Objective To establish a closed-chest pulmonary embolism-reperfusion animal model by Swan-Ganz catheter and to explore the mechanisms of pulmonary embolism(PE)-reperfusion injury(RI). Methods Experiments were made on 14 mongrel dogs,ranging in weight from 15 to 18 kg,anesthetized with 3% pentobarbital sodium.The dogs were intubated with I.D.7 endotracheal tubes.Under sterile conditions,a 7 F Swan-Ganz catheter via the external jugular vein was positioned in the unilateral pulmonary diaphragmatic lobe(DL)artery.Occlusion/reperfusion of the DL artery was controlled with 1.2 ml diluted contrast agent filled into/drawn from the balloon.After the 24 h PE,the balloon was deflated to result in 4 h reperfusion of the DL.Measurements of blood gases and tumor necrosis factor-?(TNF-?)were made at normal condition,at 24 h PE and at 4 h reperfusion.Thin-section CT scans were performed at normal condition,24 h PE,30 min,1,2,3 and 4 h reperfusion,respectively.At the end of each experiment, tissue specimens of bilateral diaphragmatic lobes were obtained for both wet/dry(W/D)weight ratio and for pathological study.Results Reperfusion pulmonary edema(RPE)was an acute,mixed,noncardiogenic edema that was observed in all 14 dogs who had been successfully established as PE/RI animal models.RPE demonstrated heterogeneous ground-glass opacifications that predominated in the areas distal to the recanalized vessels.It manifested pathologically as an edematous lung infiltrated by inflammatory cells.The mean ofPaO_2 and TNF-? of 4 h reperfusion was(81?4)mm Hg(l mm Hg=0.133 kPa)and(16.0? 2.5)pg/ml,which were significantly different(P

Objective To study the effects of nuclear factor-?B(NF-?B)decoy oligodeoxynucleotide(ODN)on the preeclamptic umbilical serum induced expression of precollagen Ⅰ,Ⅲ mRNA and tumor necrosis factor-?(TNF-?)in cultured human umbilical artery smooth muscle cells (HUASMC).Methods Primary cultured HUASMC of normal pregnancy were divided into four groups: group A(HUASMC were incubated with umbilical serum of normal pregnancy);group B(HUASMC were incubated with umbilical serum of preeclampsia);group C(HUASMC were transfected with NF-?B cis decoy ODN 48 h before incubation with umbilical serum of preeclampsia);group D(HUASMC were transfected with NF-?B scramble ODN 24 h before incubation with umbilical serum of preeclampsia).NF-?B cis decoy ODN and NF-?B scramble ODN were transfected with cationic lipofectamine to the latter two groups,respectively.The proliferation of human umbilical artery smooth muscle cells was evaluated by methyl thiazolyl tetrazolium and the apoptosis was analyzed by flow cytometry.The expression levels of precollagen Ⅰ,Ⅲ mRNA were detected by RT-PCR,the expression levels of TNF-? were detected by western blot.Results(1)The proliferation of group B(0.19?0.02)and group D(0.18?0.03)was significantly increased as compared with those of group A(0.11?0.02)and group C(0.14?20.02)(P0.05).(5)The expression of TNF-? of group B(0.74?0.11),group C(0.36?0.09)and group D(0.79?0.12)were significantly higher than that of group A(0.15?0.03)(P0.05).Conclusions NF-?B cis decoy ODN could down-regulate the proliferation,as well as the expression levels of precollagen and TNF-? of HUASMC induced by umbilical serum of preeclampsia.NF-?B may play an important role in the pathogenesis of placental artery abnormalities in preeclampsia.

