AIM: To investigate the possibility of 3T3 fibroblast growth on blood fibrin clot in culture medium with recombinant human basic fibroblast growth factor (rhbFGF). METHOD: Growth of the cells on blood fibrin clot was studied by phase-contrast, scanning and transmission electron microscopy and by Giemsa stain and MTT assay. RESULTS: The optimal concentration of rhbFGF for proliferation and survival of the cells was 100 ng/mL. The cells also grew on blood fibrin clot scaffold in the low-serum medium containing 100 ng/mL rhbFGF, and a greater number of the cells survived after 48 hours incubation compared to that after 24 hours. The elongated filopodia appeared to bridge the gaps among the fibroblasts after 24 hours incubation. Further incubation to 72 hours, a greater number of platycytes were found to be joined together by lamellopodia. CONCLUSION: 3T3 fibroblasts could grow and survive on blood fibrin clot in the low-serum medium containing rhbFGF, and a combination of blood fibrin clot and rhbFGF may have over proportional effects on wound healing.

AIM: To investigate the possibility of 3T3 fibroblast growth on blood fibrin clot in culture medium with recombinant human basic fibroblast growth factor (rhbFGF). METHOD: Growth of the cells on blood fibrin clot was studied by phase-contrast, scanning and transmission electron microscopy and by Giemsa stain and MTT assay. RESULTS: The optimal concentration of rhbFGF for proliferation and survival of the cells was 100 ng/mL. The cells also grew on blood fibrin clot scaffold in the low-serum medium containing 100 ng/mL rhbFGF, and a greater number of the cells survived after 48 hours incubation compared to that after 24 hours. The elongated filopodia appeared to bridge the gaps among the fibroblasts after 24 hours incubation. Further incubation to 72 hours, a greater number of platycytes were found to be joined together by lamellopodia. CONCLUSION: 3T3 fibroblasts could grow and survive on blood fibrin clot in the low-serum medium containing rhbFGF, and a combination of blood fibrin clot and rhbFGF may have over proportional effects on wound healing.

AIM: To investigate the behaviour of 3T3 fibroblast and macrophage co-culture on blood fibrin clot or adipose tissue with recombinant basic fibroblast growth factor (rhbFGF). METHODS: MTT method, inverted contrast microscopy, Giemsa staining as well as scanning electron microscope were used in the present study. RESULTS: The effect of rhbFGF on co-culture of 3T3 fibroblast and mouse macrophage on blood fibrin clot in low-serum DMEM with rhbFGF were monitored, and 3T3 fibroblast and macrophage growed well on the blood fibrin blot in low-serum DMEM with rhbFGF. CONCLUSION: The blood fibrin clot, with its low immunogenicity, could be used as a bionic support for three-dimensional tissue culture, and also a physiological carrier to distribute the growth factor rhbFGF for the cells.

Adult ; Air Pollutants, Occupational ; adverse effects ; Chemical Industry ; Female ; Humans ; Insecticides ; adverse effects ; Male ; Maximum Allowable Concentration ; Middle Aged ; Occupational Diseases ; chemically induced ; diagnosis ; Organothiophosphorus Compounds ; adverse effects ; Phosphorus Compounds ; adverse effects ; Sulfides ; adverse effects

Objective To design a new method to verify the position of multileaf collimator(MLC)leaf using a two-dimensional ion chamber array(2D-array).Methods 2D-array of PTW T10018 Seven29~(TM) was used to calibrate the accuracy of MLC leaf position of Elekta Precise accelerator.The edge function of the leaf position of MLC was measured and used as the reference value.The precision of MLC leaf was then evaluated through comparing the measured and reference values.Results The accuracy of MLC leaf position was found within?0.1 mm.Conclusion This method of verifying the accuracy of multileaf collimator leaf position is easy,simple and reliable

AIMTo investigate the effects of Nrf2 (Nuclear-E2 related factor) on gamma-glutamylcysteine synthase (gamma-GCS) in lung of guinea pigs with bronchial asthma.

METHODS20 adult male guinea pigs were randomly divided into two groups (n = 10): control group (C group) and asthmatic group (A group), asthmatic model was established by ovalbumin intraperitoneal and ovalbumin inhalation. The reactive oxygen piece (ROS), reduced glutathione (GSH), oxidant glutathione (GSSG) and total GSH in lung tissue were examined respectively. Inflammatory cell infiltration and index of remodeling of bronchiole were detected. In situ hybridization detected the gamma-GCS heavy subunit (gamma-GCS h) mRNA in lung tissue. Immunohistochemistry detected the expression of Nrf2 protein and gamma-GCS protein in lung tissue. RT-PCR measured the expression of Nrf2 mRNA in lung tissue. The activity of gamma-GCS was measured by coupled enzyme assay.

