Purpose To prepare a novel fusion protein of CREKA and tTF as a universal carrier targeting to cancer,and to analyze its activities.Methods CREKA and tTF gene were acquired by PCR,and inserted into plasmid pET22b(+)to construct recombinant plasmid CREKA/tTF/pET22b(+),and the fusion gene was expressed in E.coli BL21.The fusion protein Wag purified through Nickel-affinity chromatography column.After purifying,the fusion protein was refold by subsequent dialysis.The activities of the fusion proteins were measured by coagulation timing and quantitative fluorescence test in vitro.Results The recombinant plasmid CREKA/tTF/pET22b(+)with correct sequence was obtained.The fusion protein was highly expressed in E.coli BL21.The coagulation of the fusion protein Was determined by the coagulation test.And the capability of the fusion protein effectively binding to clotted plasma proteins is identified in quantitative fluorescence test.Conclusion The recombinant plasmid CREKA/tTF/pET22b(+)with correct sequence was built.The fusion protein CREKA/tTF with both TF and CREKA activity was successfully obtained.

Objective To evaluate the effects of nerve growth factor-beta (NGF-β) pretreatment on cell apoptosis induced by endoplasmic reticulum stress during ischemia-reperfusion in isolated rat hearts.Methods Male Sprague-Dawley rats,weighing 180-220 g,were anesthetized with intraperitoneal 10％ chloral hydrate 300 mg/kg.The hearts were excised and perfused in a Langendorff apparatus with K-H solution aerated with 95％ O2 and 5％ CO2 at 37 ℃.Thirty-two isolated rat hearts were randomly divided into 4 groups (n =8 each) using a random number table:control group (group C),I/R group,NGF-β pretreatment group (group N) and NGF-β combined with K252a (trkA receptor antagonist) pretreatment group (group N + K).In group C,the hearts were continuously perfused with K-H solution for 195 min.The hearts were perfused with K-H solution for 45 min in group I/R.In N and N + K groups,the hearts were perfused with K-H solution for 15 min,and then with K-H solution containing 0.1 μg/ml NGF-β and 0.1 μg/ml NGF-β mixed with 100 nmol/L K252a,respectively,for 30 min.The perfusion was suspended for 30 min followed by 120 min of reperfusion with K-H solution in I/R,N and N + K groups.HR,left ventricular end-diastolic pressure (LVEDP),left ventricular developed pressure (LVDP) and + dp/dtmax were measured at the end of 15 min equilibration (baseline) and at 5,30,60 and 120 min of reperfusion.Myocardial specimens were obtained at 120 min of reperfusion for detection of myocardial apoptosis (by TUNEL) and expression of glucose-related protein 78 (GRP78),CCAAT/enhancer-binding protein homologous protein (CHOP),and caspase-12 (by Western blot analysis).Apoptosis index was calculated.Results Compared with group C,HR,LVDP and + dp/dtmax were significantly decreased,and LVEDP,apoptosis index and expression of GRP78 and CHOP were increased in I/R and N groups,and the expression of caspase-12 was upregulated in I/R group.Compared with group I/R,HR,LVDP,and + dp/dtmax were significantly increased,and LVEDP,apoptosis index and expression of GRP78,CHOP and caspase-12 were decreased in group N,and the expression of GRP78 was down-regulated in group N + K.There was no significant difference in cardiac function indexes between group I/R and N + K.Compared with group N,HR,LVDP and + dp/dtmax were significantly decreased,and LVEDP,apoptosis index,and expression of GRP78,CHOP and caspase-12 were increased in group N + K.Conclusion NGF-β pretreatment can protect the isolated rat hearts against ischemia-reperfusion injury,and inhibition of the endoplasmic reticulum stress-triggered cell apoptosis after activating trkA receptors is involved in the mechanism.

