Objective To establish a specific fluorescence quantitative method for determining the mRNA expression of Toll-like receptor3(TLR3)in peripheral blood mononuclear cells(PBMCs).Methods Using the Beacon Designer 2.1 software,specific primers and Taqman-MGB probe were designed.The plasmid pMD18-T-TLR3 was constructed as calibrator and the amplified fragment was obtained by reverse- transcript-PCR(RT-PCR).RNA quantification based on cycle threshold values(Ct)was used to establish the standard curve.According to which,the TLR3 mRNA levels in 30 normal individuals,20 patients with primary biliary cirrhosis(PBC)and 20 ones with chronic liver cirrhosis induced by HBV were calculated automatically by software after the fluorescence of PCR product was detected continuously during amplification.Results The linear detection range of the assay for TLR3 gene and ?-actin was 10~2-10~8(r= -0.9974)and 10~3～10~8(r=-0.9984),respectively.The coefficient of variation of both intra-and inter- assay reproducibility for high concentration sample were 6.7% and 8.7%,respectively,and those for low concentration sample were 12.3% and 14.0%.The TLR3 mRNA expression level ranges from 3.46?10~2- 4.51?10~3 copies/?g RNA,4.92?10~2-1.42?10~4 copies/?g RNA and 2.58?10~2-7.17?10~3 copies/?g RNA for 30 healthy individuals,20 PBC patients and 20 ones with chronic liver cirrhosis induced by HBV, respectively.Conclusion We have successfully set up a FQ-RT-PCR method for detecting TLR3 mRNA, which may be used as an excellent tool for the clinic and basic study on the expression of TLR3 gene.

BACKGROUND: Thrombus formation and neointimal hyperplasia limit its use for revascularization of small-caliber vessels (<6mm diameter) in the coronary or peripheral circulation.OBJECTIVE: To improve hemocompatibility, the luminal surface of a small diameter expanded polytetrafluoroethylene (Eptfe) vascular prosthesis was modified with alginate and recombinant hirudin.DESIGN: Observational experiment.SETTING: This study was performed in Nanomedicine and Biosensor Laboratory, Bio-X Center, Harbin Institute of Technology from Marcy 2006 to June 2007.MATERIALS: The GORE-TEX Epife vascular grafts (W. L Gore & Associates, Inc., Flagstaff, AZ) used in this study were 4mm in internal diameter. rHir was obtained from Calbiochem, Germany. Sodium alginate (also called alginic acid sodium salt; medium viscosity) was purchased from Sigma.METHODS: A p-diazonium diphenyl amine polymer (PA) was used as an interlayer between alginate and recombinant hirudin (rHir). The diazonium moieties were capable of covalently coupling with electron-rich aromatic systems such as histidine and tyrosine residues of hirudin. No need for chemical pretreatment took all advantage by preserving the bulk properties with almost no effect on stability and elasticity of the Eptfe vascular graft. rHir amount on Epife surface Was determined by the Micro BCA (Bicinchoninic acid) Protein Assay kit.MAIN OUTCOME MEASURES: ① Determination rHir amount on Eptfe surface; ② Static water contact angles; ③ Attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) was employed to confirm the change taking place in the Eptfe graft surface modification process; ④ Characterization of the surface morphology and platelet adhesion by SEM; ⑤ APTT and PT; ⑥ Percent hemolysis.RESULTS: ① The amount of rHir adsorbed onto the Eptfe vascular was deduced to be 16.35 μg/cm2. ② Surface analysis ATR-FTTR revealed the presence of new functional groups on the modified graft surfaces. ③ The water contact angle of the modified graft surface decreased. ④ The longer APTT and PT value lower than 5% hemolysis level and dramatically decrease of platelet adhesion assay showed that rHir modified graft had great improved blood compatibility.CONCLUS10N: Cross-linked Alg/rHir onto Eptfe can improve luminal surface and hemocompatibility.

Adenocarcinoma ; pathology ; Adult ; Aged ; Female ; Humans ; Lasers ; Male ; Middle Aged ; Neuroendocrine Cells ; pathology ; Polymerase Chain Reaction ; methods ; Stomach Neoplasms ; pathology

