Future application of hair follicle stem cells: capable in differentiation into sweat gland cells.

BACKGROUND: Sweat glands (SGs) can not regenerate after complete destruction in the severe skin injury, so it is important to find a ideal stem cell source in order to regenerate functional SGs. Hair follicle stem cells (HFSCs) possess the obvious properties of the adult stem cells, which are multipotent and easily accessible. In this research, we attempted to direct the HFSCs suffered from the sweat gland cells (SGCs) special differentiation by a co-operative coculture system in vitro. METHODS: The designed co-culture microenvironment in the transwell was consist of two critial factors: heat shocked SGCs and dermis-like mesenchymal tissue, which appeared independently in the two control groups; after induction, the purified induced SGC-like cells were transplanted into the full-thickness scalded wounds of the nude mice, after 4 weeks, the reconstructed SG-like structures were identified by immunohistochemical and immunofluorescence analysis. RESULTS: A part of HFSCs in experimental group finally expressed SGCs phenotypes, by contrast, the control group 1 which just containing dermis-like mesenchymal tissue failed and the control group 2 consisted of heat shocked SGCs was in a poor efficiency; by immunofluorescence staining and flow cytometry analysis, the expression of HFSCs special biomarkers was down regulated, instead of the positive efficiency of SGCs special antigens increased; besides, the induced SGCs displayed a high expression of ectodysplasin A (EDA) and ectodysplasin A receptor (EDAR) genes and proteins; after cell transplantation, the youngest SG-like structures formed and be positive in SGCs special antigens, which never happened in untreated wounds (P < 0.05). CONCLUSION: The HFSCs are multipotential and capable in differentiating into SGCs which promise a potential stem cells reservoir for future use; our special co-culture microenvironment is promising for HFSCs differentiating; the induced SGCs are functional and could work well in the regeneration of SGs.

Transplantation of human bone marrow-derived mesenchymal stem cells transfected with ectodysplasin for regeneration of sweat glands.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 (P < 0.05). CONCLUSIONS: 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.

[A new way for isolation and cultivation of sweat gland ductal cells from human split-thickness skin in vitro].

OBJECTIVE: To explore a new method of isolation and culture of eccrine sweat gland ductal cells from human split-thickness skin graft in vitro. METHODS: Human split-thickness skin graft which was presented by volunteer (n = 10) was digested with type II collagenase, and then sweat gland duct were isolated from the split-thickness skin graft, primary cultures were incubated at 37 degrees C in humidified atmosphere of 5% CO2, 95% O2. The cultured eccrine sweat gland ductal cells were identified by analysis CEA, CK8, CK18, CK19 antigens expression with flow cytometry, RT-PCR and Western Blot, and by detecting the electrophysiology with whole cell patch clamp technology. RESULTS: The isolated eccrine sweat gland ductal cells could grow by adhering to the wall, proliferate in vitro after 48 h of adhering to the wall, and confluens after 2 - 4 weeks of adhering to the wall. The FACs analysis showed the expression of CEA was (90.26 +/- 1.12)%, (89.70 +/- 1.43)%, and CK8 was (94.41 +/- 1.84)%, (93.65 +/- 1.63)% in primary cultured sweat gland ductal cells and primary cultured eccrine sweat gland cells, respectively, and there is no significant difference between the two groups (P > 0.05). Immunocytochemistry staining showed CEA, CK8, CK18, CK19 was positive in sweat gland duct cells, RT-PCR revealed that CEA, CK8, CK18 and CK19 gene expression in sweat gland ductal cells, and Western Blot analysis showed the expression of CEA brand, CK8 brand, CK18 brand, and CK19 brand in sweat gland ductal cells, patch clamp indicated that this cells has distinct amiloride sensitive Na(+) channels. CONCLUSIONS: The cultured human eccrine sweat gland duct cells in vitro display the markers and biological characteristics of sweat gland epithelial lineage, and this method of digest the split-thickness skin graft to get the sweat gland duct cells is better than classical dissect sweat gland under dissect microscope.

[Promotive effect of adipose-derived stem cells on the wound model of human epidermal keratinocytes in vitro].

