The aim of this study is to clone the mouse T-bet promoter and enhancer, construct and identify the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS for T-bet transcription regulation study and its function in signaling of multiple sclerosis. The promoter and CNS of T-bet were predicted by bioinformatics assay. The predicted fragment of mouse T-bet promoter plus CNS was amplified by PCR and cloned into pGL4.10. The recombinant plasmid pGL4.10-TBX21pr-CNS was transferred into Escherichia coli DH5α. The positive clone was identified by double digestion with Kpn I and Sfi I and DNA sequencing. Finally, pGL4.10-TBX21pr-CNS was cotransfected with pRL-TK into 293T cells and Jurkat cells, pRL-TK and pGL4.10 as a control. The luciferase activity in 293T cells (P = 0.012 2) and Jurkat cells (P = 0.002 2) was higher than that of the control group. A fragment of 1 028 bp mouse T-bet promoter plus 1 308 bp CNS was successfully cloned and the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS was constructed. In 293T cells and Jurkat cells, pGL4.10-TBX21pr-CNS has the promoter functions. This work offers a basic material for the research of T-bet transcription.
Animals ; Gene Expression Regulation ; Genes, Reporter ; Genetic Vectors ; Luciferases ; Mice ; Multiple Sclerosis ; Plasmids ; Promoter Regions, Genetic ; T-Box Domain Proteins ; genetics

OBJECTIVE: To investigate the perioperative treatments of endoscopic sinus surgery for nasal diseases in patients with coronary heart disease after PCI. METHOD: Clinical data of 12 cases of endoscopie-assised surgery such as nasal tumors resection,functional sinus surgery,correction of deviated nasal septum,low-temperature plasma hemostasis in patients with coronary heart disease after PCI were analyzed retrospectively. RESULT: Serious bleeding did not take place with the 12 cases during surgery, and surgery progressed smoothly; one of patients had heavy nosebleed after surgery, however her condition was stable when received active treatment. Follow-up 3 months to 2 years, nasal diseases of 12 patients recovered well and symptoms were relieved; cardiovascular events such as hemorrhage, thrombosis and so on did not occur. CONCLUSION Due to physiological function of the heart dysfunction in patients with coronary heart disease after PCI,they often accompany a number of trouble issues such as medical disorders, oral antiplatelet drugs, surgery affordability loss and increase surgical risk. Correct and effective perioperative treatments, strictly surgical indications are really necessary which can keep patients safe through perioperative period.
Coronary Disease ; Endoscopy ; Epistaxis ; Humans ; Nasal Septum ; surgery ; Nasal Surgical Procedures ; Nose Deformities, Acquired ; Nose Neoplasms ; surgery ; Paranasal Sinuses ; Percutaneous Coronary Intervention ; Retrospective Studies ; Treatment Outcome

OBJECTIVE:To establish a HPLC method for the determination of chlorogenic acid and baicalin in xiaoer qingrening granula.METHODS:The determination was performed on Symmetry Shield RP C 18 ,the mobile phase of chlorogenic acid consisted of acetonitrile-0.4%phosphoric acid soluton(13∶87)and that of baicalin consisted of methanol-water-glacial acetic acid(50∶50∶1),the detection wavelengths of chlorogenic acid and baicalin were327nm and274nm,respectively,the flow rate was1.0ml/min,sample size was20?l and column temperature was25℃.RESULTS:The respective linear sample size range for chlorogenic acid and baicalin was0.1040?g～1.040?g(r=0.9998)and0.6240?g～6.240?g(r=0.9997),with average recoveries respectively at96.4%(RSD=1.4%)and98.0%(RSD=1.1%).CONCLUSIONS:The method is simple,accurate,reproducible and specific,and it can be used for the quality control of xiaoer qingrening granula.

