AIM:To study the effect of adiponectin (APN) on lipid peroxidation in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H2O2) in vitro. METHODS:The effect of H2O2 on apoptosis was measured by flow cytometry with Annexin V-FITC labeling. The concentration of malonaldehyde (MDA) in HUVECs was detected by thiobarbituric acid (TBA) method. The activities of superoxide dismutase (SOD) and catalase (CAT) were measured by colorimetric assay. The scavenging rate on superoxide anion radicals in xanthine oxidase system and OH radicals produced by Fenton reaction were also tested. RESULTS:APN significantly inhibited H2O2-induced apoptosis. H2O2 increased MDA production and reduced the activities of SOD and CAT,which were inhibited by APN. APN did not alter the activities of SOD and CAT in normal condition. APN dose-dependently decreased the superoxide anion radicals,with the scavenging rate of 11.9%,21.4%,36.9%,respectively,and scavenged OH radicals. CONCLUSION:APN inhibits apoptosis and protects endothelial cells against lipid peroxidation insult by scavenging the free radicals.

Objective To establish a TaqMan-MGB fluorescent probe characterized real-time polymerase chain reaction ( qPCR) method for detecting retinoic acid induced genes G ( RIG-G) in human acute promyelocytic leukemia ( M3 ) .Analyze RIG-G expression levels in peripheral blood of both normal persons and M3 patients and explore its diagnosis value for M 3.Methods Methodology establishment study.A detection method and standard curve of TaqMan-MGB real-time PCR were established after designing specific primers and TaqMan-MGB fluorescence probe of human RIG-G gene and using reverse transcription complementary DNA ( cDNA) as a template.The performance of this method was evaluated in specificity, accuracy, precision, analytical sensitivity and interference substances . Twenty clinical specimens with M3 were quantified RIG-G expression so as to evaluate the correlation between peripheral blood and bone marrow samples .Meanwhile , the results of RIG-G expression in peripheral blood of 40 normal specimens and 20 patients with M3 were analyzed by t-test.And receiver-operating characteristic curve ( ROC ) was used to analyze the detection efficiency of M 3.Results There was a good linear relationship between log value of RIG-G standard substance and threshold cycle number ( Ct ) ( standard curve equation:Y=-3.539X+42.952,R2 =0.999).New method was used to detect standard substance . The deviation between observed and expected values was <5% (r=0.999).Three concentration samples (107 ,104 ,101 copies/μl) were selected for precision test.Intra-assay coefficients of variation were 1.38%, 2.31% and 1.38%, respectively , and intre-assay coefficients of variation were 0.71%, 1.17% and 5.07%, separately.All were less than 10%.The sensitivity of this method was 101 copies/μl.There was a good correlation of RIG-G results between peripheral blood and bone marrow in M 3 patients(r=0.996, b=0.973).But there was no significant difference between this two group results (t=0.099, P>0.05). However , there was obvious difference of RIG-G value in peripheral blood between control group and M 3 patient group (U=18,P<0.001), 3.62 ×104(1.61 ×104 -4.90 ×104)copies/μl for controls and 7.10 ×102 (5.43 ×102 -2.21 ×103 ) copies/μl for M3 patients, respectively.Conclusions Successfully establishe a TaqMan-MGB real-time PCR method for detecting RIG-G gene in peripheral blood.The accuracy, precision, sensitivity and specificity are good .It could provide necessary help in early diagnosis and monitor treatment of clinical M3 patients.

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.
Animals ; Antigens, Viral/genetics* ; Biological Assay ; Chickens/immunology* ; Escherichia coli/genetics* ; Infectious bursal disease virus/immunology* ; Poultry Diseases ; Vaccines, Synthetic/isolation & purification* ; Viral Structural Proteins/immunology* ; Viral Vaccines/immunology*

OBJECTIVETo investigate the effect of antibacterial peptide hCAP18/LL-37 on ovarian cancer microenvironment and the regulatory mechanism of its expression.

METHODSWe assessed the effect of macrophage-promoted ovarian cancer cells invasion using BioCoat Matrigel invasion chamber. The expressions of hCAP18/LL-37 and versican V1 were determined by real-time PCR and Western blot analysis. SKOV3 cells were transfected with shRNA plasmid to abrogate the expression of versican V1, and then the expression of hCAP18/LL-37 in macrophages and the invasiveness of SKOV3 cells were assayed.

RESULTSThe Matrigel invasion assay showed that after co-culture with macrophages for 4 days, the number of penetrated SKOV3 cells was 112.8±17.1/per high power field, significantly higher than that in the SKOV3 cells cultured alone (8.2±1.9/per high power field) (P<0.05). Addition of hCAP/LL-37 neutralizing antibody into the co-cultured macrophage-SKOV3 cells markedly inhibited the macrophage-promoted SKOV3 cells invasion. The penetrated SKOV3 cells was 22.2±5.6/per high power field, significantly lower than the 100.6±25.2/per high power field in the control macrophage- SKOV3 co-cultured cells (P<0.05). The expressions of hCAP18/LL-37 mRNA and protein in macrophages were remarkably enhanced upon co-culture with SKOV3 cells, but not changed in SKOV3 cells cultured alone. The expression and secretion of versican V1 in the ovarian cancer cells were also significantly increased after co-cultured with macrophages. Knockdown of versican V1 in SKOV3 cells by small interfering RNA significantly reduced the expression of hCAP18/LL-37 mRNA and protein in the macrophages, as well as decreased the invasiveness of SKOV3 cells (P<0.05).

CONCLUSIONSIn the cancer microenvironment, the macrophage-secreted hCAP18/LL-37 promote the invasiveness of ovarian cancer cells, and the hCAP18/LL-37 expression is regulated by versican V1 protein released by ovarian cancer cells.

Antimicrobial Cationic Peptides ; metabolism ; pharmacology ; Cell Line, Tumor ; Coculture Techniques ; Collagen ; Drug Combinations ; Female ; Humans ; Laminin ; Macrophages ; metabolism ; Neoplasm Invasiveness ; Neoplasm Proteins ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; physiopathology ; Plasmids ; Proteoglycans ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Transfection ; Tumor Microenvironment ; drug effects ; Versicans ; metabolism