OBJECTIVE: To investigate the migrating effect of adipose derived stem cells (ADSCs) on the wound model of human epidermal keratinocyte (HEKa). METHODS: Rat ADSCs (rADSCs) were isolated and cultured (n = 10), rADSCs were direct co-cultured with HEKa cells in experiment group (experimental group, n = 10). In the control groups, rADSCs were indirect co-cultured with HEKa cells in transwell chamber (indirect group, n = 8), or HEKa was cultured alone (single group, n = 8). Then confluent HEKa cells were scraped to establish a wound model under invert microscope. After scraped 24, 48, and 72 h, cell numbers of which migrated across the edge of the wound was measured, the rate of wound healing was calculated by using SigmaScan Pro 5 software, and the proliferating effect of rADSCs on HEKa were examined by incorporation of [(3)H] thymidine. RESULTS: The cells migrated across the edge of wound after 24 hours in experimental group, indirect group, and single group were (9.2 + or - 0.2), (5.0 + or - 0.3), (4.2 + or - 0.3), and were (58.5 + or - 0.4), (26.5 + or - 0.3), (20.7 + or - 0.5) 48 hours after, and were (125.8 + or - 0.4), (43.0 + or - 0.5), (35.6 + or - 0.5) cells/HP 72 hours after, respectively; the numbers were all significantly higher in experimental group than those in control groups (P < 0.05). The rates of wound healing after scraped 72 hours were 61.0% + or - 3.0%, 35.0% + or - 2.5% and 32.0 + or - 2.1%, the outcome in experimental group was significantly better than in the control groups (P < 0.05). And the thymidine feeding displayed the proliferation of HEKa in the three groups were (1440 + or - 210), (1050 + or - 280) and (1130 + or - 390) cpm/10(5) cell, and there was significant difference between the experimental and the control groups (P < 0.05). CONCLUSIONS: The rADSCs can promote the migration of HEKa by direct contact with it.

[Chemotactic effects of burn rat serum on mesenchymal stem cells derived from different sources].

OBJECTIVE: To isolate and culture mesenchymal stem cells ( MSC) from different sources and to investigate the chemotactic effects of burn rat serum on MSC derived from different sources. METHODS: Seventy-two Wistar rats were randomly divided into burn( n = 36, with 30% TBSA full-thickness burns on the back) and sham burn (n = 36, without burns) groups. Bone marrow and peripheral blood of the rats in both groups were collected to isolate and culture MSC. Ratio of MSC, growth speed and cell morphology were observed with inverted microscope. Effects of different serum ( fetal bovine serum, normal rat serum and burn rat serum) on chemotaxis of MSC derived from different sources and their migration ability were subsequently examined with a transwell system. Results MSC were obtained from bone marrow of the rats in both groups. MSC were successfully obtained from bone marrow of all burn rats(100% , P <0.05) , but only from peripheral blood of 7 burn rat(58% ) , and no MSCs were obtained from peripheral blood of 12 rats in sham group( P <0.05). There was small amount of adherent cells 24 hrs after culture, and fusiform shaped adherent cells were sporadically observed in scattered distribution 2-3 days later with inverted microscope. There was no obvious difference in the cell morphology between the 2 groups. In the sham group, the number of MSC migrating to the lower surface of transwell after burn serum treatment [ (94 Il ) cells/ high power field] was significantly greater than that after the treatment with normal rat serum and fetal bovine serum [ (37 +/- 6) , (38 +/- 11) cells/high power field , P <0.01 ] , while no difference in migration ability was found after normal serum treatment compared with that after fetal bovine serum treatment ( P >0. 05). The migration rate of MSCs which were derived from bone marrow in sham group was obviously lower than those derived from bone marrow and peripheral blood from burn rats ( P <0. 05 or 0. 01). Though some difference of the migration ability existed between MSC derived from bone marrow and peripheral blood, there was no statistically significant difference ( P >0. 05). CONCLUSION: MSC can be isolated and cultured from bone marrow and peripheral blood of burn rat, but not from peripheral blood of normal rat. Burn rat serum has a stronger chemotactic effect on MSC. Moreover, the migration ability of MSC derived from burn rat is stronger than that of MSC derived from normal rat.

[Identification and cell phenotype transdifferentiation of adipose-derived stem cells].

