In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E. coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 x 10(9) cfu/microg was obtained. After amplification with helper phage, the titer of antibody library reached 5 x 10(12) cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Biopsy, Fine-Needle ; adverse effects ; methods ; Child ; Diagnosis, Differential ; Diagnostic Errors ; Female ; Hematoma ; etiology ; Humans ; Male ; Middle Aged ; Thyroid Neoplasms ; diagnosis ; pathology ; Thyroid Nodule ; pathology ; Young Adult

AIM: To discuss the effects of phytoestrogenic-genistein on cathepsin K (CK) expression stimulated by interleukin-1α (IL-1α) in osteoclast-like cells (OCLs) . METHODS: The OCLs were isolated from tissue of human giant cell tumor of bone (GCT) . The cells treated with reagents were divided into 7 groups including control (treated with phenol red-free-DMEM), vehicle (treated with 1.2 nmol· L-1 IL- 1α), 10-10-10-6genistein, genistein+ ICI 182.780, and 17[β-estrodiol (17β-E2) group. The cells were treated with 1.2 nmol· L-1IL-1α after pre-treated with genistein or 17β-E2 for 48 h (excluded the control group) . Expression of CK wasdetermined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot in OCLs stimulated by IL-1α in the presence of genistein or 17[β-E2. RESULTS: The obvious increase of expression of CK by IL1α in vehicle group was noted in comparing with control group (P ＜ 0.01 ) . Genistein down-regulated CK gene expression stimulated by IL-1α at the transcription level in a dose-dependent manner (r = 0.68, P ＜ 0.01 ) .Genistein down-regulated CK protein expression stimulated by IL-1α also in a dose-dependent manner (r = 0.61,P ＜ 0.01 ). The effects of genistein were abrogated partly after treatment with the estrogen receptor antagonist ICI 182.780. CONCLUSION: Genistein inhibits CK expression stimulated by IL-1α partly through estrogen receptor in OCLs.

Objective To assess the reproducibility of vascularization measurement in cervical carcinoma using transvaginal three-dimensional power Doppler angiography (3D-PDA) with virtualorgan computer-aided analysis ( VOCAL), and compare the reproducibility of two different contour mode (manual and automatic mode) of VOCAL. Methods Eighty patients with cervical carcinoma were examined by observer 1 using transvaginal 3D-PDA. The two acquired volume datasets were analyzed using manual and sphere automatic contour mode of the VOCAL imaging program for assessing carcinoma vascularization index( VI), flow index(FI), and vascularization flow index(VFI). Forty patients of them were examined randomly by observer 2 by the same method. Reproducibility of vascularity measurement was assessed by calculating intraclass (intra-CC) and interclass (inter-CC) , limits of agreement, 95% confidence interval definition. The contribution of various factors (examiner, measurement, contour mode and patient) to intrasubject variance was estimated using different analysis of variance models (ANOVA). Results For intraobserver,manual contour mode was more valid than sphere automatic contour mode for each observer (0. 92～0. 99 vs 0. 75～0. 94) ,and it also had smaller standard deviation,narrower limit of agreement range, 95% confidence interval and higher intra-cc than those of sphere automatic contour mode. Interobserver agreement of manual contour vascular measurements was similar to the intraobserver agreement for manual contour vascular measurements ( 0. 89 ～ 0. 97 vs 0. 92 ～ 0. 99 ) , but interobserver agreement for sphere automatic contour vascular measurements dramatically reduced (0. 52～0. 72). Conclusions Manual contour mode for 3D-PDA vascular measurement has better reproduciblity than sphere automatic contour mode, especially useful for irregular shape tissue.

The present study aims to predict the action targets of antidepressant active ingredients of Xiaoyaosan to understand the "multi-components, multi-targets and multi-pathways" mechanism. Using network pharmacology, the reported antidepressant active ingredients in Xiaoyaosan (saikosaponin A, saikosaponin C, saikosaponin D, ferulic acid, Z-ligustilide, atractylenolide I, atractylenolide II, atractylenolide III, paeoniflorin, albiflorin, liquiritin, glycyrrhizic acid and pachymic acid), were used to predict the targets of main active ingredients of Xiaoyaosan according to reversed pharmacophore matching method. The prediction was made via screening of the antidepressive drug targets approved by FDA in the DrugBank database and annotating the information of targets with the aid of MAS 3.0 biological molecular function software. The Cytoscape software was used to construct the Xiaoyaosan ingredients-targets-pathways network. The network analysis indicates that the active ingredients in Xiaoyaosan involve 25 targets in the energy metabolism-immune-signal transmutation relevant biological processes. The antidepressant effect of Xiaoyaosan reflects the features of traditional Chinese medicine in multi-components, multi-targets and multi-pathways. This research provides a scientific basis for elucidation of the antidepressant pharmacological mechanism of Xiaoyaosan.
Antidepressive Agents ; pharmacology ; Benzoates ; Bridged-Ring Compounds ; Coumaric Acids ; Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; pharmacology ; Flavanones ; Glucosides ; Glycyrrhizic Acid ; Lactones ; Medicine, Chinese Traditional ; Monoterpenes ; Sesquiterpenes ; Software

OBJECTIVETo determine whether the cardioprotection of puerarin (Pue) against hypoxia/reoxygenation injury is mediated by mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) and/or mitochondria calcium-activated potassium channel(mitoK(Ca)).

