AIM:To investigate the potential of hematopoietic stem cells(HSCs)derived from mouse embryonic stem cells(ESCs)to reconstruct hematopoiesis in vivo.METHODS:Using a three-step method,a mice embryonic stem cell line,E14.1 was induced into hematopoietic stem cells.The cell markers with CD34+/Sca-1+ were identified by flow cytometry analysis,then HSCs(1?109 cells/L)from third-step were injected into SCID mice for observing teratoma formation.To validate function of HSCs,colonogenic cell assay was conducted and the hematopoiesis in lethally irradiated mice was reconstituted.RESULTS:The method of three-step differentiation,combined to use more hematopoietic stimulating factor promoted the E14.1 cell differentiation into HSCs with highest percent of CD34+/Sca-1+ cells(as high as 58.64%?4.20%)with more CFU-E,CFU-GM and CFU-GEMM populations.The cells showed the character of hematopoietic progenitors by Wright-Giemsa staining.Positive selected CD34+/Sca-1+ cells by magnetic sorting from third-step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 d after transplantation,with 71.4%(5/7)successful engraftment rate.Three recipients showed that the cell population of the peripheral blood leukocytes,red blood cells and hemoglobin approached to normal index at 40 d after transplantation,but followed relative slow renew in platelet count.Survival rate in transplant group was 43%,compared to 100% mortality in control mice.Karyotyping assays confirmed the female mice with XY.CONCLUSION:The three-step differentiation and the culture conditions described here support the differentiation of mouse ESCs into HSCs.HSCs derived from mouse ESCs can reconstruct hematopoiesis.

BACKGROUND:E-cadhedn plays an important role in development of liver tissue in the embryonic stage.Therefore,it is importance for investigating the feasibility of dynamic expression of E-cadherin in embryonic stern cell differentiation to in vitro development of liver tissue.OBJECTIVE:To observe the dynamic expression of E-cadherin in embryonic stem cell differentiation and the effect on cell adhesion.DESIGN,TIME AND SETTING:An in vitro cytological observation was performed at Surgical Laboratory of the First Affiliated Hospital of Sun Yat-sen University from December 2007 to December 2008.MATERIALS:Embryonic stem cells of BALB/c mice were obtained from Professor Huang (Department of Ophthalmology,Sun Yat-sen University).Twenty 13-day-old pregnant BALB/c mice of clean grade were provided by the Experimental Animal Centar of Sun Yat-sen University.METHODS:Following trypsinization,embryonic stem cells were suspend-incubated in DMEM culture medium containing fetal bovine serum,2-mercaptoethanol,HEPES,penicillin,and streptomycin.Embryoid body was formed 5 days after normal development and incubated in the culture plate at day 6.Liver tissue which was obtained from 13-day-old pregnant BALB/c mice was prepared for fetal liver cells which were frozen-sectioned as the controls.MAIN OUTCOME MEASURES:E-cadherin expression was detected using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry st varying time points of 1,5,9,13,and 17 days in stages of initial differentiation of embryonic stem cells,formation of embryoid body,and formation of differentiated clusters.Additionally,the effect of E-cadherin expression on cell adhesion was also detected.RESULTS:RT-PCR showed that E-cadherin mRNA expression was not observed at day 1 but peaked at day 5;gradually,the expression was decreased until the expression was stopped at day 17.E-cadherin mRNA expression was strong in fetal liver cells in the control group.Immunocytochemistry showed a similar outcome.Morphologically,embryonic stem cells developed from unicells into compact three-embryonic layer embryoid body and into incompact cell population.CONCLUSION:E-cadherin expression correlates with differentiated cell adhesion;additionally,the lost expression in an in vitro environment may be an important cause for unable regularization of differentiated cells.

3?10 -4 mol/L), and the role was persistent if its concentration was kept. Meanwhile, the increase of intracellular free Ca 2+ induced by stretch was also inhibited by NO. Conclusion Cardiocytes hypertrophic response stimulated by stretch could be inhibited by nitric oxide. The inhibitory effect might be due to the decrease of the concentration of intracellular free Ca 2+ .

