OBJECTIVE To investigate the protective effect of urantide on myocardial apoptosis in rats induced by ischemia/reperfusion (I/R) or hypoxia/reoxygenation (H/R) injury and explore the underlying mechanism. METHODS ① In vivo test A rat myocardial I/R injury model was induced by ligating and untying the left anterior descending coronary artery with occlusion 30 min/reperfusion 60 min. Urantide 3, 10 and 30 μg·kg-1 was iv given 10 min before ischemia. TUNEL labeling was used for apoptosis measurement in myocardium. Immu-nohistochemical assay was used for Bcl-2 and Bax proteins expression detection. ② In vitro test An H/R cell model was set up by 3 h hypoxia/3 h reoxygenation. Urantide 0.1,1 and 10 nmol·L-1 was added just before hy-poxia, respectively. Hoechst33258 assay and flow cytometric techniques were used to detect apoptotic cells. RESULTS ① In vivo test Compared with sham group, the number of TUNEL-positive cells in I/R model group significantly increased (P<0.01) ; Bcl-2 protein expression slightly increased with no significant difference, Bax protein expression markedly increased ( P < 0. 01 ) , while Bcl-2/Bax ratio in I/R model group significantly decreased (P <0.01). Compared with I/R model group, the number of TUNEL-positive cells in urantide 10 and 30 μg·kg-1 groups was significantly decreased by about 36.6% and 57. 2% (P<0.05) ; Bax protein expression markedly decreased ( P <0.05 ) , while Bcl-2/Bax ratio was significantly augmented ( P <0.05 ). Urantide 30 u,g-kg1 also markedly increased Bcl-2 protein expression(P <0.05). ② In vitro test Compared with normal control group, the apoptosis rate in H/R model group significantly increased (P<0. 01). Hoechst33258 assay revealed that urantide 0.1, 1 and 10 nmol·L-1 reduced H/R-induced apoptotic nuclei by about 27.9% , 59.0% and 75. 4% , respectively (P <0.05). Flow cytometric techniques showed that the apoptosis rate was significantly reduced by about 32.8% and 64. 7% with administration of urantide 1 and 10 nmol·L-1 (P < 0. 01). CONCLUSION Urantide exerts an inhibitory effect on I/R or H/R-induced apoptosis by increasing Bcl-2 protein expression and decreasing Bax protein expression.

Objective To study the expression of tyrosine protein kinase in hepatocellular carcinoma(HCC)and observe the correlation between tyrosine protein kinase and clinical features.Methods A total of 30 cases with HCC were enrolled in this study.ERK,P38,C-jun,JAK2,STAT3 and STAT5 were detected by immunohistochemical method using tissue chip technology.Results The expression of ERK(0.220±0.033),P38(0.174±0.024),C-jun(0.183±0.064),JAK2(0.192±0.044),STAT3(0.197±0.078)and sTAT5(0.181±0.066)in HCC was significantly higher than that(0.065±0.028,0.058±0.028,0.042±0.016,0.070±0.030,0.052±0.024,0.052±0.023)in cirrhosis 1iver tissues(P＜0.01).There was significantly positive correlation of the expression between ERK,C-jun,JAK2,STAT3 and STAT5(P＜0.01 or P＜0.05).But the expression of P38 was negatively correlated with ERK in the HCC tissues(r=-0.404,P＜0.05).JAK2 had significant correlation with tumor differentiation.The expression of J AKz in stage Ⅲ was significantly higher than that in stage Ⅰ and Ⅱ cancer tissues.Conclusion There is important significance of the excessive activation of MAPK and JAK-STAT signaI transduction in hepatocellular carcinoma process.The unbalance of signal transduction might be one of the pathogenesis of tumor progress.

0.05). Conclusion Matrine could significantly down-regulate the mRNA expression of stat3 and stat5 and protein expression of STAT3 and STAT5 in SMMC-7721 cells. So matrine could inhibit the signaling transduction pathway of JAK-STAT and inhibit the proliferation of SMMC-7721 cells.