Objective To investigate the efficiency of transduction of recombinant adenovirus-mediated human endothelial nitric oxide synthase (eNOS) into lung tissue by repeated intratracheal transfection in rats.Methods Sixty 3-4 month old male Wistar rats weighing 220-280 g were randomly divided into 2 groups:control group (group C,n =10) and eNOS gene transduction group (group T,n =50).The animals were anesthetized with intraperitoneal 10％ chloral hydrate 35 mg/kg,tracheally intubated and mechanically ventilated (VT 2.5 ml,RR 60 bpm,FiO2 1.0).Recombinant adenovirus carrying human eNOS gene was given as gift by Professor Gerard from Texas University,Southwest Medical Center.In group T 50 μl of the recombinant adenovirus in concentration of 5 × 109 PFU/ml was instilled into trachea every 5 minutes for 12 times,while in group C equal volume of vector conservation solution was instilled instead.Pulmonary arterial blood samples were obtained at 2,5,7,14 and 21 d after intratracheal transfection (n =10 at each time point) for determination of serum NO concentration.The animals were immediately sacrificed after blood sample collection for determination of expression of eNOS protein in the lung tissue and RNA.The eNOS expression in the trachea,bronchus,lung,liver,spleen and kidney was detected by immuno-histochemistry.Results The serum NO concentrations were significantly higher at all time points in group T than in group C.The eNOS expression was detected in the epithelial cells of trachea and bronchi,and endothelial cells of alveoli and pulmonary blood vessels in group T but not in group C.eNOS expression was not detected in liver,spleen and kidney at 7 d after intratracheal transfection in group T.Conclusion Human eNOS gene mediated by recombinant adenovirus was transducted into rat lung tissue with normal enzyme activity by repeated intratracheal administration without being detected in distant organs.

Objective To investigate the effects of penehyclidine hydrochloride on blood-brain barrier in a rat model of cardiopulmonary bypass ( CPB) . Methods Sixty adult male SD rats, aged 4-6 months, weighing 320- 370 g, were randomly divided into 5 groups ( n = 12 each) : sham operation group (group S), CPB group, and low-, median- and high-dose penehyclidine hydrochloride groups (groups LP, MP and HP). The animals were anesthetized with intraperitoneal 10% chloral hydrate 350 mg/kg, intubated and mechanically ventilated. The femoral and jugular arteries and jugular vein were cannulated. CPB was performed for 60 min. Penehyclidine hydrochloride 0.2, 0.6 and 2.0 mg/kg were added to the priming solution in groups LP, MP and HP respectively, while the equal volume of normal saline was added in group CPB. Evans blue was injected via femoral vein at 1 h before the animals were sacrificed. Six rats in each group were sacrificed, their brains immediately removed and the hippocampi isolated for determination of Evans blue content. The other rats were sacrificed and the hippocampi isolated to determine the water content and observe the ultrastructure of blood-brain barrier. Results Compared with group S, the Evans blue content and water content were significantly increased in the other groups ( P ＜ 0.05) . Compared with groups CPB and LP, the Evans blue content and water content were significantly decreased in groups MP and HP ( P ＜ 0.05) . The Evans blue content was significantly lower in group HP than in group MP ( P ＜ 0.05). The CPB-induced changes were significantly attenuated in groups MP and HP compared with groups CPB and LP. Conclusion Penehyclidine hydrochloride can protect blood-brain barrier against the CPB-induced injury and the effect is related to the dose.

Objective To evaluate the effect of penehyclidine hydrochloride (PHC) on the level of angiopoietin-1 (Ang-1) and tyrosine kinase receptor-2 (Tie-2) during endotoxin-induced acute lung injury (ALI) in rats.Methods Forty adult male Sprague-Dawley rats,weighing 180-220 g,were randomly divided into 4 groups using a random number table (n =10 each):control group (group C),ALI group,low-dose PHC group (group L-PHC) and high-dose PHC group (group H-PHC).ALI was induced with iv injection of lipopolysaccharide 5.0 mg/kg via the tail vein.In L-PHC and H-PHC groups,PHC 0.6 and 2 mg/kg were injected,respectively,via the tail vein at 1 and 24 h after lipopolysaccharide injection.The rats were sacrificed at 48 h after the initial injection of PHC to measure the lung water content,protein concentration in bronchoalveolar lavage fluid (BALF),and the expression of Ang-1,Tie-2 and phosphorylated Tie-2 in lung tissues.The morphological changes of lung tissues were observed under light microscope and the ultrastructural changes of alveolar epithelial barrier under transmission electron microscope.Results Compared with group C,the lung water content and protein concentrations in BALF were significantly increased,and the expression of Ang-1 and phosphorylated Tie-2 was down-regulated in the other three groups (P ＜ 0.05).Compared with group ALI,the lung water content and protein concentrations in BALF were significantly decreased,and the expression of Ang-1 and phosphorylated Tie-2 was up-regulated in H-PHC group (P ＜ 0.05),and no significant changes were found in the parameters mentioned above in group L-PHC (P ＞0.05).The damage to lung tissues was significantly reduced in group H-PHC as compared with group ALI.Conclusion PHC can improve the permeability of pulmonary microvascular and reduce injury to alveolar epithelial barrier,thus ameliorating endotoxin-induced ALI in rats,and the effect is dose-related and up-regulation of Ang-1 expression and inhancement of Tie-2 activity are involved in the mechanism.

