OBJECTIVETo analyze the influence of retinoic acid (RA) on the undifferentiated state and EB formation abilities of human embryonic stem cells.

METHODSThe biological characteristics of H9 ESCs after RA treatment were characterized by real-time PCR, MTS proliferation assay and immunofluorescence staining. The expression of three germ layers markers, osteogenic differentiation markers and adipogenic differentiation markers in H9-differentiated embryoid bodies (EBs) with RA treatment were quantified by real time PCR.

RESULTSThe proliferation of H9 ESCs in the early logarithmic growth phase was accelerated by RA treatment. In addition, RA induced differentiation of H9 ESC coupled with morphology changes, decreased expression of undifferentiated markers Oct4, Nanog, Sox2 and OCT4 mRNA binding protein Lin28 at mRNA level, and reduced expression of Oct4 at protein level. RA induced formation of cavities in EBs. Real time PCR results showed that the expressions of ectodermal markers: NeuroD1, Noggin; mesodermal markers: Brachyury, Twist and endodermal markers: AFP, GATA-4 were significantly increased (P < 0.05), especially for AFP (P < 0.01), by RA treatment in a dose-dependent manner. In addition, the expression of adipogenic differentiation marker C/EBPalpha was increased while the osteogenic differentiation marker OPN was decreased in EBs after RA treatment for 5 days.

CONCLUSIONSHigh concentrations of RA induced the loss of stemness in H9 ESCs and excessive differentiation in EBs, and damaged the balance between osteogenic and adipogenic differentiation during early EB differentiation, which may be relevant to the congenital malformations.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; drug effects ; Humans ; Tretinoin ; pharmacology

BACKGROUND:Discectomy and pedicle fixation fusion are golden standard to repair lumbar degenerative disease, but the treatment would induce other complications such as degeneration of adjacent segments or severer pre-existing spinal degeneration. For the problem of lumbar fusion and fixation, lumbar elastic fixation has recently been a hot focus. ＜br> OBJECTIVE:To evaluate the short-term effectiveness of dynamic lumbar pedicle fixation in repair of lumbar spinal stenosis and lumbar disc herniation. ＜br> METHODS:From December 2010 to December 2012, 62 cases of lumbar spinal stenosis and lumbar disc herniation treated with lumbar dynamic system were included. The involved segments included:5 cases at L 3/4 , 20 cases at L 4/5 , 20 cases at L 5 S 1 , 6 cases at double segment L 3/4 and L 4/5, 8 cases at double segment L 4/5 , L 5 S 1 , 3 cases at L 3/4 and L 5 S 1 . There were 34 males and 28 females with an average age of 50.8 years (range 32 to 72 years). According to different fixation systems, they were assigned to three groups:general dynamic lumbar fixation system in 17 cases, K-Rod posterior dynamic stabilization system in 28 cases, and Dynesys system in 17 cases. The fol ow-up time was from 24 to 48 months. Evaluation indexes included visual analogue scale, Oswestry disability index, imaging analysis and excellent and good rate of curative effects. ＜br> RESULTS AND CONCLUSION:Compared with before treatment, visual analogue scale score and Oswestry disability index were significantly improved at 6 months after treatment and final fol ow-up (P＜0.01). No apparent changes were detected in the length of inserted segments and adjacent segments before treatment and during final fol ow-up. There were no significant differences in the excellent and good rate in each group after treatment (P>0.05). These data indicated that the lumbar dynamic system was an effective option for lumbar disc herniation and spinal stenosis. Although there are some differences in the structure of three kinds of flexible fixation, no obvious difference in early therapeutic effects was detected. Long-term effects deserve further investigations.

Objective To investigate the level of serum citrulline and its significance in the patients subject to intestinal transplantation. Methods Phenylisothiocyanate served as derivative reagent and the serum citrulline was determined by HPLC. Thirty-three healthy adults served as controls. Serum citrulline was measured in 2 cases of intestinal transplantation during different transplant periods. Results The serum citrulline level in 33 healthy adults was (16.87?5.97)?mol/L. Before operation, it was low (less than 10 ?mol/L) in the patients and after operation it reached to high levels and kept at higher levels. The maximium of serum citrulline was more than 30 ?mol/L in patient A and was over 50 ?mol/L in patient B. Following rejection, the serum citrulline level in patient A was decreased rapidly and simialr to that before operation and in patient B it was lower than that before operation. Conclusion The level of serum citrulline in intestinal transplantation patients was correlated with transplant rejection. More patients are reqiured for the further study.