OBJECTIVE: To investigate the transdifferentiation of the ADSCs to epidermal cells. METHODS: ADSCs were isolated and cultured from rat adipose tissue by digestion of enzyme. ADSCs was identified by immunocytochemistry and flow cytometry. ADSCs were divided into four groups in order to induce: the condition medium (containing 30% superior of homogenizing rat skin in 10% FBS/DMEM) group, 7 days; 10% FBS/DMEM with EGF (20 ng/ml) group, 7 days; the condition medium for 4 days and then 10% FBS/DEME instead of the condition medium for 3 days group; 10% FBS/DMEM for 7 days group (control group). Cytokeratin 19 and cytokeratin 10 expressions in ADSCs were detected by flow cytometry. RESULTS: (1) The results of immunocytochemistry showed that ADSCs were positive for CD49d and negative for CD106, CD34, CD19, CD10. The results of flow cytometry showed ADSCs were positive for CD49d and CD44. (2) The CK19 expression of ADSCs was 45.32% in the condition medium group, 26.58% in the condition medium with EGF group, 23.37% in te condition medium for 4 days and then 10% FBS/DMEM instead of the condition medium for 3 days gropu and 18.53% in control group, P <0.01. The CK10 expression of ADSCs was 43.56% in the condition medium group, 25.54% in the condition medium with EGF group, 18.20% in the condition medium for 4 days and then 10% FBS/DMEM instead of the condition medium for 3 days group and 2.46% in control group, P < 0.01. CONCLUSIONS: The superior of homogenizing rat skin can induce CK19 and CK10 expressing in ADSCs, and thereby demonstrating ADSCs can differentiate to epidermal cell phenotype in vitro.

Recombinant human platelet-derived growth factor enhanced dermal wound healing by a pathway involving ERK and c-fos in diabetic rats.

BACKGROUND: Platelet-derived growth factor (PDGF) has been shown to promote dermal wound healing, however, the molecular mechanisms responsible for are not fully understood. OBJECTIVE: The present study was undertaken to investigate the possible signaling mechanisms by which PDGF improved healing of cutaneous wound in diabetic rats. METHODS: Four full-thickness skin wounds were created on the dorsum of Wistar diabetic rats. Animals were treated with or without recombinant human PDGF (rhPDGF) at 7.0 microg/cm(2) wound or vehicle daily 1 day after wounding. The animals were then killed after various intervals of wounding, and the wounded skin tissues were used for histological evaluation, analysis of the phosphorylation of extracellular signal-regulated kinases (ERK) and the expression of c-fos protein, as well as the labeling indices of proliferative cell nuclear antigen (PCNA). RESULTS: Topical application of rhPDGF significantly accelerated the rate of reepithelialization compared with vehicle-treated or untreated group at 7 days after wounding. At the histological level, the significant increases in the degree of reepithelialization, the thickness of granulation tissue and the density of capillary bud were observed in the wound sites in rhPDGF-treated group at 7 and 14 days after wounding. Moreover, treatment with rhPDGF increased PCNA labeling indices, c-fos protein expression and ERK phosphorylation in the wounded tissues at the indicated time after wounding. CONCLUSION: These results suggest that application of rhPDGF increases cell proliferation, and enhances dermal tissue repair in diabetic skin lesion of rats, which might be partly mediated by ERK activation and c-fos protein expression.

Non-mitogenic acidic fibroblast growth factor reduces intestinal dysfunction induced by ischemia and reperfusion injury in rats.

BACKGROUND: Acidic fibroblast growth factor (aFGF) has potentially therapeutic uses in some diseases, but the mitogenic activity of aFGF has been found to contribute to several human pathologies, so the extensive applications of wild-type aFGF have been limited. The purpose of the present study was to explore the effects and mechanisms of wild-type (aFGF) and non-mitogenic aFGF on gut ischemia-reperfusion injury in rats. METHODS: Rat intestinal ischemia-reperfusion injury (I/R) was produced by clamping the superior mesenteric artery (SMA) for 45 min followed by reperfusion. One hundred and fourteen rats were randomly divided into four groups: sham operation (group C, n = 6), intestinal I/R + 0.1 mL saline (group S, n = 36), intestinal I/R + 4 microg/0.1 mL wild-type aFGF (group W, n = 36) and intestinal I/R + 4 microg/0.1 mL modified aFGF (i.e. non-mitogenic aFGF; group M, n = 36). According to different periods after reperfusion, groups S, W and M were further divided into 0.5-, 1-, 2-, 6-, 12- and 24-h subgroups. The contents of D-lactate and nitrite/nitrate were determined, the changes of intestinal histology were analyzed, the protein expressions of caspase-3, extracellular signal-regulated kinase (ERK)1/2, and p38 were detected by western blot, and apoptotic cells were examined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL) assay at 0.5, 1, 2, 6, 12 and 24 h after I/R, respectively. RESULTS: Compared with rats in group S, intestinal histological damage, apoptotic index, d-lactate content and nitrite/nitrate level all decreased significantly in group W and group M rats. However, there was no difference between rats treated with wild-type aFGF and those with non-mitogenic aFGF. The protein expression of caspase-3, ERK1/2, and p38 in saline-treated rats was higher than those in aFGF-treated rats. CONCLUSIONS: Both types of aFGF had protective effects on gut I/R and there was no significant difference between the two aFGF. The protective effects of aFGF may come from the non-mitogenic activity rather than the mitogenic activity of aFGF in tissue repair, indicating a potentially clinical use for the non-mitogenic effects of aFGF in preventing visceral organ injury triggered by I/R injury in the future.

