epidermis.

Objective To study expression intensity and distribution of phosphorylated forms of extracellular signal-regulated protein kinase1/2 (ERK1/2), stress-activated protein kinase (SAPK) and P38MAPK in normal skin and hypertrophic scars and their potential biological significance. Methods The morphological characteristics of hypertrophic scars in different periods after wound healing and normal skin were examined histopathologically. Protein expression of p-ERK1/2, p-SAPK and p-P38MAPK was also assessed with immunohistochemical technique. Results In normal skin, phosphorylated forms of ERK1/2, SAPK and P38MAPK were mainly located in epidermal basal cells, in which the positive cellular rates of all the three proteins were low. Along with the maturation of hypertrophic scars, protein contents of p-ERK1/2 and p-SAPK were progressively increased. In mature hypertrophic scars, positive signals of these two proteins were mostly distributed in keratinocytes and some fibroblasts. The positive cellular rate of p-P38MAPK ascended in active hypertrophic scar, then decreased to a level which was still higher than that of normal skin. Conclusion The formation of hypertrophic scars may be associated with the alteration in signaling pathways which results in the increment of protein contents of p-ERK1/2, p-SAPK and p-P38MAPK.

The purpose of this study was to investigate the localization and expression characteristics of phosphorylated form of extracellular-signal regulated-protein kinasel/2 (p-ERKl/2), Ras and C-fos in skin at different development stages and to explore their potential biological significance. Immunohistochemistry and pathological methods were used to detect the expression intensity and distribution of p-ERKl/2, Ras and C-fos in skin of 8 fetuses with different gestational ages (13 to 31 weeks) and 8 adults. Positive immunohistochemical signals of p-ERK1/2, Ras and C-fos. could be found in fetal and postnatal skins. Along with the increment in gestational age, the positive cell rates of p-ERK1/2, Ras and C-fos in the skin elevated progressively. In skins derived from the fetus of late-trimester pregnancy and adult, the positive rates of these three proteins were significantly increased in comparison with skin from the early-trimester fetus (P

Objective To investigate the inductive role of traumatic microenvironment in dedifferentiation of epidermal cells and explore the potential role of Wnt signaling pathway in this biological process. Methods The sheets of human foreskin were digested overnight after removal of adipose tissue, and then the epidermis was separated from the dermis. The separated epidermis sheets were repeatedly adhered to type Ⅳ collagen and flushed to remove the epidermal stem cells. The obtained epidermis sheets were transplanted onto the full-thickness skin wounds on the back of BALB/c nude mice, five days after which the cell lineage was evaluated by immunohistochemistry and the expressions of Wnts and downstream components in the grafted epidermal sheets examined by RT-PCR and Western blot. Results The cells in the basal layer of full-thickness epidermal sheets were positive for CK19 and β1 integrin and negative for CK10. While the cells in uhrathin epidermal sheets treated with type Ⅳ collagen were fully positive for CK10. Five days after transplantation of the ultrathin epidermal sheets, cells negative for CK10 but positive for CK19 and β1 integrin emerged at the wound-neighboring side of the skin grafts. At the same time, the expressions of Wnt-10b, Wnt-4 and Wnt-7a mRNA were increased by about 3.1-fold, 2.2-fold and 1.4-fold independently after transplantation. Furthermore, the expressions of β-catenin and β-catenin target genes (cyclin D1 and c-myc) were elevated by about 3-fold, 1.5-fold and 2-fold respectively in the grafted epidermal sheets (P < 0.01). Conclusion Traumatic microenvironment can induce epidermal cell dedifferentiation, when the Wnt/β -catenin signaling pathway may play an important role.