RESULTS(1) The number of eosinophils and lymphocytes in bronchiole of A group were significantly higher than that of C group (P < 0.05), the remodeling of bronchiole in A group was definite. (2) ROS (U/mg pro), GSSG (micromol/g pro) and total GSH in lung tissue of A group were significantly higher than that of C group (P < 0.01). The GSH/GSSG in lung tissue of A group was much lower than that of C group (P < 0.01), GSH in lung tissue showed no difference between A group and C group. (3) Immunohistochemistry indicated that Nrf2 protein and gamma-GCS protein were more positively expressed in A group than that in C group (P < 0.01). In situ hybridization discovered that the expression of gamma-GCS-h mRNA in lung tissue of A group was more positive than that of C group. (4) RT-PCR showed that the expression of Nrf2 mRNA was no difference between A group and C group (P > 0.05). (5) The activity of gamma-GCS of A group was (28 +/- 8)U which was significantly higher than that of C group (9 +/- 2)U (P < 0.01). (6) Linear correlation analysis indicated that in lung tissue of guinea pig with asthma there existed strongly positive relationship among ROS, GSSG and the expression of Nrf2, gamma-GCS mRNA, gamma-GCS protein, the activity of gamma-GCS, there existed strongly negative relationship among GSH/GSSG and the expression of Nrf2, gamma-GCS mRNA, gamma-GCS protein, the activity of gamma-GCS.

CONCLUSIONThere existed oxidative stress in lung of guinea pigs with bronchial asthma, which possibly positively regulated gamma-GCS via up regulating transcription factor Nrf2.

Animals ; Asthma ; metabolism ; Glutamate-Cysteine Ligase ; metabolism ; Guinea Pigs ; Lung ; metabolism ; Male ; NF-E2-Related Factor 2 ; metabolism ; Oxidative Stress ; RNA, Messenger ; genetics

OBJECTIVETo study the protective effect of Sanhuangyinchi Fang drug serum (SF) against hydrogen peroxide-mediated DNA oxidative damage in LO2 cells.

METHODSThe LO2 cells were randomly divided into the control group, H(2)O(2) group, SF groups (5%, 10%, and 15%) and vitE group. The morphological features of the treated LO2 cells were observed under inverted microscope. The viability of the treated cells was assessed with CCK-8 method, and the activity of SOD, CAT and GSH-PX were detected biochemically. Reactive oxygen species (ROS) levels, the content of 8-OHdG, and DNA damage of the cells were evaluated by flow cytometry, ELISA, and Comet assay, respectively.

RESULTSCompared with H(2)O(2) group, the cells in SF groups (10% and 15%) and vitE group showed higher cell survival rate (P<0.05) and higher SOD, CAT, GSH-PX (P<0.05) and ROS scavenging activities (P<0.01) with markedly decreases the content of 8-OHdG (P<0.01) and reduced tailing ratio, tail length, tail moment and Olive tail moment (P<0.05).

CONCLUSIONSF drug serum, especially at the concentration of 15%, can protect LO2 cells from H(2)O(2)-mediated DNA oxidative damage.

Cell Line ; Comet Assay ; DNA Damage ; Deoxyguanosine ; analogs & derivatives ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Hydrogen Peroxide ; toxicity ; Oxidation-Reduction ; Oxidative Stress ; Protective Agents ; pharmacology ; Reactive Oxygen Species

OBJECTIVETo investigate the mechanism of plasma bilirubin level increase after hepatic ischemia-reperfusion in rats.

METHODSRats were divided into a sham operation group (A group), a 20 min ischemia-reperfusion group (B group) and a 35 min ischemia-reperfusion group (C group). Study time points were 6 hours and 1, 3, and 5 days after the reperfusion. Pathological changes in the livers were studied with histological slides stained with hematoxilin and eosin. Routine biochemistry methods were used to detect the bilirubin level of blood plasma and the bile drained from the ischemic hepatic lobes. RT-PCR was used to analyze the expression of the multidrug resistance-associated protein 2 (MRP2) and mRNA. Immunohistochemistry was used to analyze the localization of MRP2 in the canalicular membrane.

RESULTSB and C groups showed a mild inflammatory reaction without hepatocyte necrosis. At 6 h and 1 day after reperfusion, there was a significant increase of the plasma bilirubin level and a decrease of the bilirubin level of the drained bile in B group. These changes lasted to the day 3 and day 5 in C group. MRP2 mRNA down-regulation was found at 6 h only in the B and C groups. No localization of MRP2 in the canalicular membrane was found but it appeared in "esicules" under the canalicular membrane in C group.

CONCLUSIONSAbsence of MRP2 localization in the canalicular membrane could be the cause of the blood plasma bilirubin level increase after liver ischemia-reperfusion.