METHODSCardiomyocytes were isolated from male Sprague-Dawley rats and hypoxia/reoxygenation injury was induced by myocyte pelleting model. Cell viability was assessed by trypan blue exclusion and mitochondrial membrane potential was measured by loading with TMRE. The opening of mitochondrial permeability transition pore was determined spectrophotometrically.

RESULTSPretreatment with Pue at 0.24 mmol/L for 5 min increased the cell viability against hypoxia/reoxygenation injury, while mitochondrial ATP-sensitive potassium channel inhibitor 5-hydroxydecanoate (5-HD, 100 micromol/L, 20 min) or mitochondrial calcium-activated potassium channel blocker paxilline (Pax, 1 micromol/L, 5 min) attenuated the effect of puerarin. The pretreatment with Pue at 0.24 mmol/L for 5 min attenuated collapse of delta-psim induced by hypoxia/reoxygenation injury, 5-HD and Pax abrogated the effect of Pue. In mitochondria isolated from hearts pretreated with Pue, a significant inhibition of Ca(2+)-induced swelling was observed, and this inhibition was attenuated by 5-HD and Pax.

CONCLUSIONThese findings indicate that Pue protects cardiomyocytes against hypoxia/reoxygenation injury via activating mitoK(ATP) channel and mitoK(Ca) channel, and inhibiting mitochondrial permeability transition pore opening.

Androsterone ; pharmacology ; Animals ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Indoles ; pharmacology ; Isoflavones ; pharmacology ; Male ; Mitochondrial Membrane Transport Proteins ; metabolism ; Myocardium ; cytology ; Myocytes, Cardiac ; drug effects ; metabolism ; Potassium Channels ; metabolism ; Potassium Channels, Calcium-Activated ; metabolism ; Rats ; Rats, Sprague-Dawley

Objective To establish radiation?resistant lung carcinoma cell lines, and to investigate the changes in morphology, apoptosis, invasive migration, and epithelial?mesenchymal transition ( EMT) in cells. Methods The radiation?resistant lung carcinoma cell lines were obtained by exposure of lung carcinoma cell lines, A549 and H1299, to radiation with a low dose in fractions, a sublethal dose, or a gradually increasing dose. The morphological changes in cells, radiosensitivity, survival rates after exposure, apoptosis rates, changes in invasive migration, and expression of EMT marker proteins were evaluated using microscopy, colony formation assay, CCK?8 assay, flow cytometry, transwell migration assay, and Western blot, respectively. Results Radiation with a gradually increasing dose successfully induced the radiation?resistant cell lines, A549R and H1299R. The morphological study showed that the morphology of radiation?resistant cells was converted to the morphology of mesenchymal cells. Compared with A549 and H1299 cells, the values of D0 , Dq , and SF2 were significantly increased in A549R ( P=0.017,P=0.001,P=0.000) and H1299R (P=0.033,P=0.000,P=0.008) cells, respectively;the values of α and α/β were significantly reduced in A549R (P=0.018;P=0.007) and H1299R (P=0.001;P=0.009) cells, respectively. The survival rates in A549R and H1299R cells after exposure to radiation with various doses were significantly higher than those in the control groups (all P<0.05). After exposure, the apoptosis rates were significantly reduced in A549R and H1299R cells ( P=0.02,P=0.01);the invasion and migration rates were significantly increased in A549R (P=0.000;P=0.001) and H1299R (P=0.001,P=0.002) cells;the expression of E?cadherin was significantly down?regulated in A549R and H1299R cells (P=0.00,P=0.01), while the expression of vimentin was significantly elevated in A549R and H1299R cells ( P= 0. 02, P= 0. 01 ) . Conclusions The radiation?resistant lung carcinoma cell lines are successfully established. Both cell lines show enhanced invasion and migration, which may be associated with EMT.

AIMTo determine whether the cardioprotection of puerarin (Pue) against H202-stress is mediated by mitochondrial transmembrane pores and/or channels.

METHODSCardiomyocytes were isolated from male Sprague-Dawley rats. Cell viability was assessed by trypan blue exclusion and mitochondrial membrane potential was measured by loading with Rhodamine 123. The opening of mitochondrial permeability transition pore was determined spectrophotometrically.

