Specific scAbs could be obtained through biopanning from phage antibody libraries by use of antigens as target molecules. scAbs specific to thrombin were separated from mouse antibody library by the panning strategy of alternating liquid-solid phase in this paper. Thrombin was biotinylated by photobiotin at first, then avidin-coated magnetic beads were utilized to isolate specific scAbs. The eluted phages were amplified and subject to the second round panning in microtiter plate to remove the unspecific reombinant phages. 4 specific scAbs were separated from 23 phage clones after four rounds of alternative panning.

Adult ; Chemical and Drug Induced Liver Injury ; Dimethylformamide ; poisoning ; Humans ; Male

BackgroundThe effects of extremely low frequency electromagnetic fields (ELF-EMFs) on public health have attracted wide attentions.The association of the thermal effect of ELF-EMFs with cancer and ocular tissue damage has been of concern.However,the pathological changes of scleral tissue after exposure to ELF-EMFs as well as the relationship between these changes and myopia are still poorly understood.ObjectiveThe present study was to investigate the molecular pathological changes of human fetal scleral fibroblasts (HFSFs) after exposure to ELF-EMFs in vitro and to explore the possible mechanism in the occurrence and development of myopia.MethodsHFSFs were cultured and passaged and then exposed to 50 Hz electromagnetic fields,and HFSFs that did not receive the irradiation of ELF-EMFs were used as the control group.The expression of collagen type Ⅰ (COL1A1 ) mRNA and matrix metalloproteinase-2 (MMP-2) mRNA in cultured HFSFs were detected by real-time qualitative polymerase chain reaction (real-time PCR) under different magnetic field intensites (0,0.1,0.2,0.5,1.0 mT) and different exposure time (0,6,12,24,36,48 hours).Cell proliferation assay of HFSFs was detected by the cell counting kit 8 ( CCK8 ) assay.The expression levels of COL1 A1 and MMP-2 proteins in HFSFs were further confirmed by immunofluorescence staining.Results The expression of COL1A1 mRNA was significantly down-regulated under the exposure of 0.2 mT ELF-EMFs for 6 hours,in comparison with the control group;moreover,it decreased in parallel with the increased of flux density (0.099±0.008 vs.0.050±0.004) (P =0.009 ).The expression of MMP-2mRNA was up-regulated conspicuously after exposure to 0.1 mT ELF-EMFs for 24 hours,and it increased with exposure time in comparison with the control group ( 0.009 ±0.001 vs.0.018±0.003 ) ( P =0.038 ).Proliferation of HFSFs (A450) was inhibited following the exposure to 0.2 mT ELF-EMFs for 24 hours in comparison with the control group (P =0.009 ).The expression of COL1 A1 in the experimental group was decreased,compared with the control group,but the expression of MMP-2 was increased.ConclusionsELF-EMFs inhibit the proliferation of HFSFs and expression of COL1 A1 in HFSFs,which might be one of the reasons for the development of myopia.

This study is to evaluate the therapeutic effect of fibroblast growth factor 21 (FGF21) on NAFLD in MSG-IR mice and to provide mechanism insights into its therapeutic effect. The MSG-IR mice with insulin resistance were treated with high dose (0.1 micromol.kg-1d-1) and low dose (0.025 micromol.kg-1d-1) of FGF21 once a day for 5 weeks. Body weight was measured weekly. At the end of the experiment, serum lipids, insulin and aminotransferases were measured. Hepatic steatosis was observed. The expression of key genes regulating energy metabolism were detected by real-time PCR. The results showed that after 5 weeks treatment, both doses of FGF21 reduced body weight (P<0.01), corrected dyslipidemia (P<0.01), reversed steatosis and restored the liver morphology in the MSG model mice and significantly ameliorated insulin resistance. Additionally, real-time PCR showed that FGF21 significantly reduced transcription levels of fat synthetic genes, decreased fat synthesis and promoted lipolysis and energy metabolism by up-regulating key genes of lipolysis, thereby liver fat accumulation was reduced and liver function was restored to normal levels. In conclusion, FGF21 significantly reduces body weight of the MSG-IR mice, ameliorates insulin resistance, reverses hepatic steatosis. These findings provide a theoretical support for clinical application of FGF21 as a novel therapeutics for treatment of NAFLD.
Animals ; Body Weight ; drug effects ; Dose-Response Relationship, Drug ; Dyslipidemias ; metabolism ; Energy Metabolism ; drug effects ; Fatty Liver ; chemically induced ; complications ; Female ; Fibroblast Growth Factors ; administration & dosage ; pharmacology ; therapeutic use ; Insulin Resistance ; Lipolysis ; drug effects ; Liver ; metabolism ; pathology ; Male ; Mice ; Non-alcoholic Fatty Liver Disease ; drug therapy ; Sodium Glutamate

OBJECTIVETo analyze the differential protein expression of left-sided colon cancer and right-sided colon cancer.

