Objective To construct thioredoxin and APE/ref-1 fusion protein expression vector and to investigate their subcellular localization of the fusion proteins in 293Tcells.Methods The APE/ref-1 cDNA was cloned by RT-PCR from PC12 cell.APE/ref-1-EGFP fusion protein expression vector was constructed through subcloning.Thioredoxin cDNA was subcloned into pDSred1-1 from pQE30-TRX plasmid by PCR,and thioredoxin-DsRed fusion protein expression vector was constructed in pCMV5 plasmid.The expression and subcellular localization of the fusion proteins in 293T cells transfected with the vectors by calcium phosphate was analyzed by fluorescent microscopy.Results The results demonstrated that thioredoxin——DsRed and APE/ref-1-EGFP fusion protein expression vectors were successfully constructed and expressed in 293T cells.APE/ref-1-EGFP fusion protein was located only in nucleus,and thioredoxin-DsRed fusion protein was located in cytoplasm as well as nucleus.ConclusionThis study has set up a solid base for further to study on the dynamic interaction between thioredoxin and APE/ref-1 fusion proteins.

A novel PCR-based mutagenesis method was reported, in which there is no need to purify megaprimers or design a special flanking primer. This method used one mutagenic primer and two sequencing primers (T_m≤58℃) as flanking primers. After first round PCR, 12.5 μl first PCR production was directly added into 50 μl second PCR system as template and megaprimer, and 10 rounds of asymmetrical PCR at high temperature of annealing (68 ℃ ) was to add in initiation of second PCR. This additional step greatly has increased the efficiency of mutagenesis via 600 bp or 800 bp long megaprimer. The results demonstrated that this method can achieve high fidelity, 97%～ 98% efficiency, high yield.

Objective To study the contents and distribution characteristics of trace elements in Chrysanthemum morifolium from different areas. Methods The contents of 13 trace elements in six samples of Chrysanthemum morifolium were determined by ICP-OES. The principal component analysis combined with SPSS 19. 0 software was applied to evaluate charac-teristics of elements Results Curves of trace element content of Chrysanthemum morifolium from different origins had a certain similarity. The contents of Fe, Zn and Mn were higher than others. The results of principal component analysis showed that five principal components were extracted from six samples of Chrysanthemum morifolium, and Cd, Cu, Zn, Ba and Co was the characteristic trace elements. Conclusion The contents and distribution of trace elements can reflect the characteristics of Chrysanthemum morifolium from different areas, which could be used for Chrysanthemum morifolium quality assessment and classification.

Objective To evaluate the effectiveness of a project for treating TB/HIV co-infection(phase 1,2007-2008)supported by Global Fund in Wolong district.Method Data on project implementation in 2007-2008 were collected and analyzed.Results A total of 916 TB patients were diagnosed in 2007-2008,of whom 847 cases were detected for HIV antibody,with a detection rate of 92.47%.A total of 368 people living with HIV/AIDS were identified by screening 1790 persons/times,with a screening rate of 94.36%.Among them 55 cases of active tuberculosis were co-infected with HIV,of whom 3 were smear positive.After aniti-TB chemotherapy marked symptomatic improvement was witnessed in 37 cases;the curative rate for new smear positive cases reached 66.7% and the remission rate was 70.0%.Conclusion Satisfactory results were achieved in preventing and treating TB/HIV co-infection in Wologn district,and the experience gained in implementing the phase 1 project will provide useful information for implementing the project for the next phases.

Objective To explore the expression of singal transducers and activators of transcription(STAT 3) during focal cerebral ischemic reperfusive injury in rats and the relationship between ischemic neuronal damage and it.Methods Using immunohistochemical method of avidinbiotin peroxidase complex (ABC) we observed the distribution of positive cells in STAT 3 protein immunoreaction after focal cerebral ischemic reperfusive injury in rats.Results STAT 3 immunoreactive positive cells were not found in the cortex and striatum of normal and sham operative rat brains and nonischemic hemisphere brain after cerebral ischemia,small amount of STAT 3 immunity positive cells were induced in the embolism la teral infarction area 12 h after reperfusive injury,and peaked after 24 h,especially in ischemic lateral striatum and around cortex,small number of nerve cells in around infarction still showed positive expression after one week.The difference had remarkable significance( P

Objective To investigate the prevalence of hypertension and obesity and their main influencial factors among 6～12 years old children in Shenzhen.Method 1 140 children aged from 6 to 12 years old in 4 schools in Shenzhen were sampled by random cluster sampling,and their systolic blood pressure(SBP),diastolic blood pressure(DBP),body height,weight and other morphological parameters were measured.Results The prevalent rate of hypertension was 9.4%(8.6% for boys and 10.2% for girls);Rate of overweight and obesity for boys were 13.25% and 13.72,respectively,and the girl were 9.09% and 8.10%,respectively.There were a increasing trend toward SBP and DBP with age,especially SBP.After adjusted with age and gender,the partial correlation coefficients between BMI and SBP,DBP were 0.462 and 0.357,respectively(P

