ObjectiveTo investigate the culture conditions of skin-derived precursors (SKPs) and to explore a new cell source for central nervous system cell transplantation.MethodCells from skins of juvenile and adult mice were isolated and cultured in serum -free medium, and mechanical methods were adapted to passage these cells and ce lls were identified by immunocytochemistry.ResultsA population of SKPs could be isolated from adult and neonatal skins. They co uld be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 8 pas sages, indicating their proliferative capacity. About 50?% of SKPs expressed n estin and the majorities of these cells expressed fibronectin when they were pla ted on polyornithine and laminin coated plates. About 5?% cells showed typical complicated neuronal states and expressed NF-M and NSE when SKPs were plated i n serum-containing medium. These cell could also differentiate into adipocytes and fibroblast-like cells.ConclusionsAdult skin contains stem cells capable of differentiating into neurons, adipocyt es and fibroblast-like cells. SKPs may represent an alternative autologous stem cell source for CNS cell transplantation.

Objective To investigate the effects of negative pressure wound therapy (NPWT) on the expression of EDA+ FN in granulation tissues of human diabetic foot wounds.Methods Forty patients with diabetic foot wounds fitting the inclusion criteria,admitted from Jan.2014 to Jun.2016,were randomly and equally apportioned to receive either NPWT or conventional gauze therapy (control) for 14 days.Granulated tissue biopsies were collected before (0 day) and after (14 day) treatment in both groups.All biopsies were subdivided into two parts.One part was preserved in 4％ paraformaldehyde for immunocytochemical staining of EDA+FN,and the other part was stored at-80 ℃ for Western blotting and PCR analysis of EDA+FN.Results The immunohistochemical analysis revealed that the mean area density of EDA+ FN increased in both NPWT group and control group at day 14 relative to day 0,but the change value of mean area density was higher in NPWT group than in control group (P＜0.01).Western blotting showed that the relative protein levels of EDA+FN increased in both NPWT group and control group at day 14 relative to day 0,but the change value of relative protein levels of EDA+FN was higher in NPWT group than in control group (P＜0.01).The real time PCR analysis demonstrated that the relative mRNA levels of EDA+ FN increased in both NPWT group and control group at day 14 relative to day 0,but the change value of relative mRNA levels of EDA+ FN was higher in NPWT group than in control group (P＜0.01).The results demonstrated the higher protein and mRNA levels of EDA+FN in NPWT group than that in control group.Conclusion NPWT obviously enhances EDA+FN expression in granulation tissue of diabetic foot wound,as a result promotes wound healing.

Objective To develop a high-resolution melting ( HRM ) assay for rapidly screening Gilbert syndrome ( GS) and Crigler-Najjar syndrome ( CNS) associated with UGT1A1 defects.Method Methodology was developed .Then, we applied the established method to analyze 61 clinical samples from neonatal patients with severe unexplained unconjugated hyperbilirubinemia .Neonates with known risk factors for developing hyperbilirubinemia , such as ABO hemolysis, G6PD deficiency, sepsis, hypoxic ischemic encephalopathy were excluded .Five pairs of PCR primers were designed to detect the five common mutations (G211A, C686A, C1091T, C1352T and T1456G) in Asia population.PCR and HRM Assay conditions were optimized.UGT1A1 genotyping in clinical samples was performed by using the established HRM analysis , and all results were subsequently confirmed by direct DNA sequencing .Results The mutants were readily differentiated by using HRM analysis .In this study, 42 neonates were identified with UGT1A1 mutation, and 4 different known variants were detected .Conclusion HRM analysis in this study was economical, convenient, rapid, effective for screening UGT1A1 gene mutations, which can serve as an reliable method for the clinical diagnosis of GS and CNS and the large-scale molecular epidemiological research of UGT1A1 gene-related diseases.

