Objective:To construct the prokaryotic expression vector pET28a(+)-comE, to get the highest expression of comE in E. coli BL21(DE3), and to obtain the purified ComE fusion protein. Methods: ComE gene was amplified by PCR with specific primers from genome of Streptococcus mutans UA159. After digested by two restriction endonucleases BamH Ⅰand Xho Ⅰ, the gene segment was inserted into vector pET28a(+). Then the recombinant vector was transformed into E. coli BL21(DE3). The protein expression was induced by IPTG and the result was confirmed by Western blot. The solubility of the fusion protein was examined by 12% SDS-PAGE, and the expression conditions were optimized. Finally the fusion protein was purified by a two-step purification procedure utilizing Ni2+ chelating column and S75 size exclusion column. Results: The recombinant plasmid was confirmed by PCR, restriction endonucleases digestion and sequence analysis, which showed that the inserted segment was identical to comE gene reported by GenBank without nucleotide mutation. The fusion protein which was confirmed by Western blot can be expressed in E. coli BL21(DE3) and existed both in supernatant and precipitation. The maximum expression product amount was obtained when the A600 value of E. coli BL21(DE3) was 0.4, IPTG concentration was 0.10 mmol/L and induction time was 4 h. Conclusion: The recombinant plasmid is constructed and the ComE fusion protein is purified by the optimum conditions.

To investigate the seropharmacological action of Buyang Huanwu Decoction (BHD) on hypoxia induced apoptosis of cultured cortical neurons. Models of hypoxia induced neuron apoptosis were established by Daniel method. Neuron apoptotic rate was examined by propidium iodide staining method and flow cytometry, nitric oxide (NO) and malonyl dialdehyde (MDA) contents by spectrophotometer and bcl 2 gene expression by immunohistochemical method. Effects of BHD on neuron apoptotic rate, NO and MDA contents and bcl 2 gene expression were observed in normal group (Group A), model group (Group B), normal saline group (Group C) and high dosage, moderate dosage and low dosage BHD groups (Group D, Group E and Group F). BHD could inhibit the apoptosis of cultured cortical neurons, reduce the production of NO and MDA and promote the expression of bcl 2 gene.[Conclusion]BHD has an inhibitory effect on hypoxia induced apoptosis of cultured cortical neurons and its mechanism may be concerned with the reduction of NO and MDA production and the increase of bcl 2 gene expression.

Objective To analyze the diagnosis of variant acute myocardial infarction.Methods Clinical datas were retrospectively analyzed in 27 cases with variant acute myocardial infarction.Results 27 patients were diagnosed with myodial infarction by ECG and myocardial enzymea.Conclusion Variant acute myocardial infarction is often misdiagnosed as acute abdomen,cerebral aarterial infarction the disease of neck.but its clinical manifestation especially ECG and myocardial enzymes are changeable,and it needs full analysis,early diagnosis and therapy.

Objective:To determine the level of FIB,IL-6,D-dimer and explore the differential diagnostic value and clinical significance of the tumor markers in patients with ovarian cancers.Methods: The level in serum of 65 ovarian cancer and 35 benign tumor of ovarian and 30 controls FIB and D-dimer were measured by latex agglutination assay.The IL-6 levels were measured by ELISA method.Results:Compared with the normal controls and benign tumor of ovarian cancer, there were some significantly changes in ovarian cancer patients including FIB,IL-6,D-dimer increased (P<0.05),there was no significant differences in FIB,D-dimer levels between ovarian benign tumors and normal controls(P>0.05).Companied with TNM stage increased,the levels of FIB,IL-6,D-dimer ovarian cancer groups increased.The correlation analysis showed that FIB was positively correlated with D-dimer and IL-6 (r=0.56,r=0.31).Conclusion:The level of serum FIB,D-dimer,IL-6 detection in patients with ovarian cancer are helpful in early diagnosis, dicision of clinical stages and regimen prognosis.

ObjectiveTo elucidate the association between aquaporins (AQPs) expression in kidney tissue and edema of nephrotic syndrome(NS) patients.MethodsNS patients were divided into edema group (14 cases) and non-edema group (8 cases).Ten patients without NS were used as control group.Expressions of AQP1,AQP2,and AQP4 in renal tissues of 3 groups were detectedbyimmunohistochemistrywithstandardtechniquesandsemi-quantitativeanalysis.Association between AQPs expression and edema was examined.ResultsThe positive index of AQP1 expression in proximal tubules in edema group was 0.0373±0.0110,which was significantly lower as compared to non-edema group (0.0510±0.0120) and control group 0.0574±0.0100),while the difference between non-edema and control groups was not significant.The positive index of AQP1 expression in glomerulus was 0.0106±0.0037 in edema group,which was significantly higher than that in non-edema group(0.0021±0.0013) and control group(0.0020±0.0012),while no significant difference was found between the last two groups.AQP2 mainly localized in the collecting duct system.The positive indexes of AQP2 expression were 0.0498±0.0081,0.0370± 0.0072 and 0.0255±0.0103 in edema group,non-edema group and control group,respectively.The differences were significant among 3 groups.AQP4 expression was not found in the renal cortex and collecting duct system.ConclusionsAQPs expression is different in renal tissues of NS patients.AQP2 may play an important role in the edema of NS patients,and AQP1 may involve in the occurrence of edema.

