To investigate the culture method for epidermal stem cell in vitro. Rat skin was peeled from the subcutaneous tissue gently, and cut into small pieces, then incubated for 24h in 0 25%trypsin in a 4℃ shaker. The epidermis was separated from the dermis, and shaken for 10min in 0 25% trypsin at 37℃ to dissociate into single cells. Digestion was terminated by the addition of culture medium +10% serum, and the cells were gently centrifuged and resuspended in the culture medium, which constituted EMEM without calcium, supplemented with 0 05mmol/L CaCl 2 , 9% chelexed FBS, 4ng/ml EGF, 0 5?g/ml gentamicin, and 50% fibroblast conditioned medium (CM ). The CM ( EMEM without calcium, 0 05mmol/L CaCl 2 , 9% chelexed FBS, 0 5?g/ml gentamicin ) was collected from freshly isolated primary neonate fibroblast cultures after 48h, which were passed through a filter with 0 45?m pores, and stored at -20℃ for use. 1?10 6 /ml epidermal cells were incubated for 10～15min at 37℃ on dishes coated with collagen Ⅳ, then the nonadherent cells were rinsed off. The adherent cells were grown to confluency. Cultures were subcultured in a solution of TGG for 5～10min. Once free of the dish, cells were centrifuged, resuspended in fresh medium, and then cultured at 1?10 5 /ml. Cultures were observed for colony formation under a phase contrast microscope, and the structure of the rapidly adherent cells was observed under electron microscopy; the expression of ? 1 integrin and K19 was detected in the rapidly adherent cells and colony cells with SP immunohistochemical methods. The results showed that the cells selected by rapid adherence to collagen type Ⅳ formed large colonies at 7 days, and showed a im mature feature under electron microscopy, manifesting ? 1 integrin and K19 expression. It is suggested that epidermal stem cells could be cultured in vitro .

The development of sweat glands is a very complex biological process, which involves many factors. In this study, the correlation between epidermal growth factor (EGF), matrix metalloproteinases (MMP 2,MMP 7) and development of sweat glands in human embryos was explored. Furthermore, the mechanism(s) underlying the induction of epidermal stem cells into sweat gland cells was elucidated. Skin biospies of human embryos obtained from spontaneous abortions at different gestational ages from 11 to 31 weeks were used. The dynamical expression of EGF, MMP 2, MMP 7 and keratin 7(K7) in developing sweat gland cells or extracellular stroma surrounding the sweat gland cells were examined with S P immunohistochemical methods. The localization of the cellular sources of MMP 2 and MMP 7 was examined with in situ hybridization. The results showed that at 14 20 wk of gestation, a gradual increase of EGF immunoreactivity was observed not only in developing sweat gland buds but also in extracellular stroma surrounding the buds,and the expression intensity peaked at 20～22 wk of gestational age. All mRNA positive buds or cells in developing sweat glands contained corresponding immunoreactive proteins. The immunostaining for K7 appeared in early sweat gland buds at 14～16wk of gestation, and from then on, K7 was concentrated in developing sweat gland cords or cells. It is suggested that the morphogenesis of sweat gland in human fetal skin begins at 14～16wk of gestational age, and essentially completes by 24wk. There is a close relationship among EGF ,extracellular matrix remodeling and morphogenesis of sweat glands, and EGF is one of the inducers in the development and maturity of sweat gland buds or cells.

To investigate the distribution 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 labeled 60 days previously with 5-Bromodeoxyuridine (BrdU) (4 wounds in each animal). Then these 80 wounds were randomly divided into 2 groups as follows: group A: with topical treatment of epidermal growth factor (n=40) and group B: no-treatment (n=40). BrdU, ? 1 integrin and keratin19 (K19) were employed to determine the epidermal stem cells with streptavidin-peroxidase (SP)immunohistochemical method, and the speed and quality of epithelialization were determined with routine histological methods with HE staining on the 3rd, 7th, 14th, and 21st day after the wounding. Results showed that the healing rate of wounds was 80% in group A (32/40) and 60% in group B (24/40). No cells with positive immunostaining for BrdU, ? 1 integrin, or K19 were found in the granulation tissue of all wounds in both groups during the healing process. However, a few BrdU, ? 1 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 results suggested that epidermal stem cells appearing on the wound edges were the main source of re-epithelialization of granulating wounds.

The differentiation of cell is a very important biological process. In this paper, the current understanding and advances in research on dedifferentiation are reviewed. Giving more efforts to study this subject may help us disclose the mechanisms and treatment for some diseases.

Objective To observe the effects of electrical stimulation of paraventricular nucleus(PVN) on gastric mucosal cellular apoptosis,proliferation,and expression of BCL-2,BAX induced by gastric ischemia-reperfusion(GI-R) and the potential mechanisms of protection of PVN on GI-R injury.Methods After electrical stimulation of PVN,the experimental model of GI-R were established by clamping the celiac artery for 30 min and then reperfusing the artery for 30 min,1 h,3 h,or 6 h respectively.We used immunohistochemistry to detect the gastric mucosal cells apoptosis,proliferation and the expression of BCL-2,BAX.Results Compared with GI-R group,the electrical stimulation of PVN markedly decreased gastric mucosal cellular apoptosis,increased the proliferation,and promoted the protein expression of BCL-2,but markedly inhibited the protein expression of BAX at 30 min,1 h,3 h after reperfusion respectively.Conclusion The protective effect of PVN on GI-R injury is associated with up-regulation of expression of BCL-2 and down-regulation expression of BAX,and so inhibited gastric mucosal cellular apoptosis and promoted proliferation.

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.

