Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.

Objective To identify human leucocyte antigen (HLA)-A* 0201-restricted hepatitis C virus (HCV)-cytotoxic T lymphocyte (CTL) epitopes. Methods Based on the prediction results of RANKpep and SYFPEITHI prediction programs, six candidate CTL epitopes were selected and synthesized. The affinity of candidate CTL epitopes to HLA-A* 0201 molecules of T2 cells was explored. Subsequently, enzyme-linked immunosorbent spot (ELISPOT) assay and intracellular cytokine staining (ICS) were utilized to determine whether candidate CTL epitopes could induce the recall positive response in peripheral blood mononuclear cells (PBMC) of HLA-A* 0201 positive HCV-1b-infected patients. Results Among six candidate CTL epitopes, peptides C_181(LLSCLTTPV) and NS2_172 (VLQAGLIRV) had high affinity to HLA-A* 0201 molecules. Moreover, the affinity was proportional to the concentration of peptide. Furthermore, among ten HLA-A* 0201 positive HCV-1b-infected patients, the frequencies of C_181 and NS2_172-specific interferon (IFN)-γ-producing cells were 0-19 spots forming cells (SFC)/1 × 105 PBMC and 0-20 SFC/1 × 105 PBMC, respectively.The percentages of C_ 181 and NS2_172-specific IFN-γ+ CD8+ T lymphocytes in total CD8+ T lymphocytes were 0.006%-0.065% and 0.005%-0.080%, respectively. Conclusion Peptides C_181 (LLSCLTTPV) and NS2_172 (VLQAGLIRV) are identified as novel HLA-A* 0201-restricted HCV-CTL epitopes.

Objective To investigate the level of chemotactic factors(CXCL5 and CXCL8)in hepatic fibrosis and cirrhosis tissue.Methods Hepatic tissues were obtained from 9 patients with hepatic hemangioma (hepatic hemangioma group),10 patients with liver fibrosis(liver fibrosis group)and 11 patients with liver cirrhosis(1iver cirrhosis group)at Nanfang Hospital from May 2008 to May 2009.The contents of CXCL5 and CXCL8 in hepatic tissue were assayed by ELISA.All data were analyzed by one-way ANOVA,Pearson rank correlation or Spearman correlation.Results The contents of CXCL5 and CXCL8 were(0.8±0.7)ng/g and(6.2±3.7)ng/g in hepatic hemangioma group,(2.0±2.0)ng/g and(11.6±3.5)ng/g in liver fibrosis group and (17.1±4.8)ng/g and(12.3±3.9)ng/g in liver cirrhosis group,with significant difference among the 3 groups (F=60.050,7.690,P＜0.05).The expression of CXCL5 was correlated with the content of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and prothrombin time(PT)(r=0.502,0.468,0.523,P＜0.05):the expression of CXCL8 was correlated with the content of ALT,AST.total bilirubin and PT(r=0.477,0.504,0.537,0.431,P＜0.05).Conclusions With the aggravation of hepatic fibrosis,the contents of CXCL5 and CXCL8 are increased with different patterns.The changes of CXCL5 and CXCL8 are related with the injury of liver,but the changes of CXCL5 and CXCL8 do not correspond with the degree of the injury of liver.

Objective To discuss the correlation between aortic elasticity and coronary artery calcification by CT.Methods Totally 111 patients who were diagnosed of coronary artery disease underwent coronary artery CTA.The images were qualified for aortic elasticity measurement.All patients were divided into calcification negative group (n=43) and calcification positive group (n =68).The calcification positive group was further divided into light,medium,and serious groups according to their calcification scores.The ascending aortic images were reconstructed every 5 ％ R-R intervals.The cross-sectional areas and diameters of aortic in each R-R interval were measured automatically,then diameter variation rate (％ A0),aortic distensibility (A0D),aortic compliance (A0C) and aortic stiffness (A0SI) were calculated to evaluate aortic elasticity.Correlation between aortic elasticity and coronary artery calcification were analyzed.Results ％ A0,A0 D,A0C were lower and A0SI was higher in calcification positive group than those in calcification negative group (all P＜0.05).There was no significant differences in the four reference indexes of aortic elasticity among light,medium,and serious groups in calcification positive group (all P＞0.05).Correlation analysis demonstrated negative correlations between ％ A0,A0 D,A0 C and calcification scores,and a positive correlation between A0SI and calcification scores.Conclusion Aortic elasticity is correlated with coronary artery calcification,and the combination of them will be beneficial for the early diagnosis of coronary heart disease.

Objective To evaluate the value of positive 99Tcm-MIBI tumor imaging for thyroid cancer diagnosis. Methods Fifty four suspected thyroid cancer patients underwent 99Tcm-O4 and 99Tcm-MIBI combined imaging procedure. The imaging data were confirmed by pathological findings. Results All the 54 cases had single throid nodules, and 25 of which were pathologically malignant. Fifty two cases of nodules were detected by the 99Tcm O4 thyroid static imaging, including 2 hot nodules,4 warm nodules, 10 cool nodules and 36 cold nodules;2 cases were negative by the imaging. Of the 25 malignant thyroid nodules, 16 nodules were visible by 99Tcm-MIBI uptake and were cold nodules;29 exhibited benign thyroid nodules,of which 15 could be seen by 99Tcm-MIBI uptake,including 1 warm nodules,2 cool nodules and 12 cold nodules. The sensitivity, specificity of the combined imaging of 99Tcm O4 and 99Tcm-MIBI were 64. 00% ( 16/25 ) and 48. 28% (14/29). No significant difference was found for the positivity between benign nodules and malignancy nodules by 99Tcm-MIBI tumor imaging ( χ2 = 0. 83, P ＞ 0. 05 ). Conclusion 99Tcm-MIBI tumor imaging is not specific for the diagnosis of thyroid malignancy.