Animals ; Bilirubin ; blood ; Liver Diseases ; blood ; Male ; Multidrug Resistance-Associated Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; blood

BACKGROUNDIn-stent restenosis is a common complication after stent implantation. However, the assessment of stent lumen in computed tomography (CT) coronary angiography is limited by multiple factors. Our study aimed to evaluate the accuracy and the suspected affecting factors in diagnosing coronary in-stent restenosis by dual-source CT (DSCT) compared with coronary angiography.

METHODSOne hundred and fifteen stents in 50 patients were evaluated with DSCT before coronary angiography for the detection of coronary in-stent restenosis (≥ 50% luminal narrowing). Patency of each stent was analyzed by two independent expert radiologists blinded to the results of coronary angiography. The relationship between diagnostic accuracy and the suspected factors including age, body mass index (BMI), heart rate, variation of heart rate, radiation dose, image quality, location and stent characteristics (type, material, diameter, length and strut thickness) was assessed with both univariate and multivariate analysis. The fitting of a Logistic regression model was evaluated using a receiver operating characteristic (ROC) curve.

RESULTSMean stent diameter was (2.9 ± 0.4) mm. Sensitivity, specificity, positive and negative predictive values and accuracy of DSCT in detection of in-stent restenosis were 69.2%, 91.2%, 50.0%, 95.9%, and 88.7%, respectively. In a subgroup of stents with a diameter ≥ 3.0 mm, sensitivity, specificity, positive and negative predictive values and accuracy were 100.0%, 96.5%, 75.0%, 100.0%, and 96.8%, respectively. Stent diameter < 3.0 mm and poor image quality were associated with poor diagnostic accuracy (P < 0.05). The area under curve of ROC was 0.79.

CONCLUSIONDSCT can provide high accuracy for the assessment of in-stent restenosis in stents with a diameter = 3.0 mm, and can play an important role in ruling out in-stent restenosis.

Adult ; Aged ; Aged, 80 and over ; Body Mass Index ; Coronary Angiography ; methods ; Coronary Restenosis ; diagnosis ; diagnostic imaging ; Female ; Humans ; Male ; Middle Aged ; Tomography, X-Ray Computed ; methods

Objective:To preliminarily verify the cross talk between tissue factor/active coagulation factor Ⅶ (TF/FⅦa) and epidermal growth factor receptor (EGFR) pathways in human colon cancer cells in culture.Methods:FⅦa was treated to HT-29 (KRAS-wild type) and LoVo (KRAS-mutant) colon cancer cells to activate TF/F Ⅶa pathway,qRT-PCR and Western blot were used to detect the expressions of amphiregulin (AREG) and epiregulin (EREG),ligands of EGFR on mRNA and protein levels,respectively.After knocking down expression of TF by TF-targeted siRNA transfection,FⅦa was treated and mRNA expressions of AREG and EREG were detected to see whether the FⅦa-induced effects were dependent on TF.Expressions of mRNA of TF and FⅦwere detected by qRT-PCR following the activation of EGFR pathway by treatment with epidermal growth factor (EGF) to HT-29 and LoVo cells.Results:After TF/FⅦa pathway was activated,for HT-29 cells,expressions of AREG (on mRNA level) and EREG (both on mRNA and protein level) were significantly down-regulated versus those of control group,gene expressions of AREG and EREG were 0.55 ± 0.09 vs.0.99 ± 0.09,0.67 ± 0.10 vs.1.02 ± 0.02,protein expressions of EREG were 0.54 ± 0.09 vs.1.04 ± 0.13,all P ＜ 0.05.For LoVo cells,expressions of AREG (both on mRNA and protein level) and EREG (on protein level) were significantly up-regulated versus those of control group,gene expression of AREG were 1.87 ± 0.39 vs.0.93 ± 0.23,protein expressions of AREG and EREG were 3.09 ±0.73 vs.1.11 ±0.21,1.53 ±0.19 vs.0.97 ± 0.23,all P ＜0.05.The regulating effect of AREG and EREG mRNA expression by FⅦa in HT-29 and LoVo cells could both be partly blocked by knocking down TF expression.For HT-29 cells,activation of EGFR pathway induced no significant TF mRNA expression,F Ⅶ mRNA expression was not detected.However,for LoVo cells,activation of EGFR pathway induced significantly higher mRNA expressions of both TF and FⅦ,expressions were 1.53 ± 0.23 vs.1.00 ± 0.23,53.20 ± 6.08 vs.1.00 ± 0.15,all P ＜0.05.Conclusion:In colon cancer cell LoVo,when activated,TF/FⅦa pathway and EGFR pathway could interact through upregulating the other pathway's effectors,and mutant KRAS might play a critical role in the two pathways'cross talk.