Objective To evaluate the role of 1-phosphatidylinositol 3-kinase (PI3K) signaling pathway in nerve growth factor-beta (NGF-β)-produced mitigation of cell apoptosis induced by endoplasmic reticulum stress during hypoxia-reoxygenation (H/R) in rat cardiomyocytes.Methods H9c2 cells were seeded in 96-well plates at a density of 5× 105 cells/ml (100 μl/well).The wells were randomly divided into 5 groups (n =15 each) using a random number table: control group (group C);group H/R;NGF-β group (group N);NGF-β+NGF receptor trkA antagonist K252a group (group N+K);NGF-β+ PI3K inhibitor LY294002 group (group N+L).The cells were exposed to 95％ N2-5％ CO2 for 4 h in an anaerobic incubator, followed by reoxygenation in a standard incubator for 4 h in H/R, N, N+K and N+L groups.In addition, the cells in N, N + K and N + L groups were incubated in a standard incubator containing NGF-β, the mixture of NGF-β and K252a and the mixture of NGF-β and LY294002, respectively, during reoxygenation, and the final concentrations of NGF-β , K252a and LY294002 were 50 ng/ml, 100 nmol/L and 50 μmol/L, respectively.The cell viability was detected by using CCK-8 assay, and the cell survival rate was calculated.The cell apoptosis was examined by flow cytometry, and apoptosis rate was calculated.The expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), caspase-12, Akt and phosphorylated Akt (p-Akt) was detected by Western blot.The ratio of p-Akt to Akt was calculated.Results Compared with group C, the cell survival rate was significantly decreased, and the apoptosis rate was increased in H/R and N groups, and the expression of GRP78, CHOP and caspase-12 was significantly up-regulated in group H/R, and p-Akt/Akt was significantly increased in group N (P＜0.05).Compared with group H/R, the cell survival rate was significantly increased, the apoptosis rate was decreased, the expression of GRP78, CHOP and caspase-12 was up-regulated, and p-Akt/Akt was increased in group N (P＜0.05), and no significant change was found in the parameters mentioned above in N+K and N+L groups (P＞0.05).Compared with group N, the cell survival rate was significantly decreased, the apoptosis rate was increased, the expression of GRP78, CHOP and caspase-12 was up-regulated, and p-Akt/Akt was decreased in N+K and N+L groups (P＜0.05).Conclusion NGF-β can mitigate the cell apoptosis induced by endoplasmic reticulum stress during H/R, and activation of PI3K signaling pathway is involved in the mechanism.

Acute Disease ; Adolescent ; Adult ; Animals ; Child ; Child, Preschool ; Female ; Foodborne Diseases ; diagnosis ; etiology ; therapy ; Humans ; Male ; Meat ; poisoning ; Middle Aged ; Pesticides ; poisoning ; Swine ; Treatment Outcome

Ataxia Telangiectasia Mutated Proteins ; Breast ; metabolism ; pathology ; Breast Neoplasms ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; metabolism ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; metabolism ; pathology ; Cell Cycle Proteins ; metabolism ; Checkpoint Kinase 2 ; DNA-Binding Proteins ; metabolism ; Female ; Humans ; Lymphatic Metastasis ; Neoplasm Grading ; Protein-Serine-Threonine Kinases ; metabolism ; Tumor Burden ; Tumor Suppressor Protein p53 ; metabolism ; Tumor Suppressor Proteins ; metabolism

Objective To evaluate the effect of low-dose ketamine on the efficacy of electroconvulsive therapy (ECT) under propofol anesthesia in depressed rats. Methods Sixty adult male SD rats weighing 200-250 g were used in this study. The depression model was established by chronic unpredictable mild stress (CUMS). The animals were then randomly divided into 6 groups (n = 10 each): control group (group C), depression group (group D), propofol group ( group P), propofol + ECT group ( group PE), ketamine + propofol group ( group KP), and ketamine + propofol + ECT group (group KPE). Groups P and KP received intraperitoneal propofol 100 mg/kg and ketamine 10 mg/kg + propofol 80 mg/kg respectively, and groups PE and KPE received ECT after intraperitoneal injection of propofol 100 mg/kg and ketamine 10 mg/kg + propofol 80 mg/kg respectively once a day for 7 consecutive days. All rats underwent sucrose fluid consumption and Morris water maze tests before CUMS, after CUMS, and after treatment. Results Compared with group C, the sucrose consumption percentage was significantly decreased, the escape latency was prolonged, and the time spent in the target quadrant (the original platform quadrant) was shortened after CUMS in D, P, PE, KP and KE groups ( P ＜ 0.05). Compared with group D,the sucrose consumption percentage was significantly increased (P ＜ 0.05), while no significant change in the escape latency and time spent in the target quadrant was found after treatment in group KPE ( P ＞ 0.05 ), and the sucrose consumption percentage was significantly increased, the escape latency was prolonged, and the time spent in the target quadrant was shortened after treatment in group PE ( P ＜ 0.05). Compared with group PE, the sucrose consumption percentage was significantly increased, the escape latency was shortened, and the time spent in the target quadrant was prolonged after treatment in group KPE ( P ＜ 0.05). Conclusion Low-dose ketamine can not only enhance the efficacy of ECT under propofol anesthesia in depressed rats, but also reduce cognitive impairment induced by ECT.