Aim To investigate the protective effect of the potent UT receptor(urotensin Ⅱ receptor,UTS2R)antagonist-urantide against myocardial ischemia-reperfusion(I/R)injury and its probable mechanisms in rats.Methods Rat myocardial I/R injury was induced by ligating and untying the left anterior descending coronary artery.The rats were randomly assigned into 6 groups:sham group,model group,urantide 3 ?g?kg-1 group,urantide 10 ?g?kg-1 group,urantide 30 ?g?kg-1 group and Ver(Verapamil)1.6 mg?kg-1 group.All animals except sham group were subjected to 30 min of occlusion and 60 min of reperfusion.Urantide or Ver was given ten minutes before occlusion through intravenous drug perfusion.Heart rate(HR)and the ST segment change of electrocardiogram(ECG)were recorded.The content of malondialdehyde(MDA)and nitric oxide(NO),and the activity of lactate dehydrogenase(LDH)and nitric oxide synthase(NOS)in blood serum were measured.Infarct size(IS),as a percentage of the area at risk(AAR),was determined by Evans blue and TTC double staining.The expression of iNOS protein was detected by western blotting.Results The results demonstrated that during the process of I/R,HR decreased significantly whereas ST segment of ECG markedly elevated.After I/R,MDA content and LDH activity in blood serum increased significantly,while total NO content and total NOS activity decreased sharply.Urantide(10,30 ?g?kg-1)had no significant effect on HR changes,but could markedly inhibit the elevation of ST segment of ECG,MDA content and LDH activity,and inhibit the decline of total NO content and total NOS activity,at the same time decrease I/R induced IS/AAR.But 3 ?g?kg-1 urantide had no significant effect on above indexes.Except for this,urantide(10,30 ?g?kg-1)down-regulated the I/R induced expression of iNOS.Conclusions Our findings indicate that urantide has a protective effect against myocardial I/R injury in rats.The cardio-protective involves the inhibition of lipid peroxidation and the stimulation of NO release.

Objective To investigate the effects of propofol on neuronal apoptosis in anterior horn of spinal cord in rabbits with spinal cord ischemia-reperfusion (IR) injury. Methods Sixty New Zealand white rabbits aged 4-6 months weighing 2.0-2.5 kg were randomized to receive normal saline (group C), 10% intralipid (group F) and propofol 30 mg/kg (group P1 ), 40 mg/kg (group P2), 50 mg/kg (group P3) and60 mg/kg (group P4 ). 10% intralipid was added to propofol solution to make the fluid infused equal in volume between the 6 groups ( n = 10 each). Spinal cord ischemia was induced by occlusion of abdominal aorta distal to the left renal arteries combined with simultaneous occlusion of bilateral common iliac arteries for 30 min. A catheter was inserted into abdominal aorta close to the site of occlusion via left femoral artery. Normal saline, 10% intralipid or different doses of propofol was infused through the catheter as soon as aorta was clamped at the rate of 12 ml·kg-1·h-1 for 30 min. The aorta and bilateral iliac arteries were then declamped. The L4-6 of spinal cord was removed at 48 h of reperfusion for microscopic examination and the total number of normal motor neurons in the anterior horn of spinal cord was counted. The total number of neurons and apoptosis neurons in the anterior horn of spinal cord was counted by TUNEL and the apoptosis index of neurons was calculated. The expression of caspase-3 in the anterior horn of spinal cord was determined by immunohistochemical technique. Results The number of normal motor neurons was significantly higher, and the apoptosis index and expression of caspase-3 were significantly lower in group P1-4 than in group C and F ( P < 0.05). Compared with group P1, the number of normal motor neurons was significantly increased and the apoptosis index was significantly decreased in group P2-4 and the expression of caspase-3 was down-regulated in group P3 and P4 ( P < 0.05). Compared with group P2, the number of normal motor neurons was significantly increased in group P3 while decreased in group P4, and the apoptosis index was significantly decreased and the expression of caspase-3 was down-regulated in group P3 and P4 ( P < 0.05). Compared with group P3, the number of normal motor neurons was significantly decreased and the apoptosis index was significantly increased and the expression of easpnse-3 was up-regulated in group P4 ( P < 0.05) . Conclusion Propofol 30-60 mg/kg infused through aorta during occlusion can inhibit the neuronal apoptosis and attenuate IR injury to spinal cord dose-dependently in rabbits. The underlying mechanism may be related to the down-regulation of caspase-3 expression.