BACKGROUND: During the embryonic stage, E-cadherin expression plays a critical role in the formation of hepatic tissue.OBJECTIVE: E-cadherin gene was transfected into mouse embryonic stem cells (ESCs) to observe its effects on adhesive capacity of differentiated cells.DESIGN, TIME AND SETTING: A cytological in vitro observation was performed at the Laboratory of Surgery, First Hospital Affiliated to Sun Yat-sen University between December 2007 and December 2008.MATERIALS: BALB/c mice at gestational 13 days, of clean grade, were provided by Laboratory Animal Center, Sun Yat-sen University. BALB/c mouse ESCs were preserved by professor Huang Bing from the Department of Ophthalmology, Sun Yat-sen University. CMV promoter-containing eukaryotic expression plasmid pEGFP-N1 was gifted by doctor Lu Zhi-yue from Medical College, Sun Yat-sen University.METHODS: Total RNA was extracted from BALB/c mouse fresh hepatic tissue and synthesized into cDNA by reverse transcription (RT). The synthesized cDNA was used as a template to perform a polymerase chain reaction (PCR) that amplifies a targeted fragment. Following double enzyme digestion, pEGFP-E-cadherin plasmids were reconstructed and transfected into mouse ESCs. In vitro differentiation of transfected mouse ESCs was performed.MAIN OUTCOME MEASURES: Detection of E-cadherin expression in the differentiation system using RT-PCR and immunocytochemistry and observation of adhesive capacity of differentiated cells.RESULTS: E-cadherin gene-transfected ESCs could stably express E-cadherin during differentiational 1-17 days, while non-transfected ESCs expressed a decreasing amount of E-cadherin. The adhesive capacity of differentiated cells that stably expressed E-cadherin was markedly enhanced. Compact cell connection and multi-layer growth state remained at 19 days. While non-transfected ESCs gradually changed from embryoid bodies into noncohesive cell populations.CONCLUSION: Differentiating E-cadherin ESCs exhibit markedly enhanced adhesive capacity and maintain multi-layer growth state.

Objective To investigate the effect of ambroxol pretreatment on the inflammatory response and lipid peroxidation during one-lung ventilation (OLV) .Methods Forty-five ASA I or II patients aged 37-64 yr weighing 53-65 kg undergoing thoracotomy under general anesthesia were randomly divided into 3 groups ( n = 15 each): group A two-lung ventilation (TLV); group B OLV and group C ambroxol 1 mg/kg + OLV. Anesthesia was induced with midazolam, fentanyl, propofol and atracurium and maintained with propofol infusion and intermittent iv boluses of fentanyl and atracurium. The patients were mechanically ventilated (VT8-10 ml/kg, RR 12 bpm during TLV, VT 6-7 ml/kg, RR 16 bpm during OLV, I: E 1:2, FiO2 100% ). In group C ambroxol 1 mg/kg in normal saline ( NS) 100 ml was infused at 25 min before OLV (infusion rate 4 ml/min) , while in group A and B equal volume of NS was infused instead of ambroxol. Blood samples were obtained from radial artery before induction of anesthesia and OLV (T0.1 ) and at 0.5, 1, 2 h of OLV (T2-4 ) and 1, 2 h of TLV (T5,6 ) and at 24 h after operation (T7) in group B and C for determination of serum SOD activity and TNF-α, IL-6 and IL-8 concentrations and WBC and neutrophil granulocyte counts. The same indexes were detected in group A at the corresponding time points.Results Serum SOD activity was significantly lower and serum TNF-α, IL-6 and IL-8 concentrations and WBC and neutrophil granulocyte counts were significantly higher in group B than in group A. Serum SOD activity was significantly higher and serum TNF-a, IL-6 and IL-8 concentrations and WBC and neutrophil granulocyte counts were significantly lower in group C than in group B. Conclusion Pretreatment with ambroxol 1 mg/kg can inhibit inflammatory response and lipid peroxidation during OLV.

OBJECTIVETo explore the diagnosis, treatment and long-term outcome of late acute rejection (LAR) following adult orthotropic liver transplantation (OLT).

METHODSA total of 398 consecutive adult patients who underwent OLT in Organ Transplant Center, the First Affiliated Hospital, Sun Yat-sen University between January 2007 and December 2012 were reviewed retrospectively. There were 48 patients (12. 1%) developed to LAR, including 43 male patients and 5 female patients, with an average age of (52 ± 13) years(18 - 70 years). The mean body mass index was (22.1 ± 4. 5) kg/m2 (15. 4 - 30. 4 kg/m2). The indications of the liver transplantation recipients included 16 cases of end-staged liver cirrhosis after hepatitis B or C(33. 3%), 14 cases with severe hepatitis (29. 2%), 9 cases of primary liver cancer(18. 5%), 5 cases of alcoholic liver cirrhosis (10. 4%), 1 case with autoimmune liver disease (2. 1%) , the other 3 cases (6. 3%). They were followed up by outpatient service, telephone and other means. Survival curves were generated with the Kaplan-Meier method and Cox proportional hazards modeling was used for predictors of mortality. Statistically significant variables found by single factor regression analysis were put into the Cox proportional hazards regression model of multivariate analysis.

RESULTSThe time-to-event was 23. 6 months after OLT which were more common in the first year to the third year post-transplant (26/48,52. 4%). Thirty-five cases were assessed as mild, 11 cases were assessed as moderate, and 2 cases were assessed as severe ,based on the Banff schema. After adjustment to the immunosuppressive regimen, the overall recovery rate reached to 81. 3%. The rate of steroid-resistant acute rejection was 11. 8% (4/34). Inadequate immunosuppression and steroid pulsation were two independent risk factors affecting the prognosis of LAR (P = 0. 008, P = 0. 003, respectively).

CONCLUSIONSLAR is an uncommon complication after OLT. Inadequate immunosuppression and steroid pulsation are the major risk factors for prognosis of LAR. Improving patient compliance and strengthening blood concentration surveillance can increase the patient survival.

