Objective To establish a hydrogen peroxide-impaired model of rat cerebral microvascular endothelial cells in vitro, and observe the protective effect of serum containing Naoyian. Method Rat cerebral microvascular endothelial cells were treated with six concentrations of hydrogen peroxide at four time points. The optimum injury condition of hydrogen peroxide was determined by MTT chromatometry. Then the cultured cells pretreated with serum containing Naoyian and pyrrolidine dithiocarbamate(PDTC) were interfered with hydrogen peroxide in suitable concentration and time, and OD of the treated cells was measured by MTT chromatometry. Results The cultured cells were injured obviously by hydrogen peroxide in 0 125mmol/L for 30min. The OD of the cells pretreated with 5% serum containing Naoyian and 50?mol/L PDTC were significantly higher than that of the cells without pretreatment (P

objective To determine the existence of virulence-associated invA gene in different genospeeies of Leptospira interrogans reference strains in China.and to understand the alterations of invA gene transcription and expression of L.interrogans strain Lai before or after infecting cells.Methods PCR was applied to detect the invA gene of four L.interrogans strains belonging to four different genospecies and L.biflexa strain Patoc Ⅰ.The entire invA genes from the L.interrogans strains were cloned and then sequenced.The prokaryotic expression system of invA gene of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was constructed.Using Ni-NTA affinity chromatography,the target recombinant protein rInvA was extracted and purified.Rabbits were immunized with rInvA to obtain antiserum and the titer of antiserum was determined by immunodiffusion test.A model of L interrogans strain Lai infecting human embryo kidney cell line HEK293 was established to detect the alterations of invA gene transcription and expression of the leptospiral strain before or after infecting the host cells by real-time fluorescent quantitative RTPCR and western blot assay.Results All the four tested L.interrogans strains had invA gene whereas L.biflexa strain Patoc Ⅰ not.The similarity of nucleotide and putative amino acid sequences of invA genes from the four L.interrogans strains belonging four different genospecies were 99.33%-100%and 98.66%-100%,respectively.The constructed prokaryotic expression system could efficiently express rInvA and the immunodiffusion titer of rabbit anti-rInvA serum was 1:16.After L.interrogans strain Lai infecting HEK293 cells for 30 min or above,the microbe could adhere the surface of the cells.On the 30 min after the infection,the mRNA level of invA gene of L.interrogans strain Lai was remarkably upregulated,and on the 45 min after infection the mRNA level presented a peak value and then graduated decreased.On the 45 min or 60 min after L.interrogans strain Lai infecting HEK293 cells,InvA protein could be detectable but before infection or after infection for over 90 min the InvA protein expression was negative.Conclusion The invA gene is a unique gene for pathogenic L.interrogans.The invA gene of L interrogans has characteristics of cell-touched expression and transient expression,which may have a close correlation with adhering and invading host cells.

Objective To investigate the expression of β-catenin and Oct-4 in colonal carcinoma and explore the relationship with recurrence and metastasis after operation. MethodsImmunohistochemical analysis was used to evaluate the expression of β-catenin and Oct-4.The correlation of β-catenin and Oct-4 expression with tumor cell differentiation,T stage,N stage and metastasis was analyzed.The gene expression of Oct-4 was examined by RT-PCR in 20 frozen tumor tissues and normal tissues adjacent to tumor.Results Thirty-five patients had metastasis. The positive rates of β-catenin and Oct-4 expression were significantly higher in metastasis group than in the non-metastasis group (65.71％ vs 31.11％,51.43 ％vs 13.33 ％,x2 =9.843,P =0.002,x2 =13.605,P =0.001).Expression of β-catenin and Oct-4 was not associated with differentiation,T stage or N stage.The positive expression rate of Oct-4 in colonal carcinoma tissues was significantly higher than that in normal tissues.Metastatic rates in patients with positive expression of β-catenin and Oct-4 was higher than that in negative expression.The survival analysis showed that time of metastasis was significantly different in two groups of patients (P ＜0.05).Conclusion The expression of β-catenin and Oct-4 in tumor tissues is related to metastasis of colonal cancer after surgery and might be used to predict metastasis of colonal cancer after operation.