METHODSTissue samples of left-sided colon cancer (n=7) and right-sided colon cancer (n=7) were collected. Tissue protein was abstracted and two-dimensional gel electrophoresis (2-DE) was used to examine the gel images. Peptide mass fingerprintings (PMF) was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and the proteins were identified by data searching with bioinformatics. Immunohistochemical SP method was used for the detection of glucose regulated protein 78 kD (GRP78) and heat shock protein 20 (HSP20) in left-sided colon cancer (n=50) and right-sided colon cancer (n=50) tissues.

RESULTSSixteen differentiating protein spots were identified. Compared with right-sided colon cancer, 10 proteins including GRP78 up-regulated and 6 proteins including HSP20 down-regulated in left-sided colon cancer. Immunohistochemical detection showed that in left and right sided colon cancer, the positive expression rate of GRP78 was 78% (39/50) and 56% (28/50) and the positive expression rate of HSP20 was 34% (17/50) and 72% (36/50), respectively, and the differences were statistically significant (both P<0.05). The positive rate of GRP78 was associated with tumor differentiation, infiltration layer, TNM staging, lymph node metastasis, and liver metastasis, while the positive rate of HSP20 was associated with tumor gross morphology, TNM staging, and lymph node metastasis (P<0.05).

CONCLUSIONSThere are differentially expressed proteins between left-sided colon cancer and right-sided colon cancer, especially for GRP78 and HSP20, which may be the cause leading to the biological differences between left-sided and right-sided colon cancer.

Adult ; Aged ; Colonic Neoplasms ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; HSP20 Heat-Shock Proteins ; metabolism ; Heat-Shock Proteins ; metabolism ; Humans ; Male ; Middle Aged

OBJECTIVETo observe dynamically the development of fetal long bone and detect the expression and distribution of HIF-1alpha,to investigate the expression pattern and possible effects of hypoxia inducible factor-1alpha (HIF-1alpha) in fetal long bone development of mouse.

METHODSE12.5, E13.5, E14.5, E15.5, E16.5 and E17.5 pregnant C57BL6 mice were sacrificed. After sacrifice, the embryos were delivered by caesarean section. The development of fetal long bone was dynamically observed by stereoscopic microscope, and the distributional expression of HIF-1alpha protein was detected by using method of immunohistochemistry. The expression of HIF-1alpha mRNA and osteoblast marker gene at various stage were also detected by using methods of reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe cartilaginous long bone began to form and joints outline arised at E13.5, then the primary ossification center was observed at E14.5, showing opaque ossification under stereoscopic microscope,and then the osteogenesis expanded and extended to both sides. Immunohistochemistry demonstrated lots of HIF-1alpha protein positive chondrcytes in the center of primary ossification at E14.5, then they decreased dramatically. HIF-1alpha mRNA expressed at high level from E13.5 to E15.5, and then decreased to low level.

CONCLUSIONFetal long bone development pattern appeared to be endochondral osteogenisis process, existing hypoxia microenviroment may increase HIF-1alpha mRNA expression and thus initiate the cascade of endochondral osteogenisis.

Animals ; Bone Development ; Female ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; genetics ; physiology ; Immunohistochemistry ; Male ; Mice ; RNA, Messenger ; analysis

Antineoplastic Agents ; therapeutic use ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; genetics ; Drug Delivery Systems ; Erlotinib Hydrochloride ; Female ; Genes, erbB-1 ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; Male ; Mutation ; Protein Kinase Inhibitors ; therapeutic use ; Quinazolines ; therapeutic use ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; genetics

OBJECTIVETo study the prevention of colorectal cancer liver metastasis by adenoviral transduction of the endostatin gene.

METHODSThe recombinant adenovirus expressing endostatin was constructed. Its biological activities were surveyed in vitro, as determined in human umbilicus vein endothelium cell (HUVEC) proliferation inhibition, and in vivo, by reduction of liver metastasis.

