Objective To establish a real time fluorescent quantitative revers transcripatase PCR(FQ-RT-PCR) method to detect the expression level of TNF-related apoptosis inducing ligand (TRAIL) mRNA in peripheral blood mononuclear ceils (PBMC) and determine its expression level in healthy donors, HBV-caused cirrhosis patients and PBC ones. Methods Specific primers and Taqman-MGB probe were designed and β-actin was used as endogenous control. The amplified fragment was obtained by RT-PCR. The quantitative template was constructed and then the fluorescent intensity was documented on the ABI Prism7000 analyzer. The standard curve was established, according to which, the TRAIl. mRNA levels in 30 healthy individuals, 30 patients with primary biliary cirrhosis (PBC) and 25 ones with HBV-caused cirrhosis were calculated automatically by software after the values of cycle threshold (Ct) were detected continuously during amplification. Results The linear detection range of the assay for TRAIL gene was 103 - 109 copies/ ug RNA ( r=-0.997). The coefficients of variation of both intra-and inter-assay reproducibility for high concentration samples were 5.6% and 6. 3% , respectively, and those for low concentration samples were12.5% and 14. 6%. The TRAIL mRNA expression level in PBC patients was [ (3.3±2.5)×105copies/ugRNA] significantly higher than that of healthy control [ (0.5±0.2)×105 copies/ug RNA ] (t=5.994,P <0.01). TRAIl. mRNA level of HBV-caused cirrhosis patients[ (2.1±0.9)×105 copies/ug RNA] wasalso significantly elevated (t=8.536, P<0.01). However, the difference between these two diseased groups had no significance. Conclusion We have successfully set up a FQ-RT-PCR method for detecting TRAIL gene expression and found that its expression levels of peripheral blood mononuelear cells in PBC and HBV caused cirrhosis patients are elevated, which provides a new insight into mechanism study of liver injury caused by cirrhosis.

Objective To evaluate the value of transient elastography (TE) for predicting severity of liver fibrosis in patients with chronic hepatitis B (CHB).Methods A total of 969 patients with CHB was enrolled and recruited for analysis,which had been received TE scan,including 258 patients of liver biopsy,and 117 patients of gastric endoscopy.Results A total of 35 patients was excluded from analysis due to TE failure or unreliable TE.Liver stiffness measurement (LSM) was independently influenced by bilirubin,AST,liver fibrosis and inflammation,ultrasonic score and albumin.TE predicted Child-Pugh C,B/C,liver fibrosis S4,≥S3 and ≥ S2 with respective area under receiver operating characteristics curves (AUROC)0.907 (95% CI 0.886-0.928 ),0.920 ( 95% CI 0.899-0.940 ),0.871 ( 95% CI 0.819-0.923 ),0.852(95%CI0.805-0.899) and 0.807(95% CI0.749-0.865),respectively.While LSM ＜32.2 kPa excluded Child-Pugh C with 99.4% probability,LSM ≥35.3 kPa determined Child-Pugh B/C with positive predictive value (PPV) 0.820.For compensated CHB,cut-offs of LSM 23.3,15.2 and 10.8 kPa diagnosed cirrhosis,liver fibrosis ≥S3 and ≥S2 with positive likelihood ratio nearly 10.0 and PPV 0.692,0.882 and 0.980,respectively; and cut-offs 8.8 kPa,6.6 kPa excluded cirrhosis,liver fibrosis ≥ S3 with negative likelihood ration nearly 0.1 and negative predictive value 0.977 and 0.903,respectively.Correlation coefficient between LSM and grades of esophageal varices was only 0.180,and AUROC for TE predicting EV was of no clinical value.ConclusionTE relatively make accurate prediction in the severity of liver fibrosis and classification of Child-Pugh.Patients with LSM ≥ 10.8 kPa should be considered for receiving antivirus treatment.

Objective To study the effects of radical surgical treatment for early esophageal cancer, and to investigate the prevention and cure of their complications,cancer recurrence and metastasis.Methods Treatments of 240 patients with early esophageal cancer of Chinese PLA General Hospital from January 2005 to January 2009 were retrospectively analyzed.The patients were treated by left thoracotomy,thoracic or cervical mechanical anastomosis surgical methods of treatment.Their postoperative adverse reactions,complications and 1 -,3-,5-year survival rates were observed and analyzed.Results The surgical resection rate was 1 00.00%. Complications included postoperative pulmonary infection (1 2 patients,5.00%), cardiac arrhythmias (1 patient,0.42%),delayed gastric emptying (2 patients,0.83%),pleural hemorrhage (1 patient, 0.42%),recurrent laryngeal nerve injury (2 patients,0.83%)and anastomotic fistula (1 patient,0.42%). One-year,3-year and 5-year survival rates after surgery were 1 00.00% (240 /240),97.9% (235 /240)and 95.8% (230 /240)respectively.The main causes of postoperative death were tumor recurrence and metastasis. Conclusion Timely surgery for early esophageal cancer can bring in good effect and long-term outcome,with little complication,which can obtain a good forward curative effect.

