Objective To induce mouse embryonic stem(ES)cells to differentiate into insulin-secreting cells by means of a 5-step model system.Methods E14.1 mouse ES cells were cultured in the presence of leukemia inhibitory factor(LIF)for 2 days(step 1),then the cells were cultured in hanging drops to form embryonic bodies(EBs)and the resulting EBs were cultured in suspension for 6 days in the presence of basic fibroblast growth factor bFGF(step 2).Subsequently the EBs were cultured in the medium containing glucagon- like peptide 1(GLP-1),hepatocyte growth factor(HGF),nerve growth factor(NGF)and nicotinamide for 10 days(step 3).After that,the EBs were dissociated into single cells,and the cells were cultured in monolayer in the presence of GLP-1,betacellulin,activin A,bFGF and nicotinamide for 10 days(step 4).Finally,the cells were cultured in low-glucose medium containing nicotinamide for 4 days(step 5).Insulin and some other islet- related genes expressions were investigated using RT-PCR and insulin expression was also investigated by DTZ- staining and immunohistochemistry.The percentage of insulin-secreting cells was evaluated by flowcytometry and insulin concentrations were measured by RIA.Results mRNA expression of insulin became visible at step 3 and more evident at step 5.Additionally,at step 5,mRNAs of glucagon,somatostatin,pancreatic polypeptide(PP), pancreatic duodenal homeobox 1(PDX-1),beta-cell E box transactivator 2(Beta2)and neurogenin 3(Ngn3) were detected.DTZ-staining positive cells and insulin immunohistochemical staining positive cells were observed. The percentage of insulin-positive cells was(24.0?2.5)%(n=6).In the presence of 5.6 mmol/L and 25 mmol/L glucose,insulin concentrations were(0.05?0.01)?g/L and(0.13?0.02)?g/L respectively(n= 6).Conclusion E14.1 mouse ES cells can be induced to differentiate into insulin-secreting cells by the 5-step model system.Insulin-secreting cells can release insulin into culture medium when treated with glucose,and insulin concentrations increase with rising concentration of glucose.

BACKGROUND:The high level of matrix metalloproteinase 9 (MMP9) is thought to slow down the healing of diabetic foot ulcers. Whether it can influence the biological behaviors of skin fibroblasts and affect wound healing is stillunclear. The present study aimed to observe changes in the biological behaviors of rat dermal fibroblasts induced by high expression of MMP9 and to clarify the possible mechanisms of wound healing for diabetic foot. METHODS:A cellmodel of skin fibroblast with high expression of MMP9 was established by co-culture of high glucose (22.0 mmol/L) and homocysteine (100 μmol/L). A control group was incubated with normal glucose (5.5 mmol/L). Realtime PCR, ELISA and gelatin zymography were used to detect the MMP9 mRNA, protein expression and activity of MMP9. Flow cytometry, CCK-8, ELISA assay, scratch test and transwellwere used to detect cellproliferation, viability, collagen (hydroxyproline) secretion, horizontal migration and vertical migration of cells. The data were expressed as mean±SD. P value less than 0.05 was considered statistically significant. RESULTS:The expression of MMP9 mRNA, protein levels and the activity of MMP9 were much higher in the high MMP9 group than in the control group (7.05±1.02 vs. 1.00±0.00, 206.9±33.6 pg/mL vs. 40.4±5.9 pg/mL, and 1.47±0.13 vs. 0.57±0.12, respectively,P<0.01). The proportion of S-phase cells, proliferation index, cellviability, collagen (hydroxyproline) secretion, horizontal migration rate and the number of vertical migration cells were lower in the high MMP9 group than in the control group (P<0.01). CONCLUSION:Fibroblasts with a high expression of MMP9 decreased proliferation, activity, secretion and migration of collagens, suggesting that MMP9 may inhibit the biological behaviors of fibroblasts.

OBJECTIVETo observe the changes in the myocardial ultrastructure of diabetic rats and the effect of enalapril treatment.

METHODSMale Wistar rats were divided into 3 groups, namely the control group, diabetic group and enalapril intervention group. Diabetes was induced with peritoneal injection of streptozotocin in the latter 2 groups, and in enalapril group, the rats were treated with enalapril at the daily oral dose of 2 mg/kg for 1, 3 and 5 months after streptozotocin injection. Histological analysis of the left ventricular tissue was performed with transmission electron microscope 1, 3, and 5 months after establishment of diabetes.

RESULTSOnset of myocardial damages was observed 1 month after the development of diabetes in the rats with gradual time-dependent exacerbation. Enalapril treatment could partially reverse the myocardial destruction in the diabetic rats.