To study the location and expression characteristics of epidermal stem cells in normal skin and scar epidermis of children, and to explore the relationship between the differences of these two epidermal stem cells and wound healing after burn. ?1 integrin and keratin 19 (K19) were used as the biochemical markers to identify stem cells and transit amplifying cell, keratin 14 (K14) and keratin 10 (K10) were used as the markers for post-mitotic cells and terminally-differentiated cells, respectively. Normal skin and scar tissue were obtained from children of 4 to 12 years of age. Elivision two-step immunohistochemistry was used. The results showed that in the immunostained tissue sections, the positive ?1 integrin and K19 expression cells were observed in 2～3 layers above the basal layer, whereas K10 expression cells were observed in all epidermal cells except basal cell layer in the scar tissue. Observations revealed that the number of stem cells and transit amplifying cells were less in the scar tissue than that in the normal skin, the differentiation process of scar epidermal stem cells was different from that of normal skin, and the proportion between post-mitotic cells and terminally-differentiated cells was abnormal. The results indicated that the self-renewal ability of the scar epidermis was lowered, and the differentiation process of it was deranged, and this might be considered to be a reason of abnormality of structure and function of the epidermis of scar tissue, and its poorer ability in wound healing.

To investigate the expression characteristics of fibroblast growth factor 10 (FGF10) and its receptor (Bek) underlying their effect on the formation of fetal skin appendages (SA). Expressions of FGF10, Bek, cytokeratin (CK19), Bcl-2 and proliferating cell nuclear antigen (PCNA) were detected with pathological and immunohistochemistry techniques in 130 skin specimens, which were obtained from 5 different sites (head, lower jaw, ear lobe, shoulder and presternal region) of 26 fetuses at different embryonic ages (E 8～31weeks). Results showed that FGF10, PCNA and Bcl-2 were over-expressed in interstitial cells distributed in clumps under the epidermis, and all proteins were strongly expressed in epidermal cells and pericytes at E 11weeks. In E 13weeks fetal skin, epidermal cells formed SA anlage through focal proliferation; then they developed, differentiated and migrated towards the dermis. In the skin of E 11～13weeks fetus, the expression of FGF-10, PCNA and Bcl-2 in interstitial cells in the dermis, and the expression of FGF10, Bek, PCNA, CK19, and Bcl-2 proteins in the epithelial cells in SA showed the expression characteristics of these proteins which were in accordance with growth and development of fetal SA. The results suggested that the specific binding of FGF10 from interstitial cells with Bek on the membrane of epithelial cells was the important signal to induce the proliferation and morphogenesis of embryonic SA epithelial \{cells.\ \ \ \}

Objective To investigate the dedifferentiation of epidermal cells into their progenitor stem cells induced by basic fibroblasts growth factors(bFGF) in vitro.Methods HEKa cells obtained from Cascade were found flattening and formation of cell-to-cell contacts after 6 to 7 passages,which resembled differentiated epidermal cells in vivo.To examine the effect of growth factors on the cell proliferative alterations,bFGF(100 ng/ml) was added into the culture medium for different periods(6,12,24,48,or 72 h),then the cell proliferation was measured by MTT assay.Phenotypic changes and the cell-fate determination of HEKa cells after bFGF treatment were detected by immunocytochemical assays,flow cytometry and RT-PCR analysis.HEK cells with no intervention treatment were used as a control.Results MTT assay proved that the optimal culture condition to induce the dedifferentiation of epidermal cells into their progenitors was to culture HEKa cells for 36 to 48 h when the addition of bFGF was 100 ng/ml.After treatment with bFGF for 48 h,clusters of round-shaped cells appeared around differentiated epidermal cells,and expanded progressively thereafter.These cells were smaller in shape and with larger nuclear/cytoplasm ratio,and had not only clonogenicity but also ability to form a cutaneous ridge-like structure.Immunohistochemical staining revealed that the expression levels of ?1 integrin,CK19 and CK14 were up-regulated,while the expression of CK10 was significant down-regulated after bFGF treatment.Flow cytometry indicated that there were more CK19-positive and CK14-positive cells in the treatment group than in the control(74.77% vs 15.74%,and 87.14% vs 67.26%respectively),but much lesser CK10-positive cells(4.56% vs 98.56%).Additionally,the mRNA expression levels of ?1 integrin,CK19 and CK14 were up-regulated after bFGF treatment,but that of CK10 was down-regulated.Conclusion bFGF can reverse the differentiated process of epidermal cells and induce them to produce immature,stem-like cells,which can proliferate and be used in the wound repair and regeneration of skin tissues.