Day-old chick is unique animal model in brain development and behavior study. The intermediate medial mesopallium (IMM), a region of the chick forebrain, is intimately involved in the early learning processes, which offers the ideal opportunity to study the neural changes that underlie behavioral plasticity. In this paper, the intracellular recordings were conducted from IMM neurons in chick forebrain slices, in which electrophysiological properties, synaptic responses and long-term potentiation (LTP) were observed. Coronal sections of left forebrains (500 mum thick), containing IMM, were prepared from domestic chicks, aged 2-10 days. In 69 IMM neurons, the resting membrane potential was measured to be (-59.4+/-5.3) mV, slope membrane resistance (70.8+/-27.2) MΩ, and time constant (10.2+/-4.3) ms. The amplitude, threshold, overshoot, half-width, max rise slope and max decay slope of action potential evoked by intracellular current injection were (85.2+/-9.4) mV, (-38.7+/-7.6) mV, (25.6+/-8.9) mV, (2.1+/-0.5) ms, (150.5+/-41.2) mV/ms and (-64.3+/-14.0) mV/ms, respectively. Spike-firing frequency was increased with depolarizing current intensity in 32 of 69 tested cells [linear regression slope was (21.5+/-10.9) Hz/nA, P<0.05 in all cells]. The depolarizing synaptic responses (i.e. EPSPs), with stimulus intensity- and membrane potential-dependent properties, were elicited by dorsal (n=25) or ventral (n=62) focal electrical stimuli at 0.1 Hz in all tested IMM neurons and could be nullified reversibly by perfusion with 100 mumol/L AP5 (NMDA receptor antagonist) and 3 mumol/L DNQX (non-NMDA receptor antagonist), but enlarged by 6 mumol/L bicuculline (GABA(A) receptor antagonist). The EPSPs evoked by ventral stimulation were persistently increased after tetanic stimulation (5 Hz, 300 pulses/train, 2 trains, train interval 10 min) in 6 of 12 tested IMM neurons. The amplitude of EPSPs was potentiated to more than 120% of control level (when analyzed at 45 min of enhancement, P<0.05, n=5), which lasted at least 30 min and then could be referred to as LTP. Moreover, area under curve, duration and max rise slope of EPSPs were also enhanced (P<0.05), while no significant changes were observed in the electrophysiological parameters of IMM neurons following induction of LTP (P>0.05). These results suggest that the intracellular recording techniques in the chick brain slices can be used to perform multi-parameter analysis of synaptic responses and their LTP.
Animals ; Brain ; physiology ; Chickens ; In Vitro Techniques ; Long-Term Potentiation ; Membrane Potentials ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors

OBJECTIVETo observe the effect of Shenluotong Decoction (SD) on serum levels of aldosterone, monocyte chemoattractant protein-1 (MCP-1), α-smooth muscle protein (α-SMA), and nuclear factor-KB (NF-κB) in obstructive nephropathy rats, and to explore the initial mechanism of SD for inhibiting renal interstitial fibrosis.

METHODSTotally 48 healthy Wistar rats were randomly divided into the sham-operation group (n =12) and the model group (n =36). Renal interstitial fibrosis rat model was established by unilateral ureteral obstruction (UUO). After successful modeling, 36 rats were randomly divided into the model group, the Chinese medicine group, and the Western medicine group, 12 in each group. Eplerenone was added in the forage at the daily dose of 100 mg/kg for rats in the Western medicine group. Chinese medicine was added in the forage at the daily dose of 26 g/kg for rats in the Chinese medicine group. Equal volume of normal saline was administered to rats in the sham-operation group and the model group. All medication was performed once daily. The obstructive kidneys were extracted ten days after medication. The pathomorphological changes were observed. The contents of serum aldosterone and MCP-1, and the protein or mRNA expression of MCP-1, α-SMA, and NF-KB were detected.

RESULTSCompared with the sham-operation group, infiltration of a large amount of inflammatory cells and collagen deposition significantly increased, serum contents of aldosterone and MCP-1 obviously increased (P < 0.01), the expression of MCP-1 mRNA and protein were significantly up-regulated (P <0.01), the protein expression of α-SMA and NF-KB were significantly enhanced in the model group (P <0.01). Com- pared with the model group, infiltration of inflammatory cells and renal collagen deposition were attenua- ted in the Chinese medicine group and the Western medicine group, the serum MCP-1 level were reduced, and the mRNA and protein expression of MCP-1 were significantly down-regulated (P <0.01), the protein expression of α-SMA and NF-KB were obviously inhibited (P <0. 01). At the same time, serum aldosterone level was reduced in the Chinese medicine group (P <0.01).

CONCLUSIONSinflammatory lesions of the renal tissue could promote the progress of interstitial fibrosis in rats with obstructive nephropathy. SD could attenuate interstitial fibrosis through reducing serum contents of aldosterone and MCP-1, down-regulating MCP-1/ NF-KB, and inhibiting the expression of α-SMA.