CONCLUSIONEnalapril intervention may improve the ultrastructural pathology of the myocardium in diabetic rats, which is suggestive of the action mechanisms of angiotensin-converting enzyme inhibitors in myocardium preservation.

Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Diabetes Mellitus, Experimental ; drug therapy ; pathology ; Enalapril ; pharmacology ; Male ; Myocardium ; ultrastructure ; Rats ; Rats, Wistar ; Streptozocin

BACKGROUNDPim-1 plays an important role in the apoptosis, proliferation, differentiation of cancer cells and progression of cancer. In this study we detected the expression of pim-1 mRNA in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer (PCa) and explored its diagnostic value for PCa.

METHODSThe prostate tissues were collected from 23 patients with PCa, 37 patients with BPH, and 3 healthy volunteers. Pim-1 mRNA expression levels in these samples were determined by the quantitative real-time PCR (QRT-PCR). The differences of expression were calculated based on a standard curve.

RESULTSThe ratio of pim-1 mRNA to beta-actin in the normal prostate, BPH, and PCa were 1.05 +/- 0.04, 2.57 +/- 0.74 and 4.45 +/-0.63, respectively. The differences among PCa, BPH and NT were significant (P < 0.05, respectively).

CONCLUSIONDetecting pim-1 mRNA expression by QRT-PCR provides a reliable metric for the diagnosis of PCa.

Aged ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Prostate ; metabolism ; Prostatic Hyperplasia ; metabolism ; Prostatic Neoplasms ; diagnosis ; metabolism ; Proto-Oncogene Proteins c-pim-1 ; genetics ; RNA, Messenger ; analysis ; Sensitivity and Specificity

OBJECTIVETo investigate the role of α-enolase (ENO1) in regulating glucose metabolism and cell growth in human glioma cells.

METHODSGlucose uptake and lactate generation were assessed to evaluate the changes in glucose metabolism in human glioma U251 cells with small interfering RNA (siRNA)-mediated ENO1 knockdown. MTT assay and 5-ethynyl-2'-deoxyuridine (EdU) staining were used to examine the cell growth and cell cycle changes following siRNA transfection of the cells.

RESULTSTransfection of U251 cells with siRNA-ENO1 markedly reduced glucose uptake (P=0.023) and lactate generation (P=0.007) in the cells and resulted in significant suppression of cell proliferation (*P<0.05) since the second day following the transfection. Transfection with siRNA-ENO1 also obviously suppressed cell cycle G1/S transition in the cells (P=0.0425). The expressions of HK2 and LDHA, the marker genes for glucose metabolism, were significantly down-regulated in the cells with siRNA-mediated ENO1 knockdown.

CONCLUSIONENO1 as a potential oncogene promotes glioma cell growth by positively modulating glucose metabolism.

Objective The aim of this study was to demonstrate the immunogenicity and safety of diphtheria, tetanus, pertussis (acellular, component) , poliomyelitis (inactivated) vaccine (adsorbed) and Haemophilus influenzae type b conjugate vaccine (DTaP-IPV//PRP-T) combined vaccine compared with commercially available DTaP (diphtheria, tetanus and pertussis), Haemophilus influenzae type b (Hib), tetanus conjugate and IPV monovalent vaccine. Methods Subjects were randomly divided into three groups, Group A and Group B were DTaP-IPV//PRP-T combined vaccine (PENTAXIMTM) vaccinated at 2,3,4 months of age or 3,4, 5 months of age respectively; Group C was commercially available DTaP. Hib tetanus conjugate (Act-HIBTM) and IPV (IMOVAX PolioTM) vaccines vaccinated at 3,4, 5 months of age. All groups received booster dose at 18 to 20 months of age, with antibody titers tested. Non-inferiority analysis was demonstrated in terms of seroprotection / seroconversion rates between Group A, Group B respectively and Group C. Safety information was collected after each vaccination to assess the safety of investigational vaccines. Results The non-inferiority of DTaP-IPV//PRP-T combined vaccine vaccinated at 2,3,4 or 3,4, 5 months of age versus DTaP, Hib tetanus conjugate and IPV vaccine was demonstrated for all vaccine antigens in both primary and booster phases in terms of seroprotection/seroconversion rates. DTaP-IPV//PRP-T combined vaccine was well tolerated. The rate of solicited/unsoliciated severe adverse reactions was very low and similar to the control vaccines. Conclusion DTaP-IPV//PRP-T combined vaccine was highly immunogenic with good safety profile in Chinese infants, which was comparable to the commercially available control vaccines.