OBJECTIVETo observe the expression and clinical implication of receptor for activated C kinase 1 (RACK1) in mononuclear cells and coronary atherosclerotic plaques from patients with coronary artery disease.

METHODSmRNA and protein expressions of RACK1 were detected in mononuclear cells from 29 patients with stable angina pectoris (SAP), 41 patients with acute coronary syndrome (ACS) and 30 healthy volunteers. RACK1 protein expression was also detected by immunohistochemistry in 17 coronary atherosclerotic plaques and 6 normal autopsy coronary samples.

RESULTS(1) mRNA expression of RACK1 was significantly upregulated in mononuclear cells from patients with ACS compared with those from patients with SAP (18.71 ± 5.45 vs. 12.18 ± 4.14, P < 0.05), and the latter was also significantly higher than in healthy controls (12.18 ± 4.14 vs. 3.65 ± 1.57, P < 0.05). (2) Similar changes were observed for protein expression of RACK1 for the three groups. (3) Increased expression of RACK1 was found in atherosclerotic plaques, especially in unstable plaques, positive RACK1 stain was evidenced in foam cells, inflammatory cells, smooth muscle cells and endothelial cells.

CONCLUSIONSThe expression of RACK1 is significantly upregulated in mononuclear cells from patients with coronary artery disease, especially in patients with ACS, and in coronary atherosclerotic plaques, especially in unstable plaques. Our results thus suggest that RACK1 might play an important role in the development and progression of coronary artery disease.

Adult ; Aged ; Case-Control Studies ; Coronary Artery Disease ; blood ; genetics ; pathology ; Female ; Gene Expression ; Humans ; Leukocytes ; metabolism ; Male ; Middle Aged ; Receptors for Activated C Kinase ; Receptors, Cell Surface ; genetics ; metabolism

Objective:To study the correlation between HBV Pre-S1 antigen,HBeAg levels and HBV DNA copies,so as to assess the clinical value of Pre-S1 in detection of HBV replication.Methods:A total of 363 HBsAg-positive samples were col- lected.The levels of Pre-S1 antigen,HBeAg and HBV DNA copies were determined by ELISA,time-resolved immuno-fluores- cent method and real-time fluorescent quantitative PCR(FQ-PCR),respectively.The correlation between the determination re- sults was analyzed.Results:Pre-S1 antigen level was correlated with the level of HBeAg(X~2=94.4,P

To explore the application value of bcr-abl fusion gene deterction microarray in diagnosis, typing, choosing of treatment variant and prognosis judgment, probe for fusion gene detection was designed, oligonucleotide microarray was prepared; total RNA was extracted, reverse-transcripted and labeled by fluorescence, then cDNA was hybridized with microarray in order to detect bcr-abl fusion gene in leukemia cells. The results showed that better reaction conditions were gained by exploration of hybridizotion temperature and elution conditions, bcr-abl fusion gene in leukemia cells was detected by prepared miccroarray. In conclusion, oligonucleotide microarray is effective in detecting the fusion gene and has some unique advantages and certain clinical application value, but has some deficiency too. If microarray can be improved further, it could be used widely in the field of hematology.
Fusion Proteins, bcr-abl ; genetics ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; K562 Cells ; Leukemia ; genetics ; pathology ; Oligonucleotide Array Sequence Analysis ; methods ; Reproducibility of Results

OBJECTIVETo investigate the advantage of CT and MRI image fusion in determining the target precisely during 3-dimensional conformal radiotherapy for cranial carcinoma.

METHODSTwenty-five patients received CT and MRI examination simultaneously for localizing the tumor and defining target before 3-dimensional conformal radiotherapy. The target defined by MRI image was used as gross tumor volume, whereas CT value was used to calculate dose, making plan for radiotherapy. The difference between the target defined by CT and MRI was compared.

RESULTSAll the 25 patients underwent CT and MRI image fusion for localizing the tumor and defining the target in order to make anatomic symbol and surface symbol superposed. The number of tumor nodual detected by CT was as same as that found by MRI in 23 cases except two. Compared with the GTV defined by MRI image, it was larger in 10 cases by CT image, whereas smaller in 15 cases. The response rate assessed by MRI image was 64.0% (CR + PR) at the end of radiotherapy.

CONCLUSIONCT and MRI image fusion technique is more precise than either by CT or MRI alone in defining the GTV of 3-dimensional conformal radiotherapy for cranial carcinoma.