Objective To investigate the evaluation method of processing pieces' colour and quantificationally analyse the relactionship between the pieces' colour and the internal quality. Methods Taking different processing techniques on Rhizoma Alismatis as research objects,using a camera and Adobe Photoshop software to acquire the elementary information of processing pieces' colour and determine the contents of the active principle in Rhizoma Alismatis by HPLC. The active principle in Rhizoma Alismatis and the colour of processing pieces are analyzed quantificationally by SPSS 13.0 statistical software and data mining software Clementine 8.0. Results The method for acquiring and handling colour information of processing pieces in Rhizoma Alismatis could be used to quantificationally analyze the correlation of the colour of processing pieces. There is a significant correlation between processing pieces' colour difference and the internal quality. Conclusion The colour difference of processing pieces can be one of target for the quality assessment. The method can be popularized to other precessing pieces control of techniques.

Objective To investigate the effect of via-anal ileus tube used as a drainage in left-sided acute malignant colonic obstruction.Methods Forty-seven cases of left-sided acute malignant colonic obstruction were divided into control group (25 cases) and tube group (22 cases).The patients in control group received intraoperative colonic lavage for one-stage surgery.The patients in tube group received via-anal ileus tube for decompression before the surgery.Compared the difference of bowel movement recovery time,exhaust time,fasting time,postoperative hospitalization time,hospitalization costs and complication rate between two groups.Results The technically successful rate in tube group was 90.9％ (20/22),and one-stage surgery were performed.No anastomotic leakage or postoperative stenosis occunred after operation.There were 23 cases in control group who performed coloclysis in operation and one-stage surgery.The bowel movement recovery time,exhaust time,fasting time,postoperative hospitalization time and hospitalization costs in tube group were significantly lower than those in control group [(12.78 ± 2.07) h vs.(18.01 ±3.42) h,(78.76 ± 11.43) h vs.(96.38 ±13.09) h,(3.18 ±1.76) d vs.(5.51 ±2.95) d,(10.23 ± 2.33) d vs.(15.86 ± 6.74) d,(25 437.43 ± 2 343.67) Yuan vs.(31 051.32 ± 2 542.73) Yuan](P ＜ 0.01 or ＜ 0.05).After operation,there were 2 cases of stomas fistula and 2 cases of peritoneal cavity infection in control group,and none of them in tube group,the complication rate in control group was significantly higher than that in tube group [17.4％ (4/23) vs.0,P ＜ 0.05].Conclusions The via-anal ileus tube used as a drainage in left-sided acute malignant colonic obstruction is effective and safe,and can improve the rate of one-stage anastomoses,decrease the complication rate,promote the promote.It has important clinical value.

Objective To study the material basis of hot-natured herb Euodiae Fructus. Methods Euodiae Fructus extract was separated into the chloroform fraction, ethyl acetate fraction, n-butanol fraction and water fraction. With cold syndrome rat model by reserpine, the study confirmed that n-butanol fraction of Euodiae Fructus has influence on energy metabolism in rats. With the modified magnetic polymer microspheres, Euodiae Fructus n-butanol was divided into alkaloids, phenolic acid, fat soluble and water-soluble components, and the component of Euodiae Fructus influencing energy metabolism of model rats was further confirmed.Results The Euodiae Fructus extracts and alkaloids elevated rats temperature, reduced the Na+-K+-ATP activity, increased SDH activity, the content of ATP in liver tissue, the energy charge (EC) value and body heat production, and improve the level of energy metabolism.Conclusion The alkaloid is the material basis of hot-natured herb Euodiae Fructus.

