Background The loss of perspiration after a massive deep burn hampers the survivor to lead a life of high quality, as they are deprived the function of regulating body temperature through perspiration during sultry months. With maturation of science of burn care, the number of survivors is increased, therefore, it is imperative that this problem should be tackled in order to improve their quality of life. Objective To explore the possibility of transdifferentiating bone marrow mesenchymal stem cells (MSCs) into sweat gland cells (SGCs), and implanting the latter into fresh skin wound to generate functional sweat glands. Methods Human bone marrow MSCs and SGCs were isolated from the same patients. They were identified with specific markers, and then co-cultured. The stem cells which subsequently exhibited the phenotype of sweat gland cells were implanted into scald injured paws of nude mice, and regeneration of functioning sweat glands was confirmed by perspiration test (iodine and starch) and histological examination. A male patient bearing almost iden- tical burn scars on the posterior aspect of both arms was enrolled for clinical trial. The scars were first proved to be anhydrotic with iodine and starch test. With patient's written consent, the clinical trial was carried out. Bone marrow MSCs and sweat gland cells were obtained from the patient. After being heat shocked, the SGCs were co-cultured with MSCs. Three days later, the scars of both arms were excised. MSCs having acquired the phenotype of sweat gland cells after co-culture were evenly spread onto the excision wound on the right arm. They were covered with a piece of acellular allogeneic dermis, which was perforated with numerous micropores. On top of the latter, micrografts of autologous origin were transplanted, and the wound was finally covered with a piece of allogeneic skin graft. The wound on the left side was similarly covered, but without transdifferentiated MSCs. After complete healing of the wounds, perspiration test with iodine and starch was performed, and biopsy was taken from the MSCs transplanted area. The components of the sweat collected from the implantation area were analyzed and compared with that from normal skin elsewhere on the body. The same procedure was performed in a girl patient with a chin-neck contracture. The scar was totally excised, and into one third of the excision wound in vitro transdifferentiated MSCs were implanted similar to the above patient. The examinations were repeated after wound healed. Results In the animal experiment, it was shown that there was regeneration of functional sweat glands in the burned paws of the nude mice. In human patients, all wounds healed nicely. The areas where transdifferented MSCs were implanted showed positive iodine-starch perspiration test. Histological and immunohistochemical examination confirmed that the transformed MSCs bore the specific marker carcinoembryonic antigen (CEA) of sweat gland cells. Biochemical analysis of the excreted sweat contained similar components as that of sweat collected from normal skin. Conclusions MSCs can be transdifferentiated into SGCs in vitro, and they can be implanted into a fresh wound to form functional sweat glands. However, enormous amount of work should be done before the same result would be realized in patients with massive deep burn within a short duration after the injury, so that the patients could regain the function of perspiration after surviving the massive loss of normal skin.

Aim To investigate the protective effect of non-mitogenic human acidic fibroblast growth factor (nm-haFGF) on cerebral ischemia-reperfusion injury in mice. Methods Cerebral ischemia-reperfusion model was made by ligating bilateral carotid for 20 minutes in mice. These mice were randomly divided into model group( iv NS), two doses of nm-haFGF (iv 25、50 ?g?kg-1) groups, rhaFGF group(iv 50 ?g?kg-1) and sham- operated group. Step down test and Y-type electric maze were used to examine the effect of nm-haFGF on learning and memory of mice, then Even′s Blue(EB) level and NO level in brain of these mice were measured. Results The nm-haFGF significantly decreased numbers of errors of mice in 5 min in step down test and in Y-type electric maze test; EB and NO levels in brain of these mice were lower than those of model group respectively. Conclusion The nm-haFGF can protect cerebral ischemia-reperfusion injury in mice.

OBJECTIVETo study the effects of low concentration dopamine(DA) on hydrogen peroxide-induced apoptosis in cultured rat cardiomyocytes as well as the possible molecular mechanisms.

METHODSCultured neonatal rat cardiomyocytes were randomly divided into the following groups: control group (control), hydrogen peroxide group (H2O2), pretreated with low concentration dopamine ( DA + H2O2), dopamine receptor l(DR1) antagonist group (DR1 + DA + H2O2), dopamine receptor 2(DR2) antagonist group (DR2 + DA + H2O2). The cell apoptosis was then assessed by MTT and flow cytometry. The cellular ultrastructure changes were observed by transmission electron micro- scope. The activity of lactate dehydrogenase(LDH )and superoxide dismutase (SOD) in cell medium was analyzed by colorimetry. The protein expressions of Cytochrone c, Caspase 3 and Caspase 9 were obtained by Western blot.

RESULTSCompared with hydrogen peroxide group, low concentration dopamine(10 µmol/L) decreased the apoptosis rate and the expression of protein of apoptosis related protein, enhanced SOD activity, decreased LDH activity. DR1 antagonist SCH-23390 treatment inhibited dopamine induced cardiac protective effect. DR2 antagonist haloperido treatment had no changes compared with dopamine group.

CONCLUSIONAbove findings indicate that low concentration dopanine inhibits apoptosis induced by hydrogen peroxide in neonatal rat cardiomyocytes, which is partly associated with the activation of DR1.