This study aimed at exploring the mechanism of the rapid antidepressant effects of Yueju pill on depression of Parkinson's disease (DPD).The fast antidepressant effects of Yueju pill in this study was evaluated by behavior tests,such as open field test (OFT),tail suspension test (TST),forced swimming test (FST) and sucrose preference test (SPT) according to the modeling method of subacute DPD in the literature.In vitro experiment was implemented using PC12 cells.Moreover,the protective effects of Yueju pill on 1-methyl-4-phenylpyridinium (MPP+) induced neural injury with the engagement of cAMP response element binding protein (CREB) were expounded.As a result,it was found that the immobility time of the mice in the model group was significantly longer than that in the normal group in TST and FST tests (P ＜ 0.01),and the SPT ratio of mice in the model group remarkably decreased (P ＜ 0.01).In addition,the immobility time of DPD mice was shortened in the FST test after administering Yueju pill (P ＜ 0.05),while the SPT ratio was increased (P ＜ 0.01).Yueju pill took the effects on the third day after a single administration.The phosphorylation of CREB (p-CREB) in MPP+ induced PC12 cells was decreased in comparison with the model group,while the expression of p-CREB was up-regulated with the administration of Yueju pill (P ＜ 0.01).In conclusion,DPD was quickly mitigated after the treatment of Yueju pill,the activation of CREB signaling pathway and its neuroprotective effects may be the mechanism behind it.

OBJECTIVETo discuss whether vascular endothelial growth factor (VEGF) in the peripheral blood can reflect the biological activities of pituitary adenomas.

METHODSThe concentrations of VEGF in peripheral blood were measured with ELISA in 203 patients with pituitary adenomas, 22 patients with pituitary hyperplasia, 7 patients with pituitary Rathke' pouch and 3 patients with pituitary abscess.

RESULTSThe serum VEGF levels were (366.8 +/- 211.1) pg/ml and (286.8 +/- 107.6) pg/ml in patients with pituitary adenomas and pituitary hyperhasia, respectively, which were higher than those in patients with pituitary Rathke' pouch [(180.5 +/- 61.7) pg/ml], patients with pituitary abscess [(147.5 +/- 46.3) pg/ml] and the health control [(180.8 +/- 56.2) pg/ml] (P < 0.05). In patients with pituitary adenomas, the VEGF levels were (380.0 +/- 234.5) pg/ml in macroadenomas and (380.1 +/- 2870.3) pg/ml in giant adenomas, higher than those in microadenomas [(294.6 +/- 111.6) pg/ml] and in pituitary hyperhasia respectively (P < 0.05). The serum VEGF levels were not significantly different in pituitary adenoma in terms of invasive growth, apoplexy, cyst and hormone secretory functions (P > 0.05).

CONCLUSIONSThe upregulation of serum VEGF expression may reflect the biological activities of pituitary adenoma. However, it may not be associated with pituitary Rathke' pouch, pituitary abscess, adenoma with invasiveness, apoplexy, cyst and hormone secretory function. The serum VEGF levels could be helpful in differentiating pituitary adenoma from pituitary Rathke' pouch and pituitary abscess.

Adenoma ; blood ; diagnosis ; Adult ; Diagnosis, Differential ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Hyperplasia ; blood ; diagnosis ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Pituitary Diseases ; blood ; diagnosis ; Pituitary Neoplasms ; blood ; diagnosis ; Vascular Endothelial Growth Factor A ; blood

AIMTo study the therapeutic effects of N,N'-diacetyl-L-cystine (DiNAC) on immunological liver failure.

METHODSSerum ALT, AST and T cell subsets in peripheral blood of the experimental animals during the trial period were analyzed by an automatic serum analyzer and a flow cytometer, respectively. The sectioned liver specimens were examined under a light microscope. And 24 h after the injection of Gal/LPS, the survival rate of rats was calculated.

RESULTSDiNAC (50, 200, 800 mg x kg(-1), i.p.) suppressed the elevation of serum levels of ALT and AST, markedly enhanced proliferation and differentiation of T cell subsets (CD4+, CD8+ and Th1, Th2), and improved all the histopathological features. In mice of fulminant hepatic failure (FHF), the survival time significantly prolonged and the survival rate increased 24 h after i.p. DiNAC. These effects were obviously dose-dependent.

CONCLUSIONDiNAC on mice with FHF has an inhibitory action which is related to immune mechanism.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Bilirubin ; blood ; CD4 Antigens ; metabolism ; CD8 Antigens ; metabolism ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cystine ; analogs & derivatives ; pharmacology ; Liver ; pathology ; Liver Failure, Acute ; blood ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes ; pathology