ObjectiveTo investigate the risk factors of metabolic syndrome ( MS ) in obese subjects.Methods A seven-year follow-up study was conducted in 413 simple obese subjects and 196 subjects with normal body weight who were recruited from community residents during physical examination in 2000.There was a 7 years follow-up.Anthropometrics,blood pressure,lipid profile,fasting blood glucose,and 2 h blood glucose after glucose loading were measured.Endothelium-dependent dilatation (EDD) test was also performed.Results Among 553 of 609 subjects who were followed up in 2007,there were 381 simple obese subjects ( simple obese group) and 172 normal weight subjects( normal weight group).Seven-year cumulative incidence of MS was 35.17％ in simple obese group and 8.14％ in normal weight group.In simple obese group,subjects with MS showed greater or higher levels of waist circumference( WC ),waist-hip ratio ( WHR ),triglyceride ( TG ),fasting plasma glucose ( FPG ),fasting insulin (FINS),and homeostasis model assessment for insulin resistance index (HOMA-IR) (all P＜0.05 ),and also decreased EDD( P＜0.05 ) as compared with those without MS.WC,WHR,and FINS were higher( all P＜0.05 ) and EDD was lower( P＜0.05 ) in subjects with MS of normal weight group than those without MS.Logistic analysis showed that the male gender,WC,WHR,FPG,HOMA-IR,and EDD were major risk factors of MS.Conclusion Central obesity,insulin resistance,and endothelial dysfunction are important independent risk factors for development of MS.

Objective To investigate the relationship between Trp64Arg mutation in the β3-adrenergic receptor (β3-AR) gene and the incidence of hypertension in obese subjects. Methods A seven-year follow-up study was conducted in 377 simple obese subjects who had Trp64Arg mutation in the β3-AR gene and some clinical metabolic parameters, such as body mass index, waist circumference, waist-hip ratio, blood pressure, lipid profile, fasting blood glucose, fasting insulin and insulin resistance index (HOMA-IR) were measured in the year of 2000 and 2007. Results The results of follow-up indicated that the incidence of hypertension in Trp64Arg heterozygote subjects were higher than that in Trp64Trp homozygote subjects (52.7% ,69/131 vs 37.0% ,91/246,P < 0.01), the difference was only seen in male (59.5%, 50/84 vs 45.1%, 64/142, P < 0.05). The incidence of hypertension in both Trp64Trp homozygote and Trp64Arg heterozygote subjects were higher in obese male than those in obese female (P < 0.01 or <0.05). After seven years, the blood pressure increased (9.7 ± 4.3)/(5.4 ± 4.0) mm Hg(1 mm Hg = 0.133 kPa) in Trp64Trp homozygote,and (12.8 ± 5.2)/ (7.9 ± 4.7) mm Hg in Trp64Trp heterozygote subjects. Compared with that in Trp64Trp homozygote subjects, lipid profile, fasting insulin and HOMA-IR was increased significantly in Trp64Trp heterezygote subjects (P < 0.05). Logistic analysis showed that β3-AR gene mutation, male, central obesity and insulin resistance were associated with the incidence of hypertension in obese subjects. Conclusion The β3-AR gene Trp64Arg mutation is the independent risk factor in the incidence of hypertension in male.

BACKGROUND:There are myoblasts in human embryonic skeletal muscle. It remains poorly understand whether myoblasts in vitro can form myotube and what are the corresponding markers for identifying myoblasts and myotubes. OBJECTIVE:To investigate whether in vitro cultured myoblasts from human embryonic skeletal muscle can form myotube and whether they can express neural markers. METHODS:Human embryonic muscle-derived myoblasts were cultured in serum-containing medium. When the primary culture was established, cultured cel s were identified with immunocytochemistry for neural markers, such asβ-tubulin markers (desmin, myogenin, smooth muscle actin and myosin). RESULTS AND CONCLUSION:A population of myoblasts could migrate from human embryonic muscle tissues. They could express the markers for skeletal muscle such as desmin and myogenin, and they could express neuron specific enolase, nestin and neurofilament 200. They could form myotubes in vitro, and myotubes expressedβⅢ-tubulin, neurofilament 200 and glial fibril ary acidic protein. The data support the hypothesis that myoblasts from human embryonic muscle express neural markers and muscle markers, and cultured myoblasts and myotubes expressed neuron specific enolase,β-tubulin Ⅲ, nestin, neurofilament 200 and glial fibrillary acidic protein. This indicates that these markers could not be used for cel identification of trans-differentiation study from muscle origin to nervous system.