Objective:To optimize the formula of 5-aminosalicylic acid enema in situ gel. Methods:5-Aminosalicylic acid ene-mas in situ gel was prepared using a cold dissolution method with carbomer as the gel matrix and xanthan gum as the thickener. A 32 full factorial design was used to investigate the effects of the concentrations of carbomer and xanthan gum on the viscosity before and af-ter the gelling, duration of inversion tube and sedimentation rate. Response surface methodology was used to optimize the formula. Re-sults:The quantitative relationships between the two factors and the four evaluation indices were obtained. The optimum formula was as follows:the concentration of carbopol and xanthan gum in the enema was 0. 7% and 0. 15%, respectively. The viscosity before and af-ter the gelling was 500-1 000 mPa·s and 2 200-2 700 mPa·s, respectively. The duration of inversion tube test was 40-80 min and the sedimentation rate was more than 98. 5%. Conclusion:The multi-objective simultaneous optimization of the formula of 5-aminosal-icylic acid enema in situ gel is accomplished by factorial design and response surface methodology.

Objective To discuss the preventive measures of anterior palatal fistula by modified the operation of cleft palate. Methods For 23 patients of complete cleft palate with alveolar ridge cleft, bilateral mucoperiosteum flap was moved forward as possible so as to close fistula front of hard palate, and incised junction of the hard and soft palate to prolong soft palate and the wound was repaired by buccal mucosal flap.The incidence of anterior palatal fistula and velopharyngeal closure after operation was observed. Results Twenty-three patients were rechecked 1 month after operation ,there was no anterior palatal fistula occurring,10 cases were examined by epipharyngoscope 1 year after operation,the velopharyngeal closure was 90%-100%. Six cases were followed up for 6 months,the velopharyngeal closure was 80%-85%. Conclusions For second-stage operation methods of anterior palatal fistula, there are too many discussions of selection criterias, advantages and disadvantages. If first-stage operation is taken measures to prevent anterior palatal fistula or decrease the diameter of fistula as possible. It reduces percentage of second-stage operation or decreases the difficulty. It should get more attention in the clinical works.

The visual acuity testa were performed in 8219 children(16 438 eyes)under 5 years of age with sound and light toys,electric torch and a retinoscope.The general ocular examination and mydriatic optometry were also performed for the ametropia.In 8219 children 61 were found to have dominant strabismus,including 59 cases of concomitant strabismus and 2 paralytic strabismus.AbnormaJ refraction was detected in 404 children(739 eye)accounting for 4.92%,including 68 cases of anisometropia (0.83%).In 16 438 eyes 621(3.78%)were moderate or hyper-presbyopia;53(0.32%)were myopia;65 (0.40%)were mixed astigmatism.The vision acuity test described above is a simple and convenient method suitable for children under 5 who are not able to read the conventional test chart.

Objective To assess the effects of radiofrequency ablation (RFA) and bipolar cord coagulation (BCC) on the prognosis and complication rate of complicated monochorionic twins.Methods A retrospective review was undertaken in 58 cases of complicated monochorionic twins treated with RFA or BCC at the First Affiliated Hospital of Sun Yat Sen University,from January 2008 to August 2013.Non-parametric Wilcoxon test,Chi-square test,Fisher exact test or multi-variant Logistic regression analysis were used for statistical analysis.Results Indications for selective termination in the 58 cases were:twin reversed arterial perfusion sequence in 12,severe twin to twin transfusion syndrome in 28,discordance of fetal anomalies in 10,selective intrauterine growth restriction in 7 and twin anemia-polycythemia sequence in 1.Forty-three cases were managed with BCC and 15 with RFA.Preterm labor was more common in the BCC group than in the RFA group [86.0％(37/43) vs 9/15,respectively; x2=4.598,P=0.032).Premature rupture of the membranes occurred in 48.8％(27/43) of the BCC group vs.4/15 of the RFA group (x2=2.229,P=0.135).The median procedure-todelivery time was 48 (1-150) days for the BCC group vs.101(14-138) days for the RFA group (Z=-2.245,P=0.025).Overall survival rate was 62.8％(27/43) in the BCC group vs 11/15 in the RFA group (x2=0.547,P=0.460),which was not significantly different.Neurodevelopmental delay was detected in two neonates in BCC group and in one neonate in RFA group.Logistic regression analysis showed that delivery before 28 gestational weeks was an independent risk factor for the poor prognosis of the co-twins (OR=192.720,95％CI:18.610-994.000,P ＜ 0.01).Conclusion Compared with BCC,RFA does not improve the prognosis of complicated monochorionic twins significantly.

Objective:To summarize experience of 8 lower limb deep venous thrombosis(DVT) patients who used OptEase recyclable vesa cava filter(VCF) during in operations.Methods:Through collecting the clinical data of 8 patients who used OptEase recyclable vena cava filter(VCF) ,analyzing the method of recycling and releted complications.Results:No pulmonary embolism happened during and after operations,when recycle VCF implanted,vena cava graphy shows:inperfection thrombous near the filter in 1 case and vena iliaca thromb of uninjure side in 1 case too,reclaiming successfully in 5 cases,3 cases turn to permanent placement.FolIowing-up observation for 3-8 months,recurrence of limbs vena thrombosis never happened.Conclusion:The recyclable vena cava lilter could prevent plamonary embolism effectively in lower limb deep venous thrombosis patients.