Objective To design nursing occupational safety and health curriculum reasonably.Methods With a semi-structured interview outline,focus group interviews among eight clinical nursing and nursing education specialists were conducted to develop the occupational safety and health curriculum design.Results It was important to set up nursing occupational safety and health curriculum.The main object,contents,teaching and evaluation methods were identified.Conclusions Occupational safety and health curriculum can be involved in nursing curriculum system to reduce occupational hazards and maintain physical and mental health of nursing students.

Objective To study the correlation between the percentage of intrahepatic regulatory T cell (Treg) and liver inflammatory activity in chronic hepatitis B (CHB) patients.Methods Twenty-six cases of CHB patients admitted to First Department of Internal Medicine,Nagasaki University School of Medicine,Sakamoto,Nagasaki,Japan were enrolled and performed liver biopsy in this study.CD3+cell and Foxp3+ cell in liver were detected by immunohistochemistry.the percentage of Foxp3+/CD3+cell was determined.Clinical data including alanine aminotransferase (ALT).aspartate aminotransferase(AST), hepatitis B virus (HBV) DNA level and the inflammatory activity of histological activity index score (HAD of liver pathology using Knodell evaluating system were collected.The data was analyzed by SPSS 13.0 software.Results Foxp3+Tregs in serial sections of CHB accumulated mainly in the portal area.There Was a significance correlation between the percentage of Foxp3+/CD3+ cell and liver parenchyma inflammation (P=0.007 6).Moreover,Foxp3+Treg in CHB patients with high serum ALT or AsT level presented in a higher frequency than in patients with low AIT or AST level.The difference between these tWO groups was statistically significant (rALT=O.438,PALT=0.025; fAST=O.436,Past=O.026).There Was a tendency between the percentage of Foxp3+/CD3+ cell in liver and HBV DNA level.however the correlation was not statistically significant.Conclusion It is suggested that CD4+ CD25+ Foxp3+Treg may play a major role in the pathogenesis of liver injury in the CHB patients.

Objective To investigate the effect of penehyclidine hydrochloride on brain injury induced by cardiopulmonary bypass (CPB) in rats. Methods Thirty adult male SD rats were randomly divided into 5 groups ( n = 6 each): sham operation group (group S), CPB group, and low, median and high dose penehyclidine hydrochloride groups (group PL, PM , PH). Penehyclidine hydrochloride 0.2, 0.6 and 2.0 mg/kg were added to the priming solution in group PL, PM and PH respectively, while the equal volume of normal saline was added instead in group S. Blood samples were obtained at 2 h after termination of CPB to determine the plasma concentrations of neuron specific enolase (NSE) and S-100β protein. The brain tissues were taken to observe the ultrastructure of hippocampal neurons with electron microscope. Results The concentrations of NSE and S-100β protein were significantly higher in the other groups than in group S, while lower in group PM and PH than in group CPB and PL( P＜ 0.05). The S-100β protein concentration was significantly lower in group PH than in group PM( P ＜ 0.05). The damage to hippocampal neurons was significantly attenuated in group PM and Ps. Conclusion Penehyclidine hydrochloride 0.6 or 2.0 mg/kg can reduce the CPB-induced brain injury in a dose-dependent manner in rats.