Objective To investigate the degree of cardiac function impairment, and clinical implications of BNP,TnT and TnI in patients with severe sepsis and septic shock retrospectively by comparing the differences in levels of BNP,TnT and TnI among those patients.Methods From December 2005 through December 2010,98 patients admitted with severe sepsis ( n =57 ) or septic shock ( n =41 )were enrolled to be studied.The inclusion criteria were ( 1 ) echocardiography was performed; (2) levels of serum BNP,TnT and TnI were determined on the 1st day,3rd day and 7th days after admission.These 98 patients were divided into 2 major groups:severe sepsis group (S) and septic shock group (SS) ; and each major group was further divided into 4 subgroups,including severe sepsis normal cardiac function (Sn) ;severe sepsis abnormal cardiac function (Sa); severe sepsis death (Sd),severe sepsis survival (Ss);septic shock normal cardiac function ( SSn ),septic shock abnormal cardiac function ( Ssa ),septic shock death (SSd) and septic shock survival (SSs).Then comparisonsof the plasma levels of BNP,TnT and TnI were carried out among groups respectively.The morality difference between groups S vs SS,and Savs Sn,and Ssa vs SSn were compared by using the Chi-square Test.The differences in BNP,TnT and Tnl between groups were compared by SNK-q test.Correlation between ejection fraction (EF) value and those biomarkers was calculated by Pearson correlation coefficient.The relationship between those biomarkers and outcomes was determined by using receiver operating characteristic curve (ROC). Results ( 1 ) There were no significant differences in levels of BNP and TnI between group S and SS,but TnT level in patients of SS group was significantly higher than that in S group ( P =0.001 ).( 2 ) TnT,BNP,Tnl levels in Sa group were significantly higher than those in group Sn; and BNP level in group SSd was significantly higher than that in SSs group (P =0.001 ).(3) BNP,TnT and TnI levels in group Ssa were significantly higher than those in group SSn,and BNP level in SSd group was significantly higher than that in SSs ( Pday 1 =0.001,Pday3 =0.001,Pday7 =0.016). (4) TnT and TnI levels in group SSn were significantly higher than those in group Sn,and BNP levels in SSn group on the 1st day,3rd day and 7th days were significantly higher than those in Sn group (Pday1 =0.006,Pday3 =0.006,Pday7 =0.001).(5) Higher mortality rates in group S and group SS were associated with higher BNP level.(6) Group Sa and Ssa had higher mortality rate than group Sn and SSn ( Sa vs.Sn,P ＜ 0.05,Ssa vs.SSn,P ＜ 0.05).(7) EF and serum BNP level in group Sd and group SSd at admission had negative correlation ( r =- 0.603,P ＜ 0.01 ),and BNP level higher than 0.39 ng/ml within the first 72 hours after admission may be an independent prognostic factor of mortality ( sensitivity 92.9％,specificity 80.8％ ).Conclusions Plasma BNP,TnT,TnI levels have a positive correlation with cardiac function impairment. Significant elevation in BNP,TnT,and TnI levels indicates sever cardiac dysfunction and thus a worsening prognosis is expected.Serum BNP could be a useful diagnostic indicator for the septic patients with cardiac dysfunction.

