Objective Antiangiogenesis therapy has been shown to prolong survival for patients with malignant tumor .However the present study has not been observed the clinical benefit of antiangiogenesis therapy combination with chemotherapy treated with gastric canc-er.Human recombinant vascular endothelial inhibition (endostar) as a multi-targeted anti-angiogenesis drug, the mechanism is different from other Antiangiogenesis drugs.It can block different pathways of signal transduction to inhibit angiogenesis .This study aimed to observe the effect of combined application of endostar and paclitaxel on biological behavior of gastric cancer cell lines . Methods MMT assay and Tr-answell invasion assay were respectively used to examine the inhibition rate of cell growth and invasion ability when cells were treated with va-rious concentrations of endostar and paclitaxel alone or in combination.The protein expressions of VEGF,MMP-2 and MMP-9 were examined by Western blot. Results Endostar or paclitaxel effectively inhibited the growth of MGC803 cells and the in vitro invasion of MGC803 cells in a concentration-dependent manner.The proliferation and invasion ability of combined treatment with endostar and paclitaxel was significantly lower than that of endostar or paclitaxel alone (P<0.05).Compared with con-trol group, the VEGF,MMP-2 and MMP-9 protein expressions were de-creased in experimental groups ( P <0.05).Compared with paclitaxel group, the VEGF, MMP-2 and MMP-9 protein expressions were relatively reduced in combination groups (P<0.05). Conclusion Endostar combined with paclitaxel can suppress the growth and invasion of MGC803 cells, and the decreasing VEGF , MMP-2 and MMP-9 expressions may be involved in the mechanism .

OBJECTIVETo study the cloning and expression of flagellin gene from Chinese Borrelia burgdorferi, PD91 strain and to evaluate the feasibility of using recombinant protein as diagnostic antigen when comparing the gene sequence with flagellin gene from North American Borrelia burgdorferi B31.

METHODSThe piece of genes coding flagellin from Chinese Borrelia burgdorferi PD91 by polymerase chain reaction (PCR) method was obtained, and constructed recombinant plasmid, before transformed into E. coli BL21 strain, and induced. The recombinant plasmid was identified with enzyme cutoff and gene sequence comparison. Efficient expression strain was selected and the expression product was analyzed with sodium amplified polymorphic-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot method.

RESULTSThe recombinant protein (r-flagellin) expressed in host bacteria was successful. By means of western-blot assay, the immunological response showed the same antigenicity between r-flagellin and PD91 flagellin. The piece of genes coding flagellin of PD91 was 1011 bp, but when comparing with that of North American Borrelia burgdorferi it showed 94.70% homology. Homology between the sequence of amino acid of the r-flagellin and that of B31 flagellin was 95.85%.

CONCLUSIONFlagellin gene of Borrelia garinii of Chinese Lyme disease spirochete was successfully cloned and expressed for the first time. It was proved that the immunoreactivity of r-flagellin was the same as the natural flagellin.

Amino Acid Sequence ; Base Sequence ; Borrelia burgdorferi ; genetics ; isolation & purification ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Flagellin ; biosynthesis ; genetics ; Humans ; Lyme Disease ; microbiology ; Molecular Sequence Data ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

BACKGROUNDThe antitumor role of Ras association domain family 1A (RASSF1A) gene and its potential molecular mechanisms are not well understood. The objective of this study was to observe the antitumor ability of RASSF1A in hepatocellular carcinoma, and study the mechanisms of cell apoptosis induced by RASSF1A.

METHODSAfter stably transfecting a RASSF1A (wild-type or mutant) expression vector into the BEL-7402 hepatocellular carcinoma cell line, RT-PCR and Western blotting was used to detect the RASSF1A expression levels in recombinant cells. The effects of wild-type RASSF1A on cell growth were observed in vitro by analyzing cell proliferation rate, cell colony formation, and in vivo by analyzing tumorigenesis in nude mice. In addition, the effect of RASSF1A gene expression on the chemosensitivity of human hepatocellular carcinoma cells to antitumor drugs was examined by inhibition of cell proliferation and the percentage of apoptotic cells.

RESULTSWild-type RASSF1A, not the mutant, suppressed cell growth in vitro and in vivo. Re-expression of wild-type RASSF1A could enhance the inhibition of cell proliferation and the percentage of apoptotic cells following cell treatment with mitomycin, but had no significant effect when combined with adriamycin, etoposide, 5-fluorouracil and cisplatin treatment.