Animals ; Chemokine CCL2 ; drug effects ; metabolism ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Kidney ; drug effects ; Kidney Diseases ; drug therapy ; genetics ; NF-kappa B ; drug effects ; metabolism ; RNA, Messenger ; biosynthesis ; Rats, Sprague-Dawley ; Ureteral Obstruction ; drug therapy ; genetics

Background::The use of microRNAs in the therapy of kidney disease is hampered by the difficulties in their effective delivery. Microvesicles (MVs) are known as natural carriers of small RNAs. Our prior research has demonstrated that MVs isolated from mesenchymal stem cells (MSCs) are capable of attenuating kidney injuries induced by unilateral ureteral obstruction and 5/6 sub-total nephrectomy in mice. The present study aimed to evaluate the effects of miR-34a-5p (miR-34a)-modified MSC-MVs on transforming growth factor (TGF)-β1-induced fibrosis and apoptosis in vitro. Methods::Bone marrow MSCs were modified by lentiviruses over-expressing miR-34a, from which MVs were collected for the treatment of human Kidney-2 (HK-2) renal tubular cells exposed to TGF-β1 (6 ng/mL). The survival of HK-2 cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Annexin V-Light 650/propidium iodide (PI) assays. The expression levels of epithelial markers (tight junction protein 1 [TJP1] and E-cadherin) and mesenchymal markers (smooth muscle actin alpha (α-SMA) and fibronectin) in HK-2 cells were measured using Western blot analysis and an immunofluorescence assay. In addition, changes in Notch-1/Jagged-1 signaling were analyzed using Western blotting. Data were analyzed using a Student’s t test or one-way analysis of variance. Results::MiR-34a expression increased three-fold in MVs generated by miR-34a-modified MSCs compared with that expressed in control MVs ( P < 0.01, t= 16.55). In HK-2 cells, TJP1 and E-cadherin levels decreased to 31% and 37% after treatment with TGF-β1, respectively, and were restored to 62% and 70% by miR-34a-enriched MSC-MVs, respectively. The expression of α-SMA and fibronectin increased by 3.9- and 5.0-fold following TGF-β1 treatment, and decreased to 2.0- and 1.7-fold after treatment of HK-2 cells with miR-34a-enriched MSC-MVs. The effects of miR-34a-enriched MSC-MVs on epithelial-mesenchymal transition (EMT) markers were stronger than control MSC-MVs. The effects of miR-34a-enriched MSC-MVs on these EMT markers were stronger than control MSC-MVs. Notch-1 receptor and Jagged-1 ligand, two major molecules of Notch signaling pathway, are predicted targets of miR-34a. It was further observed that elevation of Notch-1 and Jagged-1 induced by TGF-β1 was inhibited by miR-34a-enriched MSC-MVs. In addition, TGF-β1 exposure also induced apoptosis in HK-2 cells. Although miR-34a-mofidied MSC-MVs were able to inhibit TGF-β1-triggered apoptosis in HK-2 cells, the effects were less significant than control MSC-MVs (control:TGF-β1 :miR-nc-MV:miR-34a-MV = 1.3:0.6:1.1:0.9 for MTT assay, 1.8%:23.3%:9.4%:17.4% for apoptosis assay). This phenomenon may be the result of the pro-apoptotic effects of miR-34a. Conclusions::The present study demonstrated that miR-34a-over-expressing MSC-MVs inhibit EMT induced by pro-fibrotic TGF-β1 in renal tubular epithelial cells, possibly through inhibition of the Jagged-1/Notch-1 pathway. Genetic modification of MSC-MVs with an anti-fibrotic molecule may represent a novel strategy for the treatment of renal injuries.

OBJECTIVETo characterize molecular and cytogenetic abnormalities in six 46, XX males, and to investigate the clinical manifestations and underlying mechanisms in such patients.

METHODSClinical data of six XX male patients were collected. Karyotyping, multiple polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) were utilized to detect and locate the sex determining region (SRY) gene.

