Objective To investigate the proportion and function of CD_4~+ CD_(25)~+ regulatory T cells (CD_4~+ CD_(25)~+ Tr)in unexplained recurrent spontaneous abortion(URSA).Methods(1)Proportion measurement:the proportion of CD_4~+ CD_(25)~+ Tr cells in peripheral blood was measured by double-label flow cytometric analysis.The samples were taken from 15 URSA women,15 normal non-pregnancy women and 13 normal pregnancy women.(2)Function measurement:CD_4~+ CD_(25)~+ Tr ceils and CD_4~+ CD_(25)~+ T ce]ls were extracted from peripheral blood lymphocytes by the microbeads separation.The purity of CD_4~+ CD_(25)~+ Tr cells and CD_4~+ CD_(25)~+ T cells was measured by flow cytometry.The growth inhibitory effect of CD_4~+ CD_(25)~+ Tr cells on CD_4~+ CD_(25)~+ T cells was assessed in vitro.Results The proportion of CD_4~+ CD_(25)~+ Tr cells was decreased significantly in URSA women(6.9?1.8)% than that in normal non-pregnancy women[(10.8?1.1)%] (P0.05).Conclusion The results suggest that decrease in proportion and function of CD_4~+ CD_(25)~+ Tr cells may be associated with URSA.

OBJECTIVETo investigate the protective effect of phosphodiesterase type 5 inhibitors (tadalafil) on the testis following testicular ischemia-reperfusion injury in rats.

METHODSEighty-four healthy adult male SD rats were randomly and equally divided into groups A (sham operation), B (testicular torsion + low-dose tadalafil), C (testicular torsion + high-dose tadalafil), and D (testicular torsion + placebo). Models were established in the latter three groups by 7200 torsion of the right testis for 2 hours. The animals in groups A and B were treated by gavage with tadalafil at the dose of 0. 5 mg per kg per day, those in group C at 2 mg per kg per day, and those in group D with saline at the same dose. After 3, 7, and 14 days of treatment, the torsioned testes were harvested for evaluation of the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the testis tissue. The pathological changes in the testis were observed under the light microscope.

RESULTSAt 3, 7, and 14 days, the SOD activity was (254.46 +/- 7.43), (278.49 +/- 8.33), and (317.99 +/- 3.31) nU/mg prot in group B, and (277.12 +/- 8.80), (309.40 +/- 2.14), and (320.39 +/- 4.72) nU/mg prot in group C, all obviously higher than in D ([223.21 +/- 4.65], [231.45 +/- 4.16] and [248.28 +/- 5.74] nU/mg prot), while the MDA content was lower in the former two groups than in the latter. At 3 and 7 days, the SOD activity was significantly higher and the MDA level significantly lower in group C than in B (both P < 0.01) , while at 14 days, neither showed any remarkable differences between the two groups (P > 0.05). No obvious histopathological change was observed in the testis tissue of group A. At 3 and 7 days, pathological examination of the testis tissue revealed significant differences in the number of seminiferous epithelial layers, testicular histological score, and seminiferous tubule diameter in group B (P < 0.01), but the three indexes at 14 days in group B and at 7 days in group C exhibited no remarkable differences from those at 14 days in group A.

CONCLUSIONTadalafil can alleviate testicular ischemia-reperfusion injury following testis torsion/detorsion in a time- and dose-dependent manner.

Animals ; Biomarkers ; metabolism ; Carbolines ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Male ; Malondialdehyde ; metabolism ; Phosphodiesterase 5 Inhibitors ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; Seminiferous Tubules ; pathology ; Spermatic Cord Torsion ; complications ; Superoxide Dismutase ; metabolism ; Tadalafil ; Testis ; blood supply ; metabolism ; pathology ; Time Factors

BACKGROUNDPatients 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.

METHODSThe 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.

RESULTSEndogenous 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).

CONCLUSIONSOur 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.

Adult ; Animals ; Blotting, Western ; Bone Marrow Cells ; cytology ; Cell Proliferation ; Cells, Cultured ; Ectodysplasins ; genetics ; metabolism ; Female ; Flow Cytometry ; Humans ; Immunohistochemistry ; Male ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Pregnancy ; Receptors, Ectodysplasin ; Reverse Transcriptase Polymerase Chain Reaction ; Sweat Glands ; cytology ; metabolism ; Transfection ; Young Adult

This study was aimed to construct a two-dimensional culture system by using osteoblasts induced from human marrow mesenchymal stem cells (MSC) and to investigate its support effect on survival of hematopoietic stem/progenitor cells for umbilical cord blood (UCB) ex vivo. MSCs were isolated from adult human bone marrow and were cultured, the second generation of MSCs were induced into osteoblasts which were irradiated with 20 Gy gamma rays in a Cobalt 60 source and confluenced into a feeder layer. CD34(+) cells were selected from fresh umbilical cord blood samples by using Microbead Kit of MiniMACS and seeded into the two-dimensional culture system to culture ex vivo without exogenous cytokines. By using colony-forming assay, high proliferative potential colony-forming cell assay, and long-term culture initiating cell assay, the ability of the two-dimensional system to culture HSCs/HPCs was observed. The results showed that the osteoblasts induced from bone marrow MSC in constructed two-dimensional culture system displayed more significant support effect on survival of hematopoietic stem/progenitor cells from umbilical cord blood (UCB) ex vivo, compared with other culture systems, especially on long term HSCs survival ex vivo. It is concluded that the two-dimensional culture system constituted by osteoblasts induced from human MSCs has certain ability of supporting maintenance and multipotency of HSCs/HPCs from umbilical cord blood in vitro, especially sustaining survival of HSC in long-term culture. It has also been proved that osteoblasts play a crucial role in regulation of HSC growth.
Bone Marrow Cells ; cytology ; Cell Differentiation ; Coculture Techniques ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteoblasts ; cytology ; physiology

OBJECTIVETo investigate the influence of biological behavior of epithelial cells on the hair follicles and sebaceous glands (HFSG) structure in keloids (K).

