Objective To evaluate the comparability of test results of self-built human carcinoembryonic antigen(CEA)electro-chemiluminescence immunoassay(ECLIA)and imported reagent.Methods A total of different concentrations 77 fresh serum speci-mens were collected and detected CEA by two kinds of ECLIA kit.The results were analyzed with Excel2003 and SPSS1 9.0 soft-ware.Results The difference between each dose was significant (P <0.05),and the detection results between each had no signifi-cant difference (P >0.05);the sensitivity of the assay was 0.3 ng/mL,the intra coefficient of variation was 4.58%-5.83%,the inter coefficient of variation was 5.07%-5.97%,the analytical recovery was 99.13%-107.28%,the specificity of the assay had no cross reaction with CA1 99 and AFP.The correlation coefficient between two kinds of reagents determination results was greater than 0.95,with imported reagent as reference test,self-built carcinoembryonic antigen ECLIA clinical performance evaluation was acceptable.Conclusion The precision of the two kinds of ECLIA in detection of CEA accord to clinical requirement.Comparability exists in evaluating the acceptability of clinical.

Fed-batch cultures of recombinant Rchia pastoris were conducted for production of angiostatin. The whole fermentation included a growth phase on glycerol and an expression phase on methanol. When ammonium hydroxide solution was used to adjust pH, the cell growth during the expression phase was inhibited and the highest angiostatin concentration was 9.08 mg/L. Shake-flask cultures were carried out in media containing different quantities of ammonia. The results showed that ammonia had an obvious inhibition effect on the cell growth during the expression phase. Therefore KOH solution was used to adjust pH, and during the expression phase cells were able to grow and the highest angiostatin concentration reached 20 mg/L.

Objective To study the transfection effects of nuclear factor-KappaB(NF-κB)decoy oligodeoxynucleotides(ODN) to Kupffer cells (KCs) mediated by lipofectamine,and investigate it's suppression effects on KCs activation. Methods Twenty-four Wistar rats were divided into three groups (n=8).(1)Control group,in which the normal KCs were isolated.(2)LPS group,in which 1 ms/L LPs was added to the culture system.(3)NF-κB decoy ODN group,in which KCs were transduced with NF-κB decoy ODN (4μg×105KCs)prior to LPS stimulation.The transfection efficiency Was assayed,and the phagocytosis function,NF-κB(P65) translocation,CD40 mRNA expression of KCs were also detected respectively. Results Kupffer cells were obviously activated after LPS stimulation.the phagocytosis function was reinforced.the activity of NF-κB transloeated from cytoplasm into nucleus was obviosly increaced.The co-stimulatory molecules expression(CD40 mRNA)significantly increased compared with control group(t=4.01,P<0.01).NF-κB decoy oligodeoxynucleotides can efficiently transfected into KCs mediated by lipofectamine,which can obviously suppress KCs activation,and downregulate the expression of downstream gene(compared with LPS group,t=4.89,P<0.01). Condusion NF-κB decoy ODN can efficiently transfect into KCs and inhibit it's activation.

AIM: To investigate the changes of expression of SR-BI in phorbol 12-myristate 13-acetate(PMA) differentiated U937 cells.METHODS: U937 cells were cultured with 100 nmol/L PMA in order to differentiate the cells to macrophages.Immunocytochemical method,Western blotting analysis and reverse transcription polymerase chain reaction(RT-PCR) were used to detect SR-BI protein and mRNA during differentiation.RESULTS: Immunocytochemistry showed that after exposure of U937 cells to PMA for 24,48,72 hours,the values of SR-BI protein expression in U937 cells were 15.94?3.56,27.86?4.39 and 9.08?2.37,with the first two higher than that in undifferentiated cells(7.76?1.74,P0.05) increment in the expression of SR-BI protein compared with U937 monocytes.RT-PCR showed that relative SR-BI mRNA expression in different group was 0.112?0.006,0.235?0.014,0.344?0.140 and 0.138?0.010,respectively.CONCLUSION: SR-BI protein and mRNA were increased after differentiation,reached a peak at 48 hours,and decreased at 72 hours.High expression levels of SR-BI in U937 macrophages following PMA differentiation may be correlated with foam cell formation.