Objective To investigate the effect of bFGF and MSCs autografting on expression of PCNA and Ⅷ-R Ag of porcine skin defect wound. Methods Six male minipigs were used to produce 48 full-thickness skin wounds, each wound area about 2.54 cm~ 2 on both sides of the back (8 wounds per pig). These 48 wounds were randomly treated with bFGF, MSCs, bFGF+MSCs, and normal saline (12 wounds for each treatment). The tissue from the wounds was harvested on day 3, 7, 14 after surgery, then used for HE staining and immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and factor Ⅷ related antigen (Ⅷ-R Ag). Results Granulation tissue was presented in all groups on day 3 and 7 after wound, most abundant in the wounds treated with bFGF+MSCs, as well as blood capillary formation. PCNA expression increased after skin injury, reached the peak on day 7, most in the granulation tissue, and lasted to day 14, then began to decrease. On day 3, 7, 14 after skin injury, PCNA expression was the most highest in the wounds treated with bFGF+MSCs. Ⅷ-R Ag-positive cells in the wounds treated with bFGF+MSCs were significantly more than that of the other three treatments (P

Objective To investigate the possible signaling mechanisms by which recombinant human platelet-derived growth factor (rhPDGF) accelerated healing of cutaneous wound in diabetic rats. Methods Four full-thickness skin wounds were incised in the back of 26 male Wistar diabetic rats. The wounded rats were divided into 3 groups (7 or 8 rats each group). One group without treatment was used as a control, and the other 2 groups were treated with rhPDGF at a dose of 7.0 μg/cm2 wound or vehicle ( DMSO/0.9%NaCl, vol/vol 1:1) from 1 to 14 days. The wound healing was evaluated by the measurements of the wound volume and area. Immunofluorescent and immunohistochemical staining were used to examine the phosphorylation of extracellular signal-regulated kinase 1/2(ERK1/2) and the expression of proliferative cell nuclear antigen (PCNA), respectively. Results Granulation tissue appeared in the bed of wound after injury. The number of blood capillary buds and fibroblasts was greater in the rhPDGF-treated group than that in the other 2 groups. A lot of inflammatory cells infiltration and collagen deposition were observed in the wound. The wound-volume in the rhPDGF-treated group was smaller than that in control group ( P ＜ 0.05). The reepithelialization rate in rhPDGF-treated group was higher than that in the other 2 groups at 7 days after injury ( P ＜ 0.05). The expression of PCNA in reparative cells was higher in rhPDGF-treated group than in control group or vehicle-treated group at 3,7 days after injury( P ＜ 0.05). The phosphorylation of ERK1/2 was stronger in rhPDGF-treated group than that in control group or vehicle group at 7 and 14 days after injury( P ＜ 0.05). Conclusion These results suggest that rhPDGF accelerates wound healing and improves healing quality by increasing the phosphorylation of ERK1/2.

Objective To investigated the distribution of epidermal stem cells in rat full-thickness wound tissues during the wound healing process and to elucidate the roles of epidermal stem cells in wound repair in vivo. Methods Eighty circular full-thickness wounds were produced on both sides of the back in 20 male Wistar rats labeled with BrdU 60 days previously (4 wounds in each rat). BrdU, β1 integrin and keratin 19 (K19) were employed to determine the epidermal stem cells with SP immunohistochemical methods, and the epithelialization was determined with routine histological methods of HE staining on the 3rd, 7th, 14th, and 21st days after operation. Results No cells with positive immunostaining for β1 integrin, K19 and BrdU were found in granulation tissue of wound in both groups during the healing process. However, a few scattered β1 integrin and K19 positive cells were found within the stratum spinosum and stratum granulosum of the epidermis on the wound edges on the 3rd day post-injury. And these positive cells gradually became more and more in number, and mostly concentrated on the border of wound edges till the wounds healed. In addition, the number of positive cells for β1 integrin and K19 in the infected wounds was less than that in non-infected wounds. These positive cells for β1 integrin and K19 staining on the wound edge were also positively stained with BrdU in the cellular nuclei. Conclusion The above results indicate that ectopia of epidermal stem cells present a major function during wound epithelialization.