[Mechanism of signal transduction of differentiation of mesenchymal stem cells into cytokeratin-expressing epidermoid cells].

OBJECTIVE: 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. METHODS: (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. RESULTS: (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%, P < 0.05) 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 P < 0.05), 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 P < 0.05), 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 (P < 0.05), 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 P < 0.05). 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 P < 0.05), (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 P < 0.05). CONCLUSION: 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.

[Relationship between the proliferation and immunoinduction of epithelial cells and the destruction of hair follicles and sebaceous glands in keloids].

OBJECTIVE: To investigate the influence of biological behavior of epithelial cells on the hair follicles and sebaceous glands (HFSG) structure in keloids (K). METHODS: The expression of intercellular adhesion molecule (ICAM)-1, D-related human leucocyte antigen (HLA-DR), and cytokeratin (CK) 14 on epithelial cells and the amount of activity T-lymphocytes were detected in specimens of keloid edge and normal skin with immunohistochemical and histological methods. RESULTS: In comparison with normal skin specimens, epithelial cells were proliferated in K-HFSG presented structural aberration and disintegrate or abnormally to form solid-epithelial island-like structure, and the density of HSFG with hyperplasia and the ageing scar in keloids was apparently decreased. They strongly expressed ICAM-1, HLA-DR, and CK14 in the epithelial cells, there were many immunologic cells which expressed CD4, CD45RO, and interferon (IFN)-gamma around the K-HFSG. The expressed level of epithelial cells was positively correlated with the density of immunologic cells nearby K-HFSG. CONCLUSION: It could be concluded that the reactivity with hyperplasia and immunoinduction of epithelial cells might be associated with the destruction of the some HFSG structure in the keloids.

Analysis of differentially expressed genes in fetal skin of scarless and scar-forming periods of gestational rats.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

[Effect of acidic fibroblast growth factor on mitogen-activated protein kinase activity in small intestinal epithelium after ischemia/reperfusion injury in rat].

OBJECTIVE: To investigate the effect of acidic fibroblast growth factor (aFGF) on p44/p42 mitogen-activated protein kinase (p44/p42 MAPK, extracellular signal-regulated protein kinase, ERK1/2) activity in small intestinal epithelium in rat after ischemia/reperfusion (I/R) injury and its relation with the change in small intestinal epithelium in rat after I/R injury as well as the change in proliferation of epithelial cells. METHODS: Superior mesenteric artery (SMA) was occluded to produce ischemia of the intestine for 45 minutes followed by reperfusion to reproduce I/R injury. One hundred and thirty-two Wistar rats were randomly divided into sham-operation group, I/R group, aFGF treatment group (4 microg of aFGF was injected into jugular vein after reperfusion), and PD98059 (antagonist of ERK 1/2) pretreatment group (7.5 microg of PD98059 was injected via tail vein before ischemia and 4 microg of aFGF was injected via jugular vein after reperfusion). The activity of ERK1/2 and proliferation rate of small intestinal epithelial cells were determined before ischemia and 15 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours and 24 hours after reperfusion. RESULTS: The activity of ERK1/2 in small intestinal epithelium was higher in aFGF treatment group than in I/R control group, and the proliferation rate in small intestinal epithelial cells was significantly enhanced in aFGF treatment group. PD98059, the specific inhibitor of ERK1/2, inhibited ERK1/2 activity and reduced the proliferation rate of small intestinal epithelial cells in aFGF treatment group. CONCLUSION: aFGF can promote small intestinal epithelial cell proliferation in I/R injury rats, and this may be related to activation of ERK1/2 in small intestinal epithelium.

[Cellular phenotype conversion induced by co-culture of human mesenchymal stem cells cocultured with human sweat gland cells].

OBJECTIVE: To investigate the cellular phenotype conversion during human mesenchymal stem cells (hMSCs) cocultured with injured human sweat gland cells (hSGCs) in vitro. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Ontogeny of expression of basic fibroblast growth factor and its receptors in human fetal skin.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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, P<0.05). 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. CONCLUSIONS: 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.

[Apoptosis of neutrophil in peripheral blood in experimental sepsis].