OBJECTIVETo observe the effects of low-dose exogenous estrogen nonylphenol (NP) on the proliferation of human prostate cancer cell lines DU-145 and the expression of the membrane estrogen receptor GPR30 in the DU-145 cells.

METHODSWe exposed DU-145 cells to different concentrations of NP for 24 hours, followed by measurement of the half maximal inhibitory concentration (IC50) of the cells by cell proliferation assay and determination of the concentration of exposure to low-dose NP. We also observed the expressions of 3 estrogen receptors (ER), including ER-alpha, ER-beta and membrane estrogen receptor GPR30, in the DU-145 cells exposed to low-dose NP by RT-PCR.

RESULTSCell proliferation assay showed that within a certain range of doses, NP inhibited the proliferation of the DU-145 cells with an IC50 of 46 micromol/L, a much lower dose of NP than IC50, 0.01, 0.1.1 micromol/l NP, that can promote the proliferation of DU-145 cells. The results of RT-PCR indicated that the expressions of the three ERs in the DU-145 cells were similar to those in prostate epithelial cells, and that low-dose NP promoted the expression of GPR30.

CONCLUSIONMembrane estrogen receptor GPR30 may play a role in low-dose NP promoting the proliferation of DU-145 cells.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; physiology ; Estrogen Receptor alpha ; metabolism ; Estrogen Receptor beta ; metabolism ; Estrogens ; Humans ; Male ; Phenols ; administration & dosage ; pharmacology ; Prostatic Neoplasms ; metabolism ; pathology ; Receptors, Estrogen ; metabolism ; Receptors, G-Protein-Coupled ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Objective To study the effect of ursolic acid (UA) intervention on hepatocyte apoptosis induced by TGF-β1 and its potential mechanism.Methods Primary hepatocytes were extracted from healthy SD rats by in situ perfusion,cultured for 12-24h,then randomly divided into the following groups:blank control group,UA control group (UA 25μmol/L),TGF-β1 group (TGF-β1 2.5ng/ml),UA intervention group (UA 25μmol/L and TGF-β1 2.5ng/ml),DPI intervention group (DPI 0.5μmol/L and TGF-β1 2.5ng/ml).Each group was treated with drugs for corresponding time and their proliferation and apoptosis were detected by flow cytometry,the expression of CD95 (Fas) mRNA was analyzed by RT-qPCR,the expression of protein CD95 and membrane translocation of NADPH oxidase (NOX) subunit p47Phox were analyzed by Western blotting,and the reactive oxygen species (ROS) generation in primary hepatocytes was analyzed with reactive oxygen detection kit.Results UA intervention at 30min before TGF-β1 stimulating hepatocytes markedly reduced hepatocyte apoptosis (63.97 ± 3.19 vs 80.53 ± 1.56,P＜0.01) and promoted hepatocyte proliferation (18.67 ± 1.60 vs 10.83 ± 2.03,P＜0.01).UA intervention notably down-regulated the expressions of CD95 mRNA and protein (1.28 ± 0.15 vs 2.40 ± 0.25,P＜0.01;1.05 ± 0.15 vs 1.37 ± 0.18,P＜0.05),restrained membrane translocation of p47phox (1.13 ± 0.12 vs 1.76 ± 0.22,P＜0.01),and decreased ROS level in primary hepatocytes induced by TGF-β1 (2.12 ± 0.45 vs 3.23 ± 0.53,P＜0.01).Conclusion The mechanism of UA inhibiting hepatocyte apoptosis induced by TGF-β1 is likely to be that UA intervention reduced hepatocyte apoptosis by inhibiting NOX activation and decrease generation of ROS so as to down-regulate expression of CD95 in hepatocytes.