RESULTSPCR and FISH showed that all patients were SRY-positive XX males. All patients have their SRY gene located at the tip of derivative X chromosomes, which have resulted from translocation between short arms of X and Y chromosomes. High resolution karyotyping at 550-750 band level has revealed that the translocation breakpoints were at Xp22.33 and Yp11.2 in three patients. In the remaining patients, the breakpoints were either at Xp22.32 and Yp11.31 or Xp22.31 and Yp11.2. The breakpoints at Xp22.32, Xp22.31 and Yp11.31 were rarely reported. Genotype-phenotype correlation analysis indicated that the clinical manifestations were age-specific. Four adult patients have come to clinical attention due to infertility, with typical features including azoospermia and testis dysgenesis, whereas poorly developed secondary sexual characteristics and short stature were main complaints of adolescence patients, and short stature was the sole symptom in a child patient.

CONCLUSIONCombined karyotyping, PCR and FISH are important for the analysis of XX males. Particularly, high resolution karyotyping is valuable for the refinement of chromosome breakpoints and detailed analysis of genotype-phenotype correlation.

46, XX Disorders of Sex Development ; genetics ; Adolescent ; Adult ; Child, Preschool ; Chromosomes, Human, X ; Chromosomes, Human, Y ; Genetic Association Studies ; methods ; Humans ; Karyotyping ; methods ; Male ; Sex Chromosome Aberrations ; Translocation, Genetic ; Young Adult

Objective To study the influence on the tumor after percutaneous intra-tumor injection of ~(32)P-GMS in liver cancer as well as its suitable dose.Methods 24 New Zealand rabbits were used to establish the animal model of VX-2 liver cancer,and divided into A,B,C and D groups with individually 37,74,111 and 148 MBq of ~(32)P-GMS being injected,respectively;and then pathological changes of tumor were observed by light and electron microscope respectively.Result The dose of ~(32)P-GMS was obviously correlated with the radioactivity damage of tumor cells.In the A and B groups,the tumor cells were not observed to disappear completely after injection of ~(32)P-GMS,but in C group,tumor cells were almost completely disappeared and surrounded by a lot of connective tissue.Although the tumor cells were found to disappear completely in D group,normal liver tissues were also involved.Conclusion Percutaneous intra-tumor injection of ~(32)P-GMS with suitable dose that may induce the tumor tissue to be maximally damaged and may also provide some significances to prevent the tumor metastasis.

AIM:To investigate the effects of platelet-derived growth factor receptor α(PDGFRα) on melano-cyte apoptosis induced by hydrogen peroxide(H2O2). METHODS:Melanocyte PIGI was used as the research object. Af-ter exposed to H2O2at different concentrations,the cell viability was detected by MTT assay. The PIGI cells were transfec-ted with empty vector pCMV6 or PDGFRα over-expression vector pCMV6-PDGFRα. The transfection efficiency was deter-mined by RT-qPCR and Western blot. The effect of H2O2on the viability of the PIGI cells after over-expression of PDGFRα was measured by MTT assay. The cell apoptosis was analyzed by flow cytometry. The protein levels of p38, p-p38 and cleaved caspase-3 in the cells were detected by Western blot. DCDHF-DA was used to estemate the generation of reactive oxygen species (ROS) in the cells. RESULTS:The viability of PIGI cells decreased after exposed to H2O2(P<0.05), and the half maximal inhibitory concentration of H2O2was 0.7 mmol/L. Transfection with PDGFRα over-expression vector successfully induced high expression of PDGFRα at mRNA and protein levels in the PIGI cells,and increased the viability of the cells with H2O2treatment(P<0.05). Over-expression of PDGFRα decreased the apoptotic rate of PIGI cells trea-ted with H2O2(P<0.05),and the level of ROS in the cells(P<0.05). The protein levels of cleaved caspase-3 and p-p38 were also decreased (P <0.05). CONCLUSION:PDGFRα inhibits the apoptosis of melanocytes induced by H2O2,partially reverses the growth inhibition of melanocytes by H2O2,and decreases the ROS level. The mechanism may be related to regulating the protein levels of p-p38 and cleaved caspase-3 in the cells.