Objective To analyze the clinical characteristics of patients with L isteria septicemia for better clinical diagnosis and management of the disease .Methods A retrospective study was carried out in 13 patients with confirmed diagnosis of Listeria septicemia from July 2009 to November 2013 in West China Hospital of Sichuan University .The clinical features ,laboratory tests ,treatments and clinical outcomes were reviewed and analyzed . Results The vast majority of the 13 patients were immunocompromised or with critical organ dysfunction . The in vitro antimicrobial susceptibility testing indicated that penicillin ,ampicillin and levofloxacin were the most active agents against Listeria ,followed by imipenem ,erythromycin , ciprofloxacin and tetracycline .Only 33 .3% of the 13 Listeria isolates were sensitive to oxacillin .Eight patients were cured ,2 improved ,2 died after therapy .The remaining one patient gave up therapy .Conclusions The incidence of Listeria septicemia was associated with advanced age and presence of underlying diseases .Early etiology diagnosis and appropriate antibacterial therapy can improve the outcome of such patients .Actively treating underlying diseases helps reduce the mortality rate .

The stereoselective hydrolysis of esmolol in whole blood and in its separated components from rat,rabbit and human was investigated.Blood esterase activities were variable in different species in the order of rat ＞ rabbit ＞ human.Rat plasma showed the high esterase activity and had no stereoselectivity to enantiomers.Rabbit red blood cell (RBC) membrane,RBC cytosol and plasma all hydrolyzed esmolol but with different esterase activity,whereas the hydrolysis in RBC membrane and cytosol showed significant stereoselectivity towards R-(+)-esmolol.Esterase in RBC cytosol from human blood mainly contributed to the esmolol hydrolysis,which was demonstrated with no stereoselctivity.Esterase in human plasma showed a low activity,but a remarkable stereoselectivity with R-(+)-esmolol.In addition,the protein concentration affected the hydrolysis behavior of esmolol in RBC suspension.Protein binding of esmolol enantiomers in human plasma,human serum albumin (HSA) and α1-acid glycoprotein (AGP) revealed that there was a significant difference in bound fractions between two enantiomers,especially for AGP.Our results indicated that the stereoselective protein binding might play a role in the different hydrolysis rates of esmolol enantiomers in human plasma.

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

Objective To assess polymerase chain reaction(PCR)combined with restriction fragment length polymorphism(RFLP)and gene sequencing technologies in the detection and typing of HPV DNA.Methods Tissue specimens were collected from skin diseases and venereal disease in perianal or genitals.PCR was performed with HPV DNA general primers(MY09/11)in tissue samples. Positive fragments of HPV DNA were purified and digested by restriction enzymes.The digested fragments were typed by po]yacrylamide gel electrophoresis(PAGE).The Resultswere verified by direct sequencing.Results In 50 clinical samples there were 35 HPV DNA positive,including 26 from patients with condyloma acuminatum,8 from patients with bowenoid papulosis,and 1 from patients with squamous cell carcinoma.In HPV DNA positive samples,19 were HPV6,3 were HPV11,8 were HPV16,4 were HPV6 and HPV 11,and I was HPV62.Sequencing Resultswere in accordance with the PCR-RFLP Results .Conclusion PCRRFLP method is effective in the detection and typing of HPV DNA.

Objective To explore the relationship of phosphatase and tensin homology (PTEN) deleted on chromosome ten with the apoptosis of hepatic stellate cells (HSCs) in liver tissues of rats with hepatic fibrosis induced by bile stagnation.Methods Fifty adult male SD rats were randomly divided into model group (n=40) and sham operation group (n=10).The model of hepatic fibrosis was reproduced in model group by common bile duct ligation (BDL).Liver tissue of rats in model group (1,2,3 and 4 weeks after BDL) and sham operation group were obtained.PTEN expression in liver tissue was detected by immunohistochemistry.Apoptosis of HSC was determined by dual staining of terminal deoxynucleotidy transferase UTP-nick end labeling (TUNEL) and ?-SMA immunohistochemistry.Results Only a few apoptotic HSCs were found in normal livers.With the development of liver fibrosis,the expression of PTEN decreased gradually (P