BACKGROUND:Previous experiments have prepared shape memory polymer based on D,L-poly(lactic acid). According to domestic technical requirements for biological y evaluating biomaterials and medical equipments, tissue-engineered grafts must be subjected to preclinical experiment for biosecurity and cytocompatibility evaluation.
OBJECTIVE:To observe the biosecurity of the shape memory polymer based on D,L-poly(lactic acid).
METHODS:(1) Bacterial endotoxin test:polymer extract, endotoxin working standard solution and checking
standard water were added into limulus reagent, respectively. (2) Sensitization test:Polymer extract+Freund’s complete adjuvant+physiological saline and Freund’s complete adjuvant+physiological saline were injected into the scapula of Kunming mice. After induction by intradermal injection, local induction and excitation, stimulate skin erythema and edema degree were observed in animals. (3) Acute toxicity test:Kunming mice received intraperitoneal injection of 100%, 50%, 25%polymer leaching solution and physiological saline, respectively. (4) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for cel proliferation:The direct method was that human umbilical cord vascular endothelial cel s were inoculated onto the polymer film, lactic acid and glass, respectively;the indirect method was that human umbilical cord vascular endothelial cel s were inoculated into the polymer leaching solution, acrylamide solution and 1640 culture solution.
RESULTS AND CONCLUSION:This shape memory polymer based on D,L-poly(lactic acid) is free of bacterial contamination in compliance with the biosecurity standards, and it has no al ergenic and toxicity but has good cytocompatibility.

Objective This study was to understand the structure characteristics of prophages in the genome of Enterococcus hirae R17,and also to analyze their interaction relationships with the host bacterium.Methods The gene distribution and gene encoding characteristics of prophages in the genome of Enterococcus hirae R17 were identified using the PHAST software.The virulence gene,antimicrobial resistance genes,and environmental resistance genes in the prophages were also analyzed.Results Three prophages were found on the chromosome of Enterococcus hirae,including two incomplete prophage elements (Prophage-1 and Prophage-2) and one complete prophage (Prophage-3).Some function genes of bacteria were found in the sequence of three prophages,including nucleotide transportation and metabolism related genes.One incomplete prophage carrying erythromycin-and bacitracin-resistance genes was identified in the plasmid,which suggested that prophage induced gene horizontal transfer caused erythromycin-and bacitracin-resistance of Enterococcus hirae R17.Conclusion This study laid a solid foundation for the diversity analysis of prophages of Enterococcus hirae.Prophages played an important role in promotion of antimicrobial resistance of enterococci.Scientists should pay more attention to the spread of antimicrobial resistance and pathogenicity induced by prophages.

Objective To investigate the effects of galangin on gene expressions of Nrf2 andγ-GCS in oxidative damage of A375 cell;To discuss its protective mechanism for anti-oxidative damage. Methods A375 melanoma cells were induced oxidative stress to establish oxidative damage model by 700μmol/L H2O2. The study was divided into normal group, model group, positive medicine group and high-, medium-, and low-dose galangin groups. All administration groups were given relevant medicine for cultivation. Cell viability was detected by MTT;ROS content was detected by ELISA;the gene expressions of Nrf2 andγ-GCS were detected by RT-PCR.Results Compared with normal group, cell viability decreased significantly;ROS content increased significantly;the gene expressions of Nrf2 andγ-GCS decreased significantly in the model group (P<0.05,P<0.01). Compared with model group, cell viability increased, ROS content decreased, the gene expressions of Nrf2 andγ-GCS increased significantly in all administration groups (P<0.05,P<0.01). Conclusion Galangin may activate Nrf2 signal path to realize the protective effect on A375 cellular oxidation damage through upregulating the expressions of Nrf2 andγ-GCS to promote the integration of Nrf2 and antioxidant response element and relevant regulatory enzymes.

Objective To observe the effects of betulinic acid (BA) on proliferation of human hepatoma stem cell;To discuss its anti-cancer mechanism from the aspects of cell cycle and cell apoptosis. Methods HepG2 stem cells were cultivated in vitro and testified the self-renewal capacity. The effects of BA in concentration of 40, 20, 10, 5, 2.5, 1.25μmol/L on the cell vitality of cultured human liver cancer stem cells for 24 and 48 hours were measured with CCK-8 method. The human hepatoma stem cell line HepG2 was administrated by BA at concentrations of 40, 20, 10, 5μmol/L for 48 hours, and cell cycle and apoptosis rate were measured by flow cytometry. Results BA could inhibit HepG2 stem cell proliferation obviously with dose-effect relationship. BA influenced cell cycle, and induced tumor stem cell apoptosis. 40μmol/L BA blocked cell cycle in S phase, and cell apoptosis rate reached 10.86%. Conclusion BA has obvious inhibitory effects on proliferation of HepG2 liver cancer stem cell, which probably plays a part in anti-cancer by influencing cell cycle and inducing cell apoptosis.