Objective To investigate the effect of jejunal infusion of amino acids on secretion of gastrointestinal hormone in healthy dogs.Methods Six healthy adult dogs were treated with jejunal fistulas and femoral vein intubation.Twenty-four hours after the operation,solution of 8 different amino acid monomers (experimental group) or normal saline (control group) were infused into the jejunum of the dogs every 24hours.The levels of cholecystokinin (CCK),motilin,and gastrin in the peripheral plasma were measured using radioimmunoassay at the start of infusion (0 minute),and 30,60,90,and 120 minutes after infusion.Results Compared with the control group,the serum CCK level in the phenylalanine group was significantly higher 30 and 60 minutes after infusion [(1.25 ±0.19) ng/L vs.(0.66 ±0.14) ng/L,(1.23 ±0.12) ng/L vs.(0.80 ± 0.03) ng/L,both P ＜ 0.01],while that in the tryptophan group was significantly higher 30 minutes after infusion [(1.08 ±0.26) ng/L vs.(0.66 ±0.14) rig/L,P ＜0.01].The other measurement results showed no statistically significant differences.Conclusions Jejunal infusion of phenylalanine or tryptophan may stimulate the secretion of gastrointestinal hormone to some extent.Aromatic amino acids (phenylalanine and tryptophan) is more potent in triggering the release of CCK than aliphatic (leucine,isoleucine,and methionine) and charged amino acids (aspartic acid,arginine,and glutamate).The mechanism may be related to the properties of the amino acids.

OBJECTIVE:To explore the working model of developing pharmacovigilance in our hospital. METHODS:179 ADR cases reported in our hospital in 2008 were analyzed and the measures to carry out pharmacovigilance were introduced. RESULTS:Due to the practice of pharmacovigilance,the incidence of serious ADRs in our hospital was lowered significantly,down to 0 case in 2008 from 8 cases during 2005～2007; the proportion of rational drug use increased and the ADR reporting rate increased. CONCLUSION:To maintain high level of rational drug use,it is imperative to develop pharmacovigilance in our hospital.

OBJECTIVE: To evaluate the efficacy of standardized medication for patients with chronic rhinosinusitis. METHOD: According to the diagnosis and treatment guidelines on chronic rhinosinusitis formulated in 2008, by means of prospective study, we studied 54 patients suffering from chronic rhinosinusitis treated with standardized medication including, a combination of local intranasaI corticosteroids, macrolides, mucus discharging agent and nasal irrigation treatment and followed up for 3 months. Visual analogue scale (VAS), sino nasal outcome test-20 Chinese version scales (SNOT-20 CV), Lund-Mackay CT and Lund-Kennedy endoscopy methods were employed to conduct the subjective and objective assessment and comprehensively evaluate the clinical efficacy before and after treatment. RESULT: (1) After three months of standardized medication, the patients' total scores of VAS, SNOT-20 CV, CT and endoscopy were improved significantly compared with those before-treatment (P < 0.01 for all these scoring systems). (2) There was statistically significant difference between the clinical efficacies of chronic rhinosinusitis patients with and without nasal polyps groups (P < 0.01). After 3 months of standardized medication, the effective rates of the CRSwNP group evaluated by subjective assessment and CT evaluation were 66.7% and 94.4% respectively, while those of the CRSsNP groups were 91.7% and 97.2% respectively. (3) Betwecn CRSwNP and CRSsNP groups, there was no significant difference in the improvement rate or inefficiency rate in subjective assessment except for the cure rate, while there were significant differences in both cure rate and improvement rate in CT evaluation. (4) The CRS patients' self-testing-based questionnaires results showed positive correlation with objective assessments. CONCLUSION The standardized medication with combination of intranasal local glucocorticoid, macrolides (14-membered ring), the mucus discharging agent and nasal irrigation on CRS was effective.
Administration, Intranasal ; Adult ; Aged ; Chronic Disease ; Female ; Glucocorticoids ; administration & dosage ; therapeutic use ; Humans ; Macrolides ; administration & dosage ; therapeutic use ; Male ; Middle Aged ; Prospective Studies ; Quality of Life ; Rhinitis ; drug therapy ; Sinusitis ; drug therapy ; Treatment Outcome ; Young Adult

Objective To evaluate the safety, efficiency and feasibility of HLA-identical sibling using culture-expanded mesenchymal stem cells and hematopoietic stem cells in treatment for myelodysplastic syndrome (MDS). Also to investigate for valid preventive measures to avoid the infection of HBV originated from donor. Methods A 46-years-old male patient with myelodysplastic syndrome-refractory anemia (MDSRA) got a cotransplantation of culture-expanded mensenchymal stem cells (MSC) and hematopoietic stem cells (HSCs) from HLA-identical sibling donor (his sister) who was infected by hepatitis B virus (HBV). Some measures were applicated in order to avoid the recipient from getting a HBV infection. The antiviral therapy to the donor was began early at the time 1 month before transplant, and HBV vaccine inoculation was used 2 month before transplant. High titer of anti-hepatis B immunoglobulin was used 1 week before transplant and 1 month after transplant the use of prophylactic anti-hepatis B drug treatment was begun. A non-myeloablative preparative regimen included fludarabine monophosphate (Flu, 120 mg/m2), cyclophosphamide (Cy, 1200 mg/m2)and antithymocyte globulin (ATG, 15 mg/kg) was given to him before culture-expanded mesenchymal stem cell and allogeneic peripheral blood stem cell from his HLA-matched sister. Results The regimen was well tolerated, and hemopoiesis was reconstituted on day 10 after transplant, idiochromosome detected by fluorescent in situ hybridization on day 30 showed XY 47/300 and on day 90 it was 7/300. No evidence of HBV infection was detected on day 60 after transplant. Conclusion The clinical course of this patient indicate that HLA-identical sibling culture-expanded mesenchymal stem cell transplantation combined with non-myeloablative stem cell transplantation can be an effective and safe approach in treatment of MDS.