OBJECTIVE: To investigate the serum level of soluble transferrin receptor (sTfR) and its association with the degree of anemia in children with hemoglobin H (HbH) disease. METHODS: A total of 55 children with HbH disease were enrolled as the HbH group, and 30 healthy children were enrolled as the control group. The HbH group was further divided into a deletional HbH disease group and a non-deletional HbH disease group. A retrospective analysis was performed for hematological parameters and serum sTfR level in all groups. RESULTS: Of the 55 children with HbH disease, 39 had deletional HbH disease and 16 had non-deletional HbH disease. Compared with the control group, the deletional and non-deletional HbH disease groups had significantly lower hemoglobin (Hb), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) and a significantly higher serum level of sTfR. Compared with the deletional HbH disease group, the non-deletional HbH disease group had significantly lower red blood cell count (RBC) and Hb level and significantly higher MCV, MCH, and serum sTfR level. In children with HbH disease, serum sTfR level was negatively correlated with RBC and Hb level (r=-0.739 and -0.667 respectively, P<0.05) and positively correlated with MCV and MCH (r=0.750 and 0.434 respectively, P<0.05). CONCLUSIONS Serum sTfR level is associated the degree of anemia in children with HbH disease, and sTfR may be a target for the treatment of HbH disease.
Child ; Erythrocyte Count ; Hemoglobin H ; Humans ; Receptors, Transferrin ; Retrospective Studies ; alpha-Thalassemia

OBJECTIVETo extend the use of vector-derived siRNA by generating multiple shRNAs in the same plasmid.

METHODSConstruct a vector that expresses shRNAs targeting on Ku70 and Ku80 in tandem. The gene silencing efficiency of each shRNA was verified previously. After identification by restriction digestion and DNA sequencing, the reconstructed plasmid, named psiRNAKus, was transfected into the human hepatoma cell line HepG2. The tandem-shRNA-induced silencing of targeted genes was determined by RT-PCR at RNA level and Western blot at protein level.

RESULTSThe shRNAs encoded by psiRNAKus down-regulated both the expression of Ku70 and Ku80.

CONCLUSIONThe vector-derived siRNA delivery system that allows multiple shRNA species to be expressed from the same vector may be of value in experimental and therapeutic applications.

Antigens, Nuclear ; genetics ; DNA-Binding Proteins ; genetics ; Gene Knockdown Techniques ; Hep G2 Cells ; Humans ; Ku Autoantigen ; Plasmids ; RNA ; genetics ; RNA, Small Interfering

Breast Neoplasms ; pathology ; Cell Culture Techniques ; methods ; Cells, Cultured ; Female ; Humans ; Immunohistochemistry ; methods ; Quality Control

OBJECTIVETo study the clinical characteristics, diagnosis and surgical effects of thyroid-stimulating hormone pituitary adenomas (TSH-omas).

METHODSThe clinical data of 19 patients (14 female and 5 male) with TSH-omas were analyzed retrospectively in this study from January 2001 to December 2008. The patients ranged from 20 to 70 years old (average 40.5 years old) and had disease histories from 1 to 228 months (average 55 months). Among these patients, 15 of them complained of thyrotoxicosis symptoms, while the other 4 patients' symptoms were associated with headache and/or visual disturbance caused by the tumor mass effect. Initially, 12 of the 15 patients with thyrotoxicosis symptoms were misdiagnosed with Grave's disease. As a result 2 of them received (131) Iodine, and one received subtotal thyroidectomy. All of these patients underwent transsphenoidal microsurgery.

RESULTSAverage follow-up period was 3.6 years (6 months-7 years). Pathological analysis of the surgical specimen showed pituitary adenoma in all patients, immunohistochemistry were positive for TSH in 17 cases, negative for TSH in 2, positive for growth hormone in 2, positive for prolactin in 1, and positive for adrenocorticotrophic hormone in 1. Postoperative MRI revealed that the tumors in 15 patients were removed totally, though 4 patients still had residual tumors. The thyroid hormone level tests suggested that 13 patients could be considered normal 3 months after their tumors were removed, though 2 of patients with normal postoperative MRI and thyroid hormones showed increased levels of TSH. For these 2 patients, tumors did not recur and their thyroid hormone levels returned to normal after pituitary radiotherapy. The cure rate was 11/19 after surgery and 13/19 after surgery plus pituitary radiotherapy.

CONCLUSIONSThe screening test for hyperthyroidism patients with high TSH levels is a key point to improve the accuracy rate in early diagnoses of TSH-omas. The transsphenoidal microsurgery is first choice to treat TSH-omas, while pituitary radiotherapy and somatostatin analogs are beneficially adjunctive therapies.

Adult ; Aged ; Female ; Humans ; Hyperthyroidism ; metabolism ; Male ; Middle Aged ; Pituitary Neoplasms ; diagnosis ; metabolism ; surgery ; Retrospective Studies ; Thyrotropin ; metabolism ; Young Adult