CONCLUSIONWild-type RASSF1A inhibits cell growth and enhances cell chemosensitivity to mitomycin in hepatocellular carcinoma, suggesting that RASSF1A may serve as a new target for gene therapy in hepatocellular carcinoma patients.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; genetics ; Blotting, Western ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Genetic Therapy ; methods ; Humans ; Mitomycin ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Proteins ; genetics ; metabolism ; physiology

Objective To establish a method of detecting hepatitis B virus x antigen (HBxAg) and antibody to HBxAg (anti-HBx) and to demonstrate its clinical significance of HBxAg and anti-HBx in sera from patients of chronic hepatitis B (CHB),liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Methods Full length HBx gene was cloned into pET30a(+),a prokaryotic expression vector,named pET30a-X.It was transformed into Escherichia coli BL21 (DE3),followed the fusion protein of HBx-His was induced by IPTG.The purified fusion protein was used to immunize rabbit as an antigen to generate polyclonal antibody to HBx protein.The method of enzyme-linked immunosorbent assay (ELISA) was established by using purified fusion protein and generated antibody,which was used to detect HBxAg and anti-HBx in sera from patients of CHB,LC,HCC and normal healthy people.Results The positive rates of HBxAg/anti-HBx were 8.7%/10.4% for CHB,17.9%/40.6% for LC,and 9.8%/34.4% for HCC, respectively.In statistics,the positive rates of anti-HBx in LC and HCC were higher than that in CHB (P

Objective To understand the carrying status of Borrelia burgdorferi in ticks from the mountain areas from six representative provinces, including Jilin, Shanxi, Gansu, Qinghai,Guizhou and Hunan in China. Methods Flagging and trapping methods were used to collect ticks in forest area and culture medium was used to isolate the pathogen. Nested-PCR was used to detect the gem-carrying rate of ticks. Results More than 2200 ticks from six representative provinces were collected and 1000 ticks were used to isolate the pathogen. 13 Lyme disease spirochetes from ixodes persulcatus in Changbai, Jilin province and 9 Lyme disease spirochetes from ixodes granulatus in Daozhen, Guizhou province were identified. There were 1255 ticks used for PCR testing. Specific fragments of the Borrelia burgdorferi in ticks were found from the six representative provinces in China. The carrier rate was higher in Jilin (Changbai 27.08%, Tonghua 20.41% ), Qinghai (Huzhu 25.06%, Huangnan 21.11%)and Guizhou (Daozhen 25.63% ), than in Shanxi (Yuanqu 4.72%,Jiaocheng 3.64% ). Result from the sequence analysis showed that the genotype belong to Borrelia garinii in Jilin, Qinghai, Gansu, Shanxi provinces but Borrelia valaisiana in Guizhou and Hunan provinces. Conclusion Our data showed that there existed Lyme disease spirochetes in all the six representative provinces in China, but the carriying rates of ticks were different. Borrelia garinii was found in Shanxi province, and Borrelia valaisiana in Hunan province.

Objective To investigate the effect of a selective muscarinic receptor antagonist (penehyclidine hydrochloride) in three vessel occlusion model of acute global cerebral ischemia-reperfusion in rats.Method One hundred and forty-four male SD rats were randomly divided into four groups:sham operated group,vehicle treated group (saline 1 ml,i.p.),scopolamine treated group (0.01 mg/kg,i.p.) and penehyclidine hydrochloride treated group (0.01 mg/kg,i.p.) with drugs injected 40 minutes before ischemia respectively.The ischemic duration was 10 minutes.The animals were subjected to motor activity tests (open field activity test,beam-walking test and grip test) at 24 hours or on the 3rd and 7th day after reperfusion.HE staining,TUNEL staining and immunohistochemical reactions of bax and bel-2 were carried out at the time points of 2,12,24 hours,3 and 7 days after reperfusion.TTC staining was carried out in some rats for assessment of infarction volume on the 4th day after reperfusion.Results As compared with the vehicle treated group,both penehyclidine hydrochloride treatment and scopolamine treatment decreased the numbers of apeptotie neurons (P

OBJECTIVEThe study investigate the antioxidant probucol on endothelial function in patients with acute coronary syndrome (ACS).

METHODSA total of 49 ACS patients randomly received standard therapy plus probucol (P, n = 24) or standard therapy (C, n = 25). Plasma oxidized low-density lipoprotein (ox-LDL), nitric oxide (NO) and circulating endothelial cells (CEC) were measured. The brachial arterial hyperemia-induced flow mediated dilation (FMD) and sublingual nitroglycerin (NTG) mediated vasodilatations were measured by high resolution ultrasound. These variables were analyzed before and after 3 months therapy.