BACKGROUND: The proliferation and differentiation of mesenchymal stem cells (MSCs) lack of regulatory functions. Following combining with suitable vectors, MSCs cannot highly effectively proliferate and differentiate, which are keys to prevent MSCs entering the clinic. It is of great importance to effectively regulate the differentiation of stem cells into osteoblasts using pulse electromagnetic field.OBJECTIVE: To investigate the differentiation of mouse MSCs into osteoblasts in vitro following stimulation of pulse electromagnetic field.DESIGN, TIME AND SETTING: The cytological in vitro controlled study was conducted at the Laboratory of Department of Orthopaedics, Puai Hospital of Tongji Medical College, Huazhong University of Science and Technology from May 2004 to October 2007.MATEIRALS: Totally 20 BALB/C mice were supplied by the Experimental Animal Center of Tongji Medical College. Pulse electromagnetic field deviser was designed and made by the Department of Electric Machine, Naval University of Engineering.METHODS: Mouse bilateral femur was sterilely isolated. BMSCs were harvested by the Percoll density gradient centrifugation,and purified and proliferated by the adherent method. Cells at the third passage (2×10~7/L) were incubated in a 6-well plate, and then divided into 4 groups. Cells in the blank control group were incubated in the complete medium. Cells in the pulse electromagnetic field underwent pulse electromagnetic field radiation of 50 Hz, sinusoidal wave, and 1 mT, twice a day, once 30 minutes, with an interval of 12 hours, totally 10 days. Cells in the osteogenic induction group were incubated in the complete medium, supplemented with dexamethasone, sodium glycerophosphate and VitC. Cells in the pulse electromagnetic field + osteogenic induction group were subjected to the same pulse electromagnetic field radiation and then incubated in the complete medium.MAIN OUTCOME MEASURES: The differentiation of BMSCs was measured.RESULTS: Results of alkaline phosphatase staining showed that cells were negative in the blank control group, but weakly positive in the pulse electromagnetic field group, positive in the osteogenic induction group, and strongly positive in the pulse electromagnetic field + osteogenic induction group 10 days following intervention. Compared with the blank control group,absorbance value of type I collagen immunohistochemistry was significantly greater in the osteogenic induction group, pulse electromagnetic field + osteogenic induction group (P < 0.05, P < 0.01).CONCLUSION: Pulsed electromagnetism fields of 50 Hz, waves of sine, with the intensity of 1 mT could promote alkaline phosphatase and type I collagen expression and enhance the differentiation of mouse BMSCs into osteoblasts in vitro.

BACKGROUND: Biological function of leukocyte-associated immunoglobulin like-receptor 1 (LAIR 1) has clearly researched in China and abroad, but the in vivo biological function of LAIR is poorly understood. OBJECTIVE: To establish LAIR-2 (CD306) eukaryotic expression vectors and to purify and identify the fusion protein. DESIGN, TIME AND SETTING: A single sample observation experiment was performed at the Fourth Military Medical University of Chinese PLA between June 2007 and June 2008.MATERIALS: plg/3c vector was offered by Oxford University, pcDNA3.1 vector was provided by Meyaard doctor. Chinese hamster ovary (CHO) cell lines were preserved by the Department of Immunology, Fourth Military Medical University of Chinese PLA.METHODS: Two eukaryotic expression vectors plg/3c-LAIR-2 and pcDNA3.1-LAIR-2 were constructed and were transfected into CHO cells. The binding activities of LAIR-2 fusion protein to LAIR-2 mAbs were identified by Western blot, immunocytochemistry and flow cytometry assay.MAIN OUTCOME MEASURES: The construction of stably transfected cell lines, and the purification and identification of fusion protein. The activity of LAIR 2 protein combined to corresponding monoclonal antibody.RESULTS: Eukaryotic expression vectors were constructed and trasnsfected into CHO cells successfully. Two cells lines CHO/LAIR-2-Fc and CHO/LAIR-2 that steadily expressed LAIR-2-Fc fusion protein and LAIR-2 protein were established. Western blot assay showed that LAIR-2 protein could bind specially to LAIR-2 mAb 1A7, 3H12 and 4A9. Immunocytochemistry and flow cytometry assay demonstrated that 3H12 and 4A9 could bind to LAIR-2 expressed in the transfected CHO cells. CONCLUSION: Two ceils lines CHO/LAIR-2-Fc and CHO/LAIR-2 were successfully constructed, which can transfected to CHO cells. The eukaryotic expressed LAIR-2 protein has good binding activity to LAIR-2 mAbs.

Objective To investigate the effects of hyperbaric oxygen preconditioning (HBOP) on brain edema, inflammatory reaction and neuronal cell apoptosis induced by experimental hemorrhage in rats. Method Eighteen male Spraque-Dawley rats, weighing 300 - 350 g,received five successive sessions of HBOP with 3 atmosphere absolute pressure and 100% O2 one hour daily for five successive days, and other eighteen rats received five successive sessions of pretreatment with one atmosphere absolute pressure, air, one hour daily for five successive days. Twenty-four hours after the final pre-conditioning, rats received an infusion of 100 μL autologous blood into the basal ganglion. Seventy-two hours later, rats were sacrificed for brain edema measurements in 12 rats of each group. The histopathological changes around the hematoma were observed microscopically, and the neuronal cell apoptosis was detected by using the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) in six rats of each group. Data of brain water content were analyzed by using Stata 7.0 software and statistical analysis was carried out by two-tailed Student t -test. Results Compared with the control group, HBOP significantly attenuated brain edema 72 hours after intra-cerebral hemorrhage in experimental rats (81. 6± 0. 7% vs. 82. 8± 0.9%, P < 0.01). Inflammatory cell infiltration and neuronal cell apoptosis were also significantly decreased in the HBOP group. Conclusions HBOP protects the rats against brain edema formation, and quells inflammatory reaction and neuronal cell apoptosis following intra-cerebral hemorrhage in experimental rats.