Objective: To investigate the optimal culture conditio ns for adipose tissue-derived stromal cells(ADSCs) and for the induction of these cells to differentiate into osteogenic cells. Methods: ADSCs were cultured with routine methods,bFGF at 20 ng/ml was added into the medium and the proliferative of ADSCs was examined by cell counting. 0.1 ?mol /L of dexamethasone,10 mmol/L of ?-glycerophosphate and 50 ?mol/L of ascorbic acid were adapted to induce the cells to differentiate into osteogenic cells, ADSCs were identified by immunocytochemistry and differentiated osteogenic cells were identified by alkaline phosphatase(AP) staining and immunocytochemistry. Result: A population of ADSCs could be isolated from adul t human adipose tissue,the cells were fibroblast-like and could be maintaine d in vitro for extended periods with stable population doubling.The cells w ere expanded as undifferentiated in culture for more than 10 passages, indicati ng their proliferative capacity.bFGF stimulated the cell proliferation.Dexameth asone,?-glycerophosphate and ascorbic acid induced (40?8.6)% of ADSCs to ex press alkaline phosphatase(AP) ,(35?10.6)% of AP positive ADSCs were found to be collagen I positive. Calcification plaques were occasionally found in the cul tures. Conclusion:The data support the hypothesis that adu lt human adipose tissue contains stem cells capable of diffferentiating into ost eogenic cells.

Objective To study the changes of sodium currents(I_(Na))of ventricular cells in rats after acute pancreatitis.Methods Rats'models of acute panereatitis were produced by injecting sodium taurocholate into the pancreatic duet.After 24 hours,single ventrieular cells was isolated enzymatieally,and I_(Na),were recorded by using patch clamp techniques.Results I_(Na) peak from ventricular cells in the acute panereatitis group was significantly reduced(- 6.80?2.03)pA/pF compared with that in control group(-13.55?5.33)pA/pF,P＜0.001.The steady-state inactivation curve was shifted to upward direction,the half-maximal voltage dependence of inactivation(V_(0.5))was(-121?26)mV in ventrieular cells from panereatitis group and(-105?21)mV form the control group.I_(Na) returned to normal more slowly in ventrieular cells from panereatitis group than that from control group.Conclusion Inhibitation of I_(Na) was found in ventrieular cells,which might cause arrhythmias in rats with acute pancreatitis.

BACKGROUND:Human skin-derived precursors can be cultured for a long term in vitro, and differentiated into neurons, glial cel s, smooth muscle cel s, Schwann cel s and cel s with peripheral neurons phenotype.
OBJECTIVE:To investigate the culture conditions and multiple differentiation capacity of multipotential stem cel s from human skin, especial y the potentials of differentiating into neurons and osteoblasts.
METHODS:Human skin-derived precursor cel s were cultured with trypsin digestion method, and identified with immunocytochemistry. Cel s at passages 3-4 were induced to differentiate into neurons and osteoblasts, and underwent von Kossa staining protocol for calcium, chondrocyte induction, toluidine blue staining, immunohistochemical staining and Sudan black staining. The expression of nestin, vimentin,βIII-tubulin, S100 and col agen II in the human skin-derived precursors was detected.
RESULTS AND CONCLUSION:The human skin-derived precursor cel s cultured with trypsin digestion method could proliferate and form suspending spheres, and nestin positive cel s were detected at any time point of the culture. Al the cultured cel s expressed vimentin, and some adherent cel s expressedβIII-tubulin. Human skin-derived precursor cel s were induced with Salvia miltiorrhiza to differentiate into neuron-like cel s, and expressed marker of nerve cel s. Skin-derived precursors could be induced to differentiate into osteoblasts and von Kossa staining displayed black calcified nodules in the culture dish. Skin-derived precursors could also be induced to differentiate into chondrocytes, and toluidine blue staining was strongly positive, and some cel s expressed col agen II, which suggested that, the differentiated cel s contained chondrocytes. Experimental findings indicate that, skin contains multipotential stem cel s that are capable of differentiating into osteoblasts, chondrocytes, Schwann cel s and oligodendrocytes.