RESULTSPretreatment with Pue at 0.24 mmol/ L for 5 min increased the cell viability against H2O2-stress, while mitochondrial calcium-activated potassium channel blocker paxilline (Pax, 1 micromol/L, 5 min) or PKC inhibitor(chelerythrine, 5 micromol/L, 30 min) attenuated the effect of puerarin. The pretreatment with Pue at 0.24 mmol/L for 5 min or mitochondrial calcium-activated potassium channel opener NS 1619 (10 micromol/L, 10 min) attenuated mitochondrial depolarization induced by H2O2-stress, while mitochondrial permeability transition pore opener atractyloside (20 micromol/L, 20 min) abrogated the effect of Pue. In mitochondrial isolated from hearts pretreated with 0.24 mmol/L Pue for 5 min, a significant inhibition of Ca2+ -induced swelling was observed, which inhibition was attenuated by Pax (1 micromol/L, 5 min).

CONCLUSIONThese findings indicate that Pue protects cardiomyocytes against H2O2-stress via inhibiting mitochondrial permeability transition pore opening and activating mitochondrial calcium-activated potassium channel.

Animals ; Cardiotonic Agents ; pharmacology ; Cell Separation ; Cell Survival ; drug effects ; Hydrogen Peroxide ; Isoflavones ; pharmacology ; Male ; Membrane Potential, Mitochondrial ; physiology ; Mitochondria, Heart ; physiology ; Mitochondrial Membrane Transport Proteins ; antagonists & inhibitors ; Myocytes, Cardiac ; cytology ; metabolism ; physiology ; Potassium Channels, Calcium-Activated ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stress, Physiological

AIMTo determine whether the cardioprotection of puerarin (Pue) against ischemia/reperfusion (I/R) is mediated by mitochondrial transmembrane pore or channels.

METHODSMale Sprague-Dawley rats were used for Langendorff isolated heart perfusion. The hearts subjected to global ischemia for 30 min followed by 120 min of reperfusion. Formazan, a product of 2,3,5-triphenyltetrazolium chloride (TTC), which is proportional to myocardial viability, was measured at 490 nm, and the level of lactate dehydrogenase (LDH) in the coronary effluent was measured to evaluate the cardiac injury.

RESULTSThe pretreatment with Pue at 0.24 mmol/L for 5 min before ischemia increased formazan content of myocardium, reduced LDH release, improved the recovery of the left ventricular developed pressure, maximal rise/fall rate of left ventricular pressure, left ventricular end-diastolic pressure and rate pressure product (left ventricular developed pressure multiplied by heart rate) and attenuated the decrease of coronary flow during reperfusion. Administration of atractyloside (20 micromol/L), an opener of mitochondrial permeability transition pore, for 20 min (first 20 min of reperfusion) and 5-hydroxydecanoate (100 micromol/L), the mitochondrial specific K(ATP) blocker, for 20 min before ischemia attenuated the protective effects of Pue.

CONCLUSIONThe findings indicate that in the isolated rat heart, Pue protects myocardium against ischemia/ reperfusion injury via the opening of mitochondrial ATP-sensitive potassium channel and the inhibition of mitochondrial permeability transition pore opening.

Animals ; Decanoic Acids ; metabolism ; Hydroxy Acids ; metabolism ; Isoflavones ; pharmacology ; Male ; Mitochondria, Heart ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocardial Reperfusion Injury ; metabolism ; Rats ; Rats, Sprague-Dawley

Objective To assess iodine nutrient levels of vulnerable population in Linxia Hui Autonomous Prefecture of Gansu. Methods Local pregnant, lactation, child-bearing age women and 0 - 3 years old infants were selected. The urinary iodine (UI) were determined by arsenic-Ce catalytic spectrophotometry (WS/T 107-1999) and salt iodine content was determined using direct titration method (GB/T 13025.7-1999). The development quotient (DQ) among 0 - 3 years old infants were surveyed by the scales of neuropsychologists development among 0 - 6 years old, and their body height and weight were then investigated. Results The consumption and coverage rates of iodized salt at household level were all below than 90% in 5 counties among 8 counties. The median urine iodine (MUI) among pregnant, lactation, child-beating age women and 0 - 3 years old infants were 90.17, 89.28, 84.85 and 107.28 μg/L, respectively. The UI proportion less than 50 μg/L were 29.6%(278/938), 30.7%(239/778), 32.2% (265/824), and 23.9%(252/1056), respectively. The UI proportion equal or more than 50 μg/L and less than 100 μg/L were 25.1%(235/938), 24.0%(187/778), 23.7%(195/824), 23.7%(250/1056). MUI in pregnant were all less than 150 μg/L in 8 counties, and MUI in lactation women were less than 100 μg/L in 6 counties. The DQ median of infants 0 - 3 years old was 93.0, and 59.9%(395/659), 34.4%(227/659) and 5.6%(37/659)had a DQ below middle, at middle and above middle, respectively. Forty point seven percent (266/654), 50.2%(328/654) and 9.1% (60/654) had a body height below middle,at middle and above middle, respectively. Thirty-five percent (232/663), 51.3%(340/663) and 13.7%(91/663) had a body weight below middle, at middle and above middle, respectively. Conclusions The local qualified iodized salt consumption rate and iodized salt coverage rate are still low. The urine iodine level is below than the reference value recommended. The iodine nutrient is not enough among iodine deficiency disorder vulnerable population, especially in pregnant and lactation women. Infants growth development is retarded.