RESULTSPlasma NO and FMD was significantly increased after 3 months therapy than before therapy [(80.46 +/- 10.24) micromol/Lvs (48.46 +/- 12.24) micromol/L, P < 0.01; (13.46 +/- 1.20)% vs (7.45 +/- 1.02)%, P < 0.05, respectively], while the number of CEC and ox-LDL were significantly decreased (P < 0.01) in P group. These values were similar before and after 3 months in C group. The linear correlation analysis showed that plasma ox-LDL negatively correlated with NO (r = -0.574, P < 0.01) and FMD (r = -0.517, P < 0.01) and positively correlated with CEC (r = 0.385, P < 0.01) in patients received 3 months probucol therapy.

CONCLUSIONSChronic antioxidant probucol therapy could improve endothelial function in patients with ACS.

Adult ; Aged ; Angina, Unstable ; blood ; drug therapy ; Anticholesteremic Agents ; therapeutic use ; Endothelial Cells ; drug effects ; physiology ; Endothelium, Vascular ; drug effects ; physiopathology ; Female ; Humans ; Lipoproteins, LDL ; blood ; Male ; Middle Aged ; Myocardial Infarction ; drug therapy ; physiopathology ; Nitric Oxide ; blood ; Probucol ; therapeutic use

OBJECTIVETo develop a method to obtain and identify human coronary artery endothelial cells obtained during percutaneous coronary interventions (PCI).

METHODSCoronary guide wires were used to obtain endothelial cells from coronary arteries in 28 patients undergoing PCI. The cells were eluted from the wire tips and then purified by magnetic beads coated with anti-CD146 antibody. von Willebrand factor (vWF) was used as an immunocytochemical marker for endothelial cells. The cellular viability was evaluated by observing cell membrane integrity and energy-dependent uptake of DiI-labeled acetylated low-density lipoprotein.

RESULTSAn average of 96 coronary artery endothelial cells with good viability per patient were obtained by one guide wire. vWF identification showed their endothelial morphology and immunoreactivity.

CONCLUSIONThe viable coronary endothelial cells could be obtained during routine percutaneous coronary interventions combined with magnetic beads isolation technique. These cells may be used for further cellular functional analyses (such as immunocytochemistry and molecular biology) and expand our understanding on mechanisms of coronary artery diseases.

Biopsy ; methods ; Coronary Vessels ; cytology ; pathology ; Endothelium, Vascular ; cytology ; pathology ; Female ; Humans ; Male ; Middle Aged

OBJECTIVETo investigate the correlation between circulating endothelial progenitor cells (EPCs) and the risk factors of coronary heart disease (CHD) as well as the severity of coronary lesions, and its clinical significance.

METHODS42 patients with CHD and 36 patients excluding CHD (control) were studied. Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and were cultured in M199 medium supplemented with 20% fetal bovine serum, 50 ng/ml vascular endothelial growth factor (VEGF). After 14 days cultured, the numbers of colony-forming units of EPCs were counted by phase-contrast microscope. The relationship between the number of colony-forming units of EPCs and the risk factors of CHD (such as age, gender, hypertension, hypercholesterolemia, diabetes, smoking, positive family history of CHD) as well as the severity of coronary lesions were assessed.

RESULTSThe number of risk factors of CHD was significantly correlated with a reduction of EPCs levels (r = -0.436, P = 0.014). Smoking was associated with significantly lower EPCs levels, whereas a minor but nonsignificant reduction of EPCs levels was detected in the presence of gender, hypertension, and a positive family history of CHD. It was observed that low density lipoprotein (LDL) and uric acid were negatively correlated with the number of colony-forming units of circulating EPCs (P < 0.05). A correlation existed between age, high density lipoprotein, apoprotein A and levels of circulating EPCs, however, this relation was not statistically significant. The number of colony-forming units of circulating EPCs in CHD groups was significantly lower than those in control group (12.8 +/- 6.34 versus 37.0 +/- 5.5, P < 0.001); and the circulating EPCs level of coronary artery lesion group (including single, double, triple vessels disease) was significantly lower than that of control group (P < 0. 01).

CONCLUSIONSThe level of circulating EPCs was inversely associated with the risk factor scores of CHD and the severity of coronary artery lesion. These finding imply that endothelial injury in the absence of sufficient circulating EPCs may affect the degree of the heart disorder and the clinical situation.

Aged ; Coronary Disease ; blood ; pathology ; Endothelial Cells ; cytology ; Female ; Humans ; Male ; Middle Aged ; Risk Factors ; Stem Cells ; cytology

OBJECTIVETo study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure.

METHODSFP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index.

RESULTSCriteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.

CONCLUSIONEstablishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China.

Blotting, Western ; methods ; Borrelia burgdorferi Group ; pathogenicity ; China ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Humans ; Lyme Disease ; diagnosis ; microbiology