Objective: To develop a method in the determination of somatostatin level in blood for clinical medication. Methods: A method for the determination of somatostatin(Octreotide) in serum by high performance liquid chromatography with diode-array detector(DAD) was developed.After ultrafiltration,the pH values of blood samples were adjusted to 4,and samples were analyzed by Kromasil C18 column(4.6 mm? 250 mm,5 ?m i.d.) with a large pore(300) by acetonitrile/water/trifluoroacetic acid(20/80/0.01%,v/v) as mobile phase. Results:The recovery rate of Octreotide was 85%,and the calibration curve showed good linearity in the range of 0.006-0.5 mg/mL. Conclusion: The method is suitable for the detection of Octreotide content by liquid chromatography.

Objectives：To introduce the author's experiences in bioassay of platelet activating factor with thin layer chromatographic separation and platelet aggregation method,and to discuss probable problems in the process.Methods and Results：The method was used in determining portal venous blood samples from swine models of acute severe pancreatitis,control value were 0.79～1.65 pmol/L,in acute severe pancreatitis,mean blood level of platelet activating factor may be as high as 14.75 pmol/L.Conclusions:Thin layer chromatography separation and platelet aggregation assay were suitable for the determination of platelet activating factor,the results were satisfactory in case of standardized operation.

Objective To study the alterations of serum amino acid level in patients with short bowel syndrome (SBS). Methods Blood levels of 17 amino acids were measured in 17 SBS patients on admission to the hospital, and 15 healthy volunteers. Results Compared with the control group, serum concentrations of Val, Leu, Ile, Lys, Met, Ser, His, Cys in patients with SBS significantly decreased ( P

Objectives:To determine 3-methylhistidine(3-MH) and tyrosine(Tyr) in standard solution and preparations by high performance liquid chromatography(HPLC) with phenylisothicyanate(PITC) pre-column derivation. Methods:PITC was used as pre-column derivative of HPLC and stored in refrigerator after drying.Two phases gradient elution was performed with acetic sodium and cyanide for 50 minites. Results:The linearity of the measurement was very good for 3-MH and Tyr(r= 0.999 1 and 0.999 2).The recovery rate was 82.6% and 87.6%,respectively. Conclusions:The method of HPLC with PITC pre-column derivation for determination of 3-MH and Tyr is stable,efficient,and sensitive.PITC is an ideal derivative.

Objective: To compare the difference of Vitamin B in serum in healthy adults and patiens with Crohn's disease.Methods: 24 healthy adults and 20 patients were measured for Vitamin B1,B6,B12 in serum.A HPLC-DAD method was developed to determine vitamin B extracted directly from human serum.Purospher RP-18 column was used for the gradient elution with mobile phase: 0.04 mol phosphate buffer: methanol(60∶40).Results: The results proved to be linear in measurements of VB1,VB6 and VB12 and the correlation coefficients were 0.999 827,0.985 097 and 0.999 994,respectively.The contents of Vitamin B(VB1,VB6,VB12) in Cronhn's patients were significantly lower than in healthy adults.Conclusion: The measurements of Vitamin B in human serum by HPLC-DAD method are clinically helpful to diagnosis and treatment for Crohn's disease.

Objectives:To observe the effect of metabolic intervention of anti TNF antibody on the hypermetabolism occurred in intra abdominal infection(IAI) complicated by multiple organ dysfunction syndrome(MODS). Methods:Twenty rabbits were operated on with cecal ligation plus puncture(CLP) inducing IAI and MODS and were randomly divided into two groups, one receiving the anti TNF serum raised against TNF ?(anti TNF group) at 0.5?h after CLP and another receiving the non specific serum (control group). All animals were placed in metabolic cages and maintained with intravenous infusion for the observation period of one week. Serum levels of cytokines(TNF, IL 6), hormones (cortisol, insulin, glucagon), biochemical indexes (glucose, cholesterol, triglyceride, albumin) and daily excretions of urea nitrogen (UN),creatinine (Cr) and 3 methylhistidine (3 MH) were dynamically determined for 7 days. The death of animals was also recorded. Results:Compared with the control group, the levels of serum TNF, IL 6 and cortisol were significantly decreased and the levels of insulin and glucagon were kept normal after the injection of immune serum in anti TNF group, with significant improvements of biochemical indexes and decreased excretions of UN, Cr and 3 MH in urine. The survival rate was significantly increased in the anti TNF group. Conclusions:The anti TNF antibody can attenuate the metabolic abnormalities of IAI and MODS, being of the metabolic intervention on the hypermetabolism.

Objectives:To observe the metabolism of glutamine in the rat Kupffer cells and the effect of endotoxin. Methods:The changes of glutamine concentrations were measured before and 24 h after the in vitro cultures of the rat Kupffer cells with or without the stimulations of lipopoly saccharide. Results:Consumption and utilization rate of Gln in Kupffer cells with no LPS were (225?24)?mol/L and (9 33?0 99)% repectively.When the concention of LPS was 10.00 mg/L,consumptoin and utilization of Gln were (4 937?289)?mol/L and (73.29? 4.72 )%. Conclusions:Glutamine is an important substrate of Kupffer cell metabolism in the rest and LPS activated conditions.