Adolescent ; Adult ; Aged ; Brain Neoplasms ; diagnostic imaging ; pathology ; radiotherapy ; Humans ; Imaging, Three-Dimensional ; Magnetic Resonance Imaging ; methods ; Middle Aged ; Radiotherapy Planning, Computer-Assisted ; Radiotherapy, Conformal ; methods ; Remission Induction ; Tomography, X-Ray Computed ; methods ; Tumor Burden ; Young Adult

OBJECTIVETo investigate the nasal epithelium toxicity of adjuvants and rHV2 nasal spary(HVS).

METHODCiliary movement were evaluated with in situ toad palate model; The histology assessment of nasal epithelium were carried out after long-lasting and repeated use of HVS.

RESULT AND CONCLUSIONAdjuvants included SDS, Brij 35, azone, lecithin, EDTA, menthol, nipagin and thiomersal were able to significantly inhibited the ciliary movement, while tween80, glycyrrhizic acid monoammonium salt, benzalkonium bromide, sodium benzoate and adhensive materials investigated had less influence on it. HVS was able to damaged the nasal epithelium, but this effect recovered soon after stopping administration. It was demonstrated that SDS, Brij 35, azone,lecithin, EDTA, menthol, nipagin and thiomersal. It had significant cilitoxity, while tween80, glycyrrhizic acid monoammonium salt, benzalkonium bromide, sodium benzoate and adhensive materials investigated had no significance; Chitosan co-administration with some adjuvants may make the cillitoxity severer; It is available that rHV2 be administered by nasal spary.

Adjuvants, Pharmaceutic ; administration & dosage ; toxicity ; Administration, Intranasal ; Animals ; Bufo bufo ; Chitosan ; administration & dosage ; toxicity ; Cilia ; drug effects ; Epithelium ; drug effects ; Female ; Hirudins ; administration & dosage ; toxicity ; Male ; Nasal Mucosa ; drug effects ; Palate ; drug effects ; Rabbits ; Recombinant Proteins ; administration & dosage ; toxicity

AIM: To explore the role of PI3K/Akt/nNOS in Zhouluotong extract resisting diabetic peripheral neuropathy.METHODS:The Schwann cells were divided into normal group ( D-glucose 25 mmol/L) , model group ( D-glucose 100 mmol/L) , Zhouluotong extract Z-6 +high glucose group, Zhouluotong +high glucose group, mecobalamine+high glucose group.The viability, nitric oxide content and the Ca2+-ATPase activity in Schwann cells were determined by Cell Counting Kit-8 , nitric oxide assay kit and Ca2+-ATPase assay kit, respectively.The apoptosis of Schwann cells were analyzed by flow cytometry.The expression of Bcl-2, Bcl-xL, Bax, Bak and caspase-3, and the phosphorylation levels of nNOS and Akt were determined by Western blotting.The signal pathway of PI3K/Akt was explored by dominant negative PI3K and Akt (δp85 and DN-Akt) transient transfection assay.RESULTS:Under high-glucose culture, the cell viability, nitric oxide content in culture supernatant, the expression of Bcl-2 and Bcl-xL, and the phosphorylation levels of Akt and nNOS in the Schwann cells were significantly increased.The cell apoptosis, the expression of Bax, Bak and caspase 3 in the Schwann cells were significantly decreased by Zhouluotong extract Z-6, compared with model group.In-creased nitric oxide content and the up-regulation of nNOS were observed.However, the effects of blocking PI3K/Akt, the upstream pathway of nNOS , by transfection with DN-δp85 on Akt phosphorylation in the Schwann cells was still unclear. CONCLUSION:Zhouluotong extract Z-6 changes the phosphorylation of nNOS, and the expression of anti-apoptotic fac-tors , caspase-3 and pro-apoptotic factors in Schwann cells under high-glucose culture, thus reducing apoptosis and elevating viability.The relationship to PI3K/Akt/nNOS pathway needs further investigation.

AIMTo investigate the pharmacokinetics and the anticoagulation action of recombinant hirudin-2 (rHV2) nasal spray after administration of the preparation.

METHODSrHV2 concentration in plasma was determined by chromogenic substrate method and the relative bioavailability was calculated. The anticoagulation action of rHV2 spray after intranasal administration in normal rats and DIC model rabbits after intranasal administration of rHV2 spary were studied.

RESULTSThe in vivo course of rHV2 in rats fitted to the one-compartment model after intranasal administration of rHV2 spray and the relative bioavailability was 28.53%. Coagulating times of APTT and TT were significantly prolonged in normal rats, and APTT in DIC model rabbits was significantly shortened and was close to the normal values after administration of rHV2 nasal spray.

CONCLUSIONrHV2 spray could be an effective nasal preparation of rHV2.

Administration, Intranasal ; Animals ; Area Under Curve ; Biological Availability ; Disseminated Intravascular Coagulation ; physiopathology ; Hirudins ; administration & dosage ; pharmacokinetics ; pharmacology ; Male ; Partial Thromboplastin Time ; Rabbits ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; administration & dosage ; pharmacokinetics ; pharmacology ; Thrombin Time