Bovine adrenal medulla 22 (BAM22), an endogenous opioid peptide, is one of the cleavage products of proenkephalin A. It potently activates opioid receptors and sensory neuron-specific receptor (SNSR). The present study was aimed at investigating the effect of BAM22 on morphine tolerance. Intrathecal (i.t.) administration of morphine for 7 d produced morphine tolerance in rats. Then the rats were divided into three groups in which morphine, saline or BAM22 were administered i.t., respectively, on day 8, and morphine was given to all of the animals on day 9. It was found that morphine administered on day 9 resumed antinociceptive effects in BAM22 group, but not in saline or morphine group. The potency of morphine in BAM22 group was 48.5% of the maximal possible effect (MPE) detected by paw withdrawal test and the antinociception persisted for approximately 1 h. Following the similar treatment, morphine administered on day 9 reduced nocifensive behaviors by 3.2 min and 24 min in BAM22 group in the first and second phases, in the formalin test, respectively. The decreases were 45% and 82% of the corresponding values observed in saline group. Furthermore, following the treatment with BAM22 (10 nmol) on day 8 in morphine-tolerance rats, morphine administered on day 9 decreased the expressions of the heat-evoked c-Fos-like immunoreactivity (FLI) protein by approximately 80% in laminae I-II, III-IV and V-VI in the spinal cord at L4-L5 compared with that in saline or morphine group. The present study provided evidence at behavioral and cellular levels showing that BAM22 resumed antinociception of morphine. The results that the reversal effect of BAM22 on morphine tolerance was more efficient in persistent pain model than in acute pain may indicate that BAM22 differentially modulates morphine tolerance. The present study suggests that SNSR is involved in the modulation of morphine tolerance.
Animals ; Drug Tolerance ; Enkephalins ; pharmacology ; Morphine ; pharmacology ; Pain ; drug therapy ; Peptide Fragments ; pharmacology ; Rats ; Receptors, G-Protein-Coupled ; metabolism

OBJECTIVEHaemophilus (H.) influenzae is a gram-negative bacillus that is a common commensal organism of the human upper respiratory tract and an important cause of human diseases such as pneumonia, meningitis, septicemia, epiglottitis and cellulitis. Strains of H. influenzae are classified according to their capsular polysaccharide. There are six serotypes, designated as a through f. In addition, there are nonencapsulated strains. Although the type of infectious diseases caused by H. influenzae has changed considerably in recent years because of the widespread and routine immunization of children against type b H. influenzae (Hib), Hib remains an important pathogen. Ampicillin is the drug of choice for treating many infections caused by H. influenzae, but its usefulness has been compromised by the increasing prevalence of ampicillin-resistant strains. The continued monitoring of resistant strains by using genotyping methods may provide insights into the epidemiology of transmission. A molecular epidemiological study of ampicillin-resistant H. influenzae derived from nasopharyngeal swabs specimens of children less than 5 years of age with respiratory tract infection were investigated in this study.

METHODSA total of 899 isolates were collected from Beijing, Shanghai, and Guangzhou during 2000-2003. Susceptibility to ampicillin was determined by using E-test. Ampicillin-resistant H. influenzae strains were selected according to National Committee for Clinical Laboratory Standards (NCCLS) 2002 breakpoints. Nested PCR method with primers specific for bexA gene and b capsulate type-specific gene was established. Genotyping by pulsed-field gel electrophoresis (PFGE) and multiplex PCR assay was performed for all ampicillin-resistant H. influenzae strains.

RESULTSSeventy-four ampicillin-resistant H. influenzae strains were obtained. Two strains were positive by nested PCR, characterized as b genotype. The incidence of Hib in ampicillin-resistant H. influenzae strains was 2.7%; 38 genotypes were detected by PFGE. Detection of five types strains of clonal dissemination by PFGE accounted for 55.4% in all ampicillin-resistant H. influenzae strains. Among them eighteen H. influenzae strains belonged to one type, accounted for 24.3% in all ampicillin-resistant H. influenzae strains. Thirty one genotypes were identified by multiplex PCR assay for ampicillin-resistant H. influenzae. The identity ratio of PFGE and multiplex PCR was 63.5%.

CONCLUSIONIn Beijing, Shanghai and Guangzhou areas 55.4% of ampicillin-resistant H. influenzae strains had clonal dissemination during the 4 years.

Ampicillin Resistance ; genetics ; Anti-Bacterial Agents ; pharmacology ; Child, Preschool ; China ; epidemiology ; DNA, Bacterial ; genetics ; Drug Resistance, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Genotype ; Haemophilus Infections ; epidemiology ; microbiology ; Haemophilus influenzae ; classification ; genetics ; isolation & purification ; Humans ; Microbial Sensitivity Tests ; Molecular Epidemiology ; Nasopharynx ; microbiology ; Polymerase Chain Reaction ; Respiratory Tract Infections ; microbiology