OBJECTIVE: To evaluate the changes in apoptosis of neutrophil in peripheral blood in sepsis in rats. METHODS: The rat sepsis model was reproduced by cecum ligation and puncture (CLP). One hundred and forty-four rats were randomly divided into normal control group, sham operation group and 2, 6, 12, 24, 48, 72 hours after CLP groups. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to identify neutrophil apoptosis. RESULTS: In early period after CLP, neutrophil apoptosis in peripheral blood was limited with a positive rate of less than 5.00%. The positive rate rose to (48.33+/-12.53)% at 48 hours, and it began to lower, approaching the normal level at 72 hours after CLP. CONCLUSION: Death is the main pathway of loss of neutrophils which are produced in the acute phase of sepsis, and apoptosis is the main pathway of loss of neutrophil in the later phase of sepsis.

[Investigation of mesenchymal-epithelial transdifferentiation in the morphogenesis mechanism of embryonic epidermic cells].

OBJECTIVE: To study the relationship between the morphologic mechanism of human embryonic epidermic cells and mesenchymal-epithelial transformation (MET) and its modulation factor. METHODS: Morphological occurrence of epidermis was detected with histologic methods in earlier period [estimated gestational age (EGA) 6-14 weeks] human embryonic skin samples. At the same time, the characteristic expression and their distribution markers of mesenchymal cells [vimentin and alpha-smooth muscle actin (alpha-SMA)], embryonic specific epidermic protein CK8&18, specific protein of epidermic stem cell CK19, transforming growth factor-beta1) (TGF-beta1) and its receptor (TGFbetaRI) in embryonic epidermis were examined with immunohistochemistry and indirect-immunofluorescent doble-labelling method. RESULTS: During EAG 6-8 weeks, ectodermal cells containing Vim+/alpha-SMA(-) were found to transform into epidermal stem cells with CK8&18+/CK19+. In ectodermal cells, protein expression density of TGFbetaRI was moderate (+ +), while positive signal of TGFbeta1 was weak (+/-). After EGA10 weeks, epidermal cells showed typical morphological characteristics. CONCLUSIONS: At EGA 6-8 weeks, human embryonic skin epidermal cells began to form through MET, in which the signal pathway mediated by TGFbetaRI might play important roles, but the role of TGFbeta1 need to be further studied.

Intravenous acid fibroblast growth factor protects intestinal mucosal cells against ischemia-reperfusion injury via regulating Bcl-2/Bax expression.

AIM: To detect the effect of acid fibroblast growth factor (aFGF) on apoptosis and gene expression of bax and bcl-2 gene in rat intestine after ischemia/reperfusion (I/R) injury, and to explore the protective mechanisms of aFGF. METHODS: One hundred and eight Wistar rats were randomly divided into sham-operated control group (C) (n = 6), intestinal ischemia group (I) (n = 6), aFGF treatment group (A) (n = 48) and intestinal ischemia-reperfusion group (R) (n = 48). In group I, the animals were killed after 45 min of superior mesenteric artery (SMA) occlusion, while in groups R and A, the rats sustained 45 min of SMA occlusion and were then treated with normal saline and aFGF, respectively, sustained 15 min, 30 min, 1, 2, 6, 12, 24, or 48 h of reperfusion, respectively. In group C, SMA was separated, but without occlusion. Apoptosis in intestinal villus was determined with terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling technique (TUNEL). Intestinal tissue samples were taken not only for detection of bax and bcl-2 gene expression by RT-PCR, but also for detection of bax and bcl-2 protein expression and distribution by immunohistochemical analysis. RESULTS: The rat survival rates in aFGF treated group were higher than group R (P<0.05) and the improvement of intestinal histological structures was observed at 2, 6, and 12 h after the reperfusion in group A compared with group R. The apoptotic rates were (41.17+/-3.49)%, (42.83+/-5.23)% and (53.33+/-6.92)% at 2, 6 and 12 h after reperfusion, respectively in group A, apparently less than those of group R at matched time points (50.67+/-6.95, 54.17+/-7.86, 64.33+/-6.47, respectively) (P<0.05). The bax gene transcription and translation were significantly decreased in group A vs group R, while mRNA and protein contents of Bcl-2 in group A were obviously higher than those in group R during 2-12 h period after reperfusion. CONCLUSION: The changes in histological structure and the increment of apoptotic rate indicated that the intestinal barrier was damaged after intestinal I/R injury, whilst intravenous aFGF could alleviate apoptosis induced by ischemia and reperfusion in rat intestinal tissues, in which genes of bax and bcl-2 might play important roles.