RESULTSHUVEC proliferation was obviously inhibited by the infecting supernatant of recombinant adenovirus. Persistent high serum levels of endostatin in peripheral blood, especially in the liver vein were observed. The production of liver metastasis was intervened.

CONCLUSIONSThe single injection in the vein of the recombinant adenovirus realizes the high effective and stable expression of endostatin in general body and liver, which brings about the ideal prevention of liver metastasis.

Adenoviridae ; genetics ; Animals ; Cell Division ; drug effects ; Collagen ; genetics ; therapeutic use ; Colorectal Neoplasms ; pathology ; Disease Models, Animal ; Endostatins ; Endothelium, Vascular ; drug effects ; pathology ; Genetic Therapy ; Genetic Vectors ; Liver Neoplasms ; prevention & control ; secondary ; Mice ; Mice, Inbred BALB C ; Neoplasm Metastasis ; prevention & control ; Neoplasm Transplantation ; Peptide Fragments ; genetics ; therapeutic use ; Transduction, Genetic

OBJECTIVEBrain natriuretic peptide (BNP) levels are elevated in patients with atrial fibrillation (AF). The aim of this study is to investigate the relation between the pre-procedural BNP level and the incidence of recurrence AF after circumferential pulmonary vein ablation (CPVA).

METHODSPlasma BNP level was measured before CPVA in 69 consecutive symptomatic paroxysmal AF (PAF) patients without heart failure symptom. Atrial thrombus was detected by transesophageal echocardiography in 15 patients and CPVA was not performed in these patients. CPVA was successful in the remaining 54 patients and followed up for 3 months. All patients were asked to keep a log of the duration and frequency of their symptoms and underwent 24 h ECG monitoring at least once per month after the ablation.

RESULTSAt the end of follow up, 39 patients were free of AF recurrence (successful group) and 15 patients experienced AF recurrence (failure group). BNP concentration was below the heart failure range (< 500 ng/L) in 69.6% patients, but exceeded the normal range (0 - 144 ng/L) in 59.4% patients. Median baseline BNP level was significantly higher in failure group than in successful group (371.6 ng/L vs. 97.4 ng/L, P = 0.001). Left atrial (LA) dimension was also larger in failure group than in successful group [(53.3 ± 15.1) mm vs. (45.2 ± 11.2) mm, P = 0.036]. Moreover, BNP level was positively correlated with LA dimension (r = 0.574, P < 0.01).

CONCLUSIONThe pre-procedural BNP level and LA dimension are predictive of successful CPVA for PAF patients.

Adult ; Aged ; Atrial Fibrillation ; blood ; surgery ; Catheter Ablation ; Female ; Humans ; Male ; Middle Aged ; Natriuretic Peptide, Brain ; blood ; Treatment Outcome

OBJECTIVETo study the mutation patterns of epithelial growth factor receptor (EGFR) exon 18, 19 and 21 in Chinese non-small-cell lung cancers (NSCLC).

METHODSSomatic mutation in samples of 32 cases without Iressa-treatment were compared with that in 10 volunteers blood control. The mutations were identified for the forward and reverse sequence chains for the tyrosine kinase domain of the EGFR gene, followed by DNA template abstraction and Touchdown PCR.

RESULTSNine types of mutation were found in sequences of 7 cases among the 32 non-small cell lung carcinoma tissues, namely, five reported mutation within exon 19, and two new heterozygous mutations, L833V and H835L within exon 21, and two intron polymorphism. These results showed a mutation rate of 9/32 (28.1%) in Chinese with NSCLC, and of 31.6% in lung adenocarcinomas.

CONCLUSIONEGFR mutation rate in Chinese with NSCLC is consistent with those of Asian women reported in the literature but new mutation points in Chinese were presented as L833V and H835L. The mutation rate is in concordance with release rate of NSCLC obtained by Gefitinib treatment in Chinese.

Adenocarcinoma ; genetics ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Carcinoma, Non-Small-Cell Lung ; ethnology ; genetics ; China ; DNA Mutational Analysis ; Exons ; genetics ; Female ; Humans ; Lung Neoplasms ; ethnology ; genetics ; Male ; Middle Aged ; Mutation ; Receptor, Epidermal Growth Factor ; genetics