Acute Disease ; Adolescent ; Adult ; Aged ; Female ; Graft Rejection ; diagnosis ; mortality ; therapy ; Hepatitis B ; Humans ; Immunosuppression ; Liver Cirrhosis ; Liver Neoplasms ; Liver Transplantation ; mortality ; Male ; Middle Aged ; Multivariate Analysis ; Prognosis ; Proportional Hazards Models ; Retrospective Studies ; Risk Factors ; Steroids ; Young Adult

Objective Labial gland biopsy is one of major diagnostic methods for Sj(o)gren's syn-drome(SS).Meanwhile,99TcmO-4 parotid scintigraphy has been proven useful for the clinical evaluation of SS.This study was performed to investigate the correlation between the two examinations and evaluate the semi-quantitative parotid scintigraphy in the early diagnosis and staging for SS patients.Methods There were 135 SS patients and 30 normal subjects as control group in this study.They all underwent 99TcmO-4 pa-rotid scintigraphy.Semi-quantitative analyses of parotid scintigraphy were conducted with parameters inclu-ding maximum accumulation ratio (MAR),maximum secretion ratio(MSR),time interval from stimulation to minimum count(tparotid),prestimulatory oral activity index (PRI) and poststimulatory oral activity index (POI).For comparison, the biopsy of labial gland was performed in each patient and the pathological se-verity was classified into grade 0,1,2,3,4 (also defined as subgroups).One-way ANOVA and q-teat were applied for the correlation analyses between the two examinations.Results There was significant difference between pathological subgroup 3 or subgroup 4 and the control in all the semi-quantitative parame-ters (q=6.79-38.64,P<0.O1).In subgroups 1 and 2,only PRI and POI showed significant changes compared with the control(q=8.33,8.63,all P<0.01).The pathological stages were negatively correla-ted with MAR(r=-0.679,P<0.01),MSR(r=-0.601,P<0.01),PRI(r=-0.724,P<0.01)and POI(r=-0.751,P<0.01),but only positively correlated with tparotid(r=0.364,P<0.01).Con-clusions Most semi-quantitative parameters of 99TcmaO-4 parotid scintigraphy may be well correlated with the pathological severity of labial gland biopsy in SS patients.Further,the semi-quantitative indices espe-eially PRI and POI may be helpful for the early diagnosis and staging of SS patients.

Objective To investigate the preventive effect of nano-powder of Wugong-sanqi (NW),the rhizome of Anemone flaccid,on postoperative intestinal adhesion in rats.Methods Fifty SD rats were subjected to operation with Ellis' method for establishing intestinal adhesion models,then randomly divided into 5 groups (n=10),namely model,positive (Dexamethasone,i.m.10 mg/kg),NW high,medium and low dose group (p.o.450,225 and 112 mg/kg,respectively).Another ten normal rats were selected as normal control group.After administration 3 days pre-operation and 7 days post-operation,all of rats were killed,the intestinal adhesion was graded and the tissues were observed by optical microscope.Results NW evidently reduced the severity of postoperative adhesion (P＜0.05 or P＜0.01),compared with model group.The histopathologic changes such as proliferation of fibroblast cells and capillary,interstitial granulomas and inflammatory cells infiltration in intestinal tissues were also improved significantly in NW groups.Conclusion NW could inhibit the formation of postoperative intestinal adhesion effectively.

Objective To explore the change of serum Apelin level and its relationship with insulin resistance (IR) in infertility patients with polycystic ovarian syndrome (PCOS). Methods Ninety-eight infertility patients with PCOS (PCOS group) and 72 infertility patients without PCOS (non-PCOS group) visiting our hospital for the first time from January 2011 to June 2012 were selected. The BMI , serum Apelin level (detected by ELISA), fasting blood glucose (FPG, detected by glucose oxidase method), fasting insulin (Fins, detected by chemiluminescence), and homeostasis model assessment of insulin resistance index (HOMA-IR) of the two groups were detected. Results The serum Apelin level and HOMA-IR in PCOS patients were higher than those in non-PCOS patients (3.28 ± 1.24 vs. 1.94 ± 0.78, P < 0.05; 3.84 ± 1.23 vs. 2.14 ± 0.77,P < 0.05). Pearson correlation analysis showed that serum Apelin level was positively correlated with HOMA-IR in PCOS patients (r=0.65, P<0.01). Conclusions The serum Apelin level in infertility patients with PCOS increased evidently, and was positively correlated with HOMA-IR; which indicated that Apelin may involve in the development of IR in infertility patients with PCOS.

Ergosterol is the important component of the fungal membrane, and having stable structure. This makes it a suitable indicator for growth of fungi. In the paper, isolation and determination techniques of ergosterol as the indicator of the fungal biomass were reviewed. The methods of extracting ergosterol include traditional saponification and refluxing, rapid physical disruption, rapid ultrasonication, supercritical fluid extraction and so on. The ergosterol determination methods are high performance liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and thin-layer chromatography, et al. The application of these techniques was also introduced. Finally, the paper prospected the feasibility of applying the ergosterol as the indicator of fungal biomass in composting.