METHODSThe 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.

RESULTSIn 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.

CONCLUSIONIt 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.

Adolescent ; Adult ; Cell Proliferation ; Child ; Child, Preschool ; Epithelial Cells ; pathology ; Female ; HLA-DR Antigens ; metabolism ; Hair Follicle ; pathology ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Keloid ; immunology ; pathology ; Keratin-14 ; metabolism ; Male ; Middle Aged ; Sebaceous Glands ; pathology ; Young Adult

OBJECTIVETo investigate the migrating effect of adipose derived stem cells (ADSCs) on the wound model of human epidermal keratinocyte (HEKa).

METHODSRat 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.

RESULTSThe 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).

CONCLUSIONSThe rADSCs can promote the migration of HEKa by direct contact with it.

Adipose Tissue ; cytology ; Animals ; Cell Count ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Epidermis ; cytology ; Humans ; Keratinocytes ; cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; Wound Healing

The promoter of NAR gene in Magnaporthe grisea was isolated and sequenced. The promoter sequences contained the "TATA" box, the "CAAT" box, and binding sites for fungal regulatory proteins. Programs that predict promoter sequences indicated that promoter sequence lies between locations 430 and 857 of the NAR promoter fragment. GFP expression under the NAR promoter and NAR transcript analysis revealed that this promoter is activated primarily at the mycelial stage in the rice blast fungus and could be used to express native or extrinsic genes in the mycelia of the rice blast fungus.
Base Sequence ; Cloning, Molecular ; Fungal Proteins ; genetics ; Gene Expression ; genetics ; Hyphae ; genetics ; Magnaporthe ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics ; Transcriptional Activation ; genetics

OBJECTIVETo 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.

METHODSThe 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.

RESULTSThere 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.

CONCLUSIONThere 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.

Adult ; Epithelial Cells ; metabolism ; ErbB Receptors ; metabolism ; Female ; Fetus ; metabolism ; Humans ; Immunohistochemistry ; Receptor, ErbB-2 ; metabolism ; Skin ; metabolism ; Wound Healing

OBJECTIVETo observe the changes in matrix metalloproteinase-2,7 (MMP-2,7) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in deep partial thickness burn during the process of wound healing, and the effects of bFGF on wound healing.

METHODSThe rats inflicted by 30% TBSA deep partial thickness burn were randomly divided into simple scald and bFGF treatment groups. Biopsies from wound skin were harvested at 3 and 6PBHs and 1, 3, 7, 14 PBDs for the detection of the epithelialization rate and collagen content. The above indices were also detected in the skin of another 6 normal rats as normal control.

RESULTS(1) The epithelialization rate in bFGF treatment group was higher than that in simple scald group during 3PBH to 14 PBD. (2) The collagen contents in both bFGF treatment group and simple scald group were continually decreased during 3 PBH to 3 PBD, and increased from 7 to 14 PBD, but still lower than that in normal control (P < 0.05). (3) The expression of MMP-2,7 and TIMP-2 in simple scald group enhanced from 1 to 14 PBD, and peaked on 7 PBD. (4) The expression of MMP-2,7 in bFGF treatment group was similar to that in simple scald group from 3 to 6 PBH, while the expressions of MMP-2,7 and TIMP-2 was higher than those in simple scald group from 1 to 14 PBD.

CONCLUSIONThe collagen deposition would be affected by the activities of extracellular matrix in scald wound in rats. Changes in MMP-2,7 and TIMP-2 expressions were an important process of wound repair, which was closely related to the acceleration of wound healing by the application of bFGF.

Animals ; Burns ; metabolism ; Collagen ; analysis ; Epithelium ; physiology ; Fibroblast Growth Factor 2 ; pharmacology ; Male ; Matrix Metalloproteinase 2 ; analysis ; Matrix Metalloproteinase 7 ; analysis ; Rats ; Rats, Wistar ; Tissue Inhibitor of Metalloproteinase-2 ; analysis ; Wound Healing

OBJECTIVETo investigate the different location and the expression characteristics of epidermal stem cells in normal adult skin and scar tissue.

METHODSSkin 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.

RESULTSThe 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.

CONCLUSIONThe 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.

Adult ; Burns ; complications ; metabolism ; pathology ; Cicatrix ; etiology ; metabolism ; pathology ; Epidermis ; chemistry ; cytology ; Humans ; Immunohistochemistry ; Integrin beta1 ; analysis ; Keratin-10 ; Keratin-14 ; Keratins ; analysis ; Middle Aged ; Skin ; chemistry ; cytology ; Stem Cells ; chemistry ; cytology