Objective: To assess the regional systolic and diastolic function of left ventricle by pulsed wave Doppler tissue imaging ( PW-DTI) in patients with coronary artery disease ( CAD ) . Methods : Seventy-seven cases of coronary angiography were adopted for study and grouped in terms of severity of coronary arterial lesion. Peak systolic, peak early diastolic, and peak late diastolic motion velocities (Sm, Em and Am, respectively) were recorded at 6 different sites on the basal and medial segments corresponding to the anteroseptal, posteroseptal, lateral, anterior, inferior, and posterior walls of the left ventricle with PW-DTI. Results: Am in segments corresponding to normal coronary arteries (group A) was lower than in segments corresponding to slightly stenosed coronaries (group B). There was no significant difference in Sm and Em between group A and group B. Compared with group A, Sm, Em and Am were significantly decreased in segments corresponding to severely stenosed coronary arteries ( group D). Conclusion : PW-DTI could be used to evaluate quantitatively and accurately the regional mvocardial function of left ventricle in patients with CAD.

AIM: To study the effect of mesenchymal stem cells (MSCs) infusion on hematopoietic recovery after peripheral blood stem cell transplantation in mice. METHODS: BALB/c mice conditioned by high dose chemotherapy/radiotherapy were infused with 10 6 peripheral blood mononuclear cells (PBMC) mobilized by granulocyte colony-stimulating factor (PBSCT group), 10 4 MSCs culture-expanded in vitro and 10 6 PBMC(experimental group 1), 10 6 MSCs and 10 6 PBMC(experimental gruop 2). Survival rate within 4 weeks, white blood cell count, bone marrow nucleated cells (BMNC), granulocyte-macrophage colony forming unit(GM-CFU) and fibroblast colony forming unit (F-CFU) were examined. RESULTS: Survival rate, BMNC, GM-CFU, F-CFU were significantly higher in experimental group 2 than that in PBSCT group ( P

Objective: To observe the effect of ZuoGuiWan and YouGuiWan on induced experimental autoimmune encephalomyelitis(EAE) in Lewis rats,compare the therapeutic effect between ZuoGuiWan and YouGuiWan,and explore the underlying immunoregulatory mechanism between warming-yang and nourishing-yin therapy.Methods : 120 male Lewis rats were bred at Capital Medical University under specifi c pathogen-free conditions.24 rats were selected as normal group before immunization.The other rats were randomly devided into 4 groups after immunization,including EAE group,prednisone group,ZuoGuiWan group and YouGuiWan group.Rats of normal group were injected with normal sodium in the hind feet pad on both sides,the other rats were injected with the antigen consisted of myelin basic protein(MBP) and complete Freund’s adjuvant(CFA) to induce EAE.Rats of normal group and EAE group were administered with normal sodium after immunization,rats of prednisone group with prednisone(5mg/kg),rats of ZuoGuiWan group with ZuoGuiWan(2g/kg),rats of YouGuiWan group with YouGuiWan(3g/kg).On the day 15 and 27 after immunization,the peripheral blood was collected,lymphocyte subgroups and NK cells were detected by ? ow cytometer.Results: On day 15 after immunization: In peripheral blood,the number of CD4+lymphocyte in ZuoGuiWan group was signifi cantly higher(P

Objective: To observe the immunohistochemical expression of interferon-? (IFN-?), matrix metalloproteinase -9(MMP-9) in brain and spinal cord tissue, and explore the action mechanism of Zuogui Pills and Yougui Pills on rats with experimental autoimmune encephalomyelitis(EAE). Methods: Lewis rats were immunized with the myelin basic protein (MBP).120 Rats were grouped randomly into nomal group, EAE group, prednisone group, Zuogui Pills group and Yougui Pills group after post immunization (PI). Rats in normal group and EAE group were administered sodium, each 3ml/d, rats in prednisone-group were administered suspension of prednisone after developed clinical signs, each 5mg/kg, rats in Zuogui Pills group were administered suspension of Zuogui Pills, each 2g/kg, and rats in Yougui Pills group were administered suspension of Yougui Pills each 3g/kg. On days 15 and 27 PI, rats were killed and the immunohistochemical staining was performed on the sections of brain and spinal cord. Results: On days 15 and 27 PI, the expression of IFN-? and MMP-9 in central nervous system of EAE group were higher than those in nomal group (P

To confirm the etiology of a dead case for a 6 year-old female Ailurus fulgens,one strain of the predominant bacteria from pathologic tissues(heart,liver,spleen,lung and other samples) of the dead Ailurus fulgens were examined and isolated.The isolate was named R1 and no other bacteria were isolated.The bacterial etiological examination(morphological characteristics,biochemical characteristics and 16S rDNA gene detection)of R1 showed that it was identifed as K.peneumoniae.Artificial infection to mice about R1 was also conducted in this study.R1 had strong pathogenicity to mice and the LD50 is 6.5 × 104 CFU/mL.Moreover,the clinical and pathological features of the dead mice were consistent with that of the Ailurus fulgens.To find effective therapeutic drugs of curing other Ailurus fulgens,antibiotic sensitivity test of R1 was conducted,and the results revealed that R1 was highly sensitive to cefotaxime et al,moderately sensitive to amikacin and resistant to penicillin.These data showed that K.peneumoniae was bacterial pathogen leading to death of the Ailurus fulgens and it had strong resistance to penicillins,macrolides and virginiamycin and it had broad drug resistance spectrum.However,R1 is sensitive to cephalosporins and aminoglycoside antibiotics.