BACKGROUND:At present,the most frequently agents used for neural induction of bone marrow stromal cells(BMSCs)in vitro are anti-oxidants,such as beta-mercaptoethanol and all trans-retinoids.The majorities of induction from BMSCs are neuron-like cells in these protocols;however,whether it has neuronal function or not should be further studied.OBJECTIVE:TO investigate the differentiated characteristics of inducing human BMSCs into neural cells in serum-free medium.DESIGN:Observational study.SETTING:Chaozhou Central Hospital.MATERIALS:The experiment was carried out in the Chaozhou Central Hospital from April 2004 to December 2005.Adult bone marrows were derived from femoral and tibial bone marrow of three patients with fracture.All patients provided the confirmed consent and were approved by the Ethics Committee of Chaozhou Central Hospital.DMEMIF12 medium(1:1),fetal bovine serum (FBS), glutamine, N2 supplements and B27 Supplements were from GIBCO/BRL Company;recombinant basic fibroblast growth factor(bFGF)and recombinant epidermal growth factor(EGF)from Sigma Company;monoclonal antibody for vimentin(1:100),monoclonal antibody for myelin basic protein(MBP) (1:100),monoclonal antibody for S1 00(1:1 00),monoclonal antibody for neuron specific enolase(NSE)(1:1 00),and monoclonal antibody for neurofilament 200(NF200)(1:1 00)from Beijing Zhongshan Company;monoclonal antibody for glial fibrillary acidic protein (GFAP)(1:200)and polyclonal antibody for nestin(1:100)from Boster Company(Wuhan);mouse monoclonal antibody for beta-tubulin 3(1:1 000)from Sigma Company;SP-9000 kits and quick AEC from Beijing Zhongshan Company; culture dishes and flasks from Coming Company.METHODS:BMSCs from human bone marrow were cultured in serum-containing medium.When the primary culture was established, BMSCs were transferred into serum-free medium containing N2 or B27 supplement with 20 μg/L basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF),and cells were cultured in an incubator containing C02of 0.05 volume fraction at 37℃. Morphological changes of BMSCs in serum-free medium were observed under phase contrast microscope. And two days after culture. Expression of relative markers of BMSCs was detected withimmunocytochemistry.MAIN OUTCOME MEASURES:Morphological changes of BMSCs and expression of relative markers of nerve cells.RESULTS:A population of BMSCs could be isolated from adult human bone marrow,and they were processed to obtain a fibroblast-like population and were expanded as undifferentiated cells in culture for more than 10 passages.indicating their proliferative capacity.They could form spheroid state when they were sub-cultured in serum-free media supplemented with bFGF and EGF.these cells could express the markers for neural stem cells such as vimentin and nestin;they could expressed neuron specific enolase(NSE),beta-tubulin 3,TrkC and neurofilament 200(NF200)when they were plated on dishes with serum-containing medium; some cells exhibited the phenotypes for astrocytes.expressing gilal fibrillary acidic protein(GFAP)and S100 protein.CONCLUSION:The morphology,protein expression and differentiation ability of BMSCs in serum-free medium was similar to those of neural stem cells.The data support the hypothesis that adult bone marrow contains stem cells capable of differentiating into neural cells,the serum-free media make BMSCs overcome their mesenchymal commitment,showing the phenotypes for neural stem cells.