ObjectiveTo investigate the effect of propofol on cyclooxygenase-2 (COX-2) expression in hippocampal neurons in depressed rats after electroconvulsive therapy (ECT).MethodsFifty 2-3 months old male SD rats weighing 200-250 g were randomly divided into 5 groups (n =10 each):group control (group C) ; group depression (group D); group propofol (group P); group ECT (group E) and group propofol + ECT (group PE).Depression was induced by separation and chronic unpredictable mild stres in groups D,P,E and PE.Groups P and PE received intraperitoneal pro pofol 80 mg/kg.Groups E and PE received ECT at 5 min after IP normal saline 8 ml/kg and propofol 80 mg/kg respectively once a day for 7 consecutive days.The learning and memory function was assessed by using Morris water maze test before (baseline) and after depression was induced and ECT.The animals were then sacrificed and their brains removed for detection of COX-2 mRNA expression in hippocampus (by RT-PCR).Results In group D depression significantly prolonged evasive latency and decreased swimming time percentage in platform quadrant and up-regulated COX-2 mRNA expression as compared with group C.In group E ECT further prolonged evasive latency and up-regulated COX-2 mRNA expression in depressed rats.In group PE propofol pretreatment attenuated ECT-induced impairment of learning-memory function and increase in COX-2 mRNA expression as compared with group E.ConclusionPropofol can ameliorate the decrease in learning and memory function induced by ECT in depressed rats by inhibiting COX-2 expression in hippocampal neurons.

Objective To investigate the effect of propofol anesthesia on electroconvulsive therapy (ECT)-induced hyperphosphorylation of Tau protein in hippocampus in depressed rats.Methods Thirty-two female WYK rats in which the total score was 30-120 after Open-field test,aged 24 weeks,weighing 200-250 g,were randomly divided into 4 groups ( n =8 each):control group (group C),propofol group (group P),ECT group (group E)and propofol + ECT group (group PE).In groups C and E,the animals received intraperitoneal normal saline 5 ml,and in addition the animals received ECT 15 min later in group E.In groups P and PE,the animals received intraperitoneal 100 mg/kg propofol 5 ml,and in addition the animals received ECT 15 min later in group PE.The learning and memory function was assessed by Morris water maze test at 24 h after ECT.The animals were sacririced at 6 h after Morris water maze test and the hippocampal tissues were removed for determination of the expression of phosphorylated Tau protein.Results Compared with group C,the escape latency was significantly prolonged,the swimming time was significantly shortened in groups P,E and PE,the expression of phosphorylated Tau protein in hippocampus was down-regulated in group P,and the expression of phosphorylated Tau protein in hippocampus was up-regulated in group E ( P ＜ 0.05).Compared with group E,the escape latency was significantly shortened,the swimming time was significantly prolonged,and the expression of phosphorylated Tau protein in hippocampus was down-regulated in group PE (P ＜0.05).Conclusion The mechanism by which propofol anesthesia improves cognitive impairment induced by ECT may be related to inhibition of hyperphosphorylation of Tau protein in hippocampus in depressed rats.

Objective To evaluate the effects of sepsis on pharmacodynamics of rocuronium in rats.MethodsForty SD male rats weighing 250-350 g were randomly divided into 3 groups: control group(group C,n =10) ; sham operation group(group S,n =10)and sepsis group (group Sep,n =20).Cerum was ligated and perforated to produce sepsis model in Sep group,rocuronium 3.81 mg/kg was intravenously injected at 6 or 16 h after operation,each time contains 10 rats.Cecum was not ligate and perforate in group S,but rocuronium 3.81 mg/kg was intravenously injected at 6 h after operation.Onset time,TOF no reaction period,duration of peak effect,clinical duration,totel duration,time for recovery of T1 to 10％,25％,50％,75％,90％ and recovery index were recorded by RM6240B signal acquisition and processing system.ResultsCompared with groups C and S,onset time was significantly prolonged,TOF no reaction period,duration of peak effect,clinical duration,total duration and time for recovery T1 to 10％,25％,50％,75％,90％ and recovery index were shortened in group Sep ( P ＜ 0.05).Onset time was significantly prolonged,time for recovery of T1 to 75％ was shortened when rocuronium injection at 16 h after operation as compared with that at 6 h after operation in group Sep( P ＜ 0.05).ConclusionSepsis can attenuate the effects of nondepolarizing neuromuscular blocker rocuronium,the degree is related to the stage of sepsis.