Animals ; Apoptosis ; Benzazepines ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cells, Cultured ; Dopamine ; pharmacology ; Hydrogen Peroxide ; L-Lactate Dehydrogenase ; metabolism ; Myocytes, Cardiac ; drug effects ; Rats ; Rats, Wistar ; Receptors, Dopamine D1 ; metabolism ; Superoxide Dismutase ; metabolism

Adolescent ; Anti-Bacterial Agents ; therapeutic use ; Calcitonin ; blood ; Calcitonin Gene-Related Peptide ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Protein Precursors ; blood ; Respiratory Tract Infections ; blood ; drug therapy

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 purposed to investigate the efficacy of different whole flow lysis reagents for lysis of red blood cells in flow cytometric analysis. The expression of immunocytes was detected by flow cytometry after lysis of red blood cells using commercial reagents (Optilyse C, FACS Lysing Solution) and self-made red blood cell lysis reagents (RBC Lysis Buffer), the detection results were analyzed comparatively. The results showed that there was no significant difference in the percentage of CD3e(+), CD3e(+)CD4(+), CD3e(+)CD8a(+), CD3e(-)CD19(+), CD3e(-)NK1.1(+) and Gr-1(+) cells between 3 different lysis reagent groups. However OptiLyse C solution was suitable to Gr-1(+) cell detection, but did not suit to Foxp3(+) Treg detection. The self-made RBC Lysis Buffer and FACS Lysing Solution were suited to Foxp3(+) Treg detection. It is concluded that the use of self-made RBC Lysis Buffer for flow cytometry can get the lysis efficiency of commercially available lysis solutions when samples are prepared in accordance with standardized procedure. The self-made RBC Lysis Buffer not only can satisfy experimental requirements, but also can reduce the experimental costs.
Animals ; Erythrocyte Count ; Erythrocytes ; immunology ; metabolism ; Flow Cytometry ; instrumentation ; methods ; Immune System ; immunology ; Indicators and Reagents ; analysis ; Mice ; Mice, Inbred C57BL

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

Effects of FEMY-R7, composed of fucoidan and evening primrose extract, on the bacterial growth and intragastric infection of Helicobacter pylori as well as gastric secretion were investigated in comparison with a proton-pump inhibitor pantoprazole. For in vitro anti-bacterial activity test, H. pylori (1x10(8) CFU/mL) was incubated with a serially-diluted FEMY-R7 for 3 days. As a result, FEMY-R7 fully inhibited the bacterial growth at 100 microg/mL, which was determined to be a minimal inhibitory concentration. In addition, 6-hour incubation with H. pylori, FEMY-R7 inhibited urease activity in a concentration-dependent manner, showing a median inhibitory concentration of 1,500 microg/mL. In vivo elimination study, male C57BL/6 mice were infected with the bacteria by intragastric inoculation (5x10(9) CFU/mouse) 3 times at 2-day intervals, and simultaneously, orally treated twice a day with 10, 30 or 100 mg/kg FEMY-R7 for 7 days. In Campylobcter-like organism-detection test and bacterial identification, FEMY-R7 exerted a high bacteria-eliminating capacity at 30-100 mg/kg, comparably to 30 mg/kg pantoprazole. In contrast to a strong antacid activity of pantoprazole in a pylorus-ligation study, FEMY-R7 did not significantly affect gastric pH, free HCl, and total acidity, although it significantly decreased fluid volume at a low dose (10 mg/kg). The results indicate that FEMY-R7 eliminate H. pylori from gastric mucosa by directly killing the bacteria and preventing their adhesion and invasion, rather than by inhibiting gastric secretion or mucosal damage.
Animals ; Bacteria ; Gastric Mucosa ; Helicobacter pylori ; Homicide ; Humans ; Hydrogen-Ion Concentration ; Male ; Mice ; Oenothera biennis* ; Urease

As the request of the authors, Acknowledgments section has been changed.
Oenothera biennis*

OBJECTIVETo explore the inhibitory effect of combined administration of bear bile powder (BBP) and cyclophosphamide (Cytoxan, CTX) on colorectal cancer liver metastasis by regulating tumor promotion inflammation microenvironment.

METHODThe CRC liver metastasis mode in mice was established through in situ spleenic injection of SL4 tumor cells into spleens. The mice were randomly divided into 5 groups: the model group, the CTX (80 mg x kg(-1)) treatment group, the CTX + BBP high dose (300 mg x kg(-1)) group, the CTX + BBP middle dose (150 mg x kg(-1)) group and the CTX + BBP low dose (75 mg x kg(-1)) group. Mice were orally administered with drugs for 12 days, and sacrificed on the 13'h day for weighing their spleens and lives, HE staining, and immunofluorescence analysis. Their peripheral blood, and metastatic tumor in spleens and lives were analyzed with flow cytometry.

RESULTSpleen and liver weights of the: CTX treatment group and other doses groups were significantly lower than that of the model group. HE staining and immunofluorescence analysis showed that lymphocyte infiltration was detected in normal tissues, and macrophages infiltration was observed around the tumor tissues. Flow cytometry analysis showed that the number of T-lymphocytes in peripheral blood of different doses groups were much higher than that of the CTX treatment group (P < 0.05), with the rise in the ratio of CD4/CD8; the total number of lymphocytes in spleen cell suspension increased in different doses groups, compared to the CTX treatment group, with notable increase in B cells (P < 0.05) and significant decrease in CD11b, F4/80 cells (P < 0.05). The combined treatment showed less monocyte macrophages in liver metastasis than that of the CTX treatment group.

CONCLUSIONThe combined treatment of bear bile powder and cyclophosphamide has the effect in not only protecting liver and increase immunity, but also in anti-inflammation and antitumor by regulating tumor microenvironment and reducing the collection of mononuclear macrophages. Particularly, the combined administration of low dose of bear bile powder and CTX shows the most significant effect in reducing inflammatory cell infiltration.

Animals ; Bile ; chemistry ; Colorectal Neoplasms ; drug therapy ; mortality ; pathology ; Combined Modality Therapy ; Cyclophosphamide ; administration & dosage ; Humans ; Liver Neoplasms ; drug therapy ; mortality ; physiopathology ; secondary ; Male ; Mice ; Mice, Inbred C57BL ; Tumor Microenvironment ; drug effects ; Ursidae