Objective To approach a simple identification method for clinical Burkholderia cepacia isolates.Methods Thirty eight clinical isolates and 5 referenc strains were identified by phenotypic and genotypic methods. A simple method presented here included TSI agar, oxidase test, pigmentation test, catalase test and antibiograms.Results All but one B. cepacia isolate identified by phenotypic and genotypic methods were identified correctly by our method. One non B. Cepacia isolate identified by the genotypic method was identified as Burkholderia spp. by phenotyic and our methods.Conculsion The method we presented here was simple, practical for identification of clinical B. cepacia isolates.

Objective To screen the efficient antigenic epitopes in genus-specific envelope proteins OmpL1 and LipL21 of Leptospira interrogans for further development of multiple antigenic peptide (MAP)vaccine.Methods Based on bioinformatic technique,the combined epitopes of T and B lymphcytes in OmpL1 and LipL21 molecules were screened.Nucleotide fragments of each epitopes were amplified by PCR and then constructed their phage display systems.Using antisera against rOmpL1,rLipL21,L.interrogans serogroup Icterohaemorrhagiae strain Lai and leptospirosis patients' sera as the first antibodies.respectively,Western blot assays were performed to determine the immunoreaetivity and reactive ability of the epitopes with different antisera.Resuits Four combined epitopes of OmpL1 and two combined epitopes of LipL21 were selected out by the predicting procedure.All the amplified epitope fragments were accurately inserted into the region at N end of phage PⅢ protein and successfully expressed.All of the antisera could recognize each of the epitopes.Based on the results of Western blot,the two LipL21 epitopes at 97-112 and 176-184 showed similar strong hybridization signals with any of the antisera,and the hybridization signals of four OmpL1 epitopes with the three antisera were 173-191,87-98,297-320 and 59-78,from strong to weak.Conclusion The six combined epitopes in this study are efficiently antigenic.And the epitopes at positions 97-112 and 176-184 in LipL21 as well as the epitopes at position 87-98 and 173-191 in OmpL1 have a potential for developing leptospiral MAP vaccine.

Objective To observe the expression of mRNA of podocin,nephrin,CD2AP and α - actin - 4 in Doxorubicin - induced nephrotic(ADN)rats,and explore the possible mechanisms of podocyte molecule during the de-velopment of proteinuria. Methods Forty - eight Sprague - Dawley(SD)rats were divided into ADN model group(in-jected with 6. 5 mg/ kg Doxorubicin in tail vein,n = 24)and control group(injected with saline solution in tail vein,n =24). After the nephropathy model was established,6 rats were killed at the end of 1st ,2nd ,4th ,6th week in each group. The changes of the following indicators were observed:(1)24 - hour urinary protein,serum albumin and cholesterol were detected;(2)mRNA expression of nephrin,podocin,CD2AP and α - actin - 4 in cortex of kidney were examined by real time fluorescence quantification PCR. Results The model group came out massive proteinuria(15. 66 ± 1. 50) mg/ 24 h,(45. 98 ± 1. 45)mg/ 24 h,(65. 58 ± 4. 68)mg/ 24 h,(82. 83 ± 8. 43)mg/ 24 h in 1,2,4,6 weeks respec-tively,hypoalbuminemia(27. 4 ±2. 5)g/ L,(23. 6 ±2. 9)g/ L,(20. 6 ±1. 5)g/ L,(6. 9 ± 2. 3)g/ L in 1,2,4,6 weeks respectively and hypercholesterolaemia(2. 00 ± 0. 25)mmol/ L,(2. 16 ± 0. 44)mmol/ L,(4. 02 ± 0. 81)mmol/ L, (7. 54 ± 1. 12)mmol/ L in 1,2,4,6 weeks respectively,and the differences of proteinuria,plasma albumin and total cholesterol compared with control group at each time point had statistical significance(all P ﹤ 0. 01). Compared with the control group,podocin mRNA expression in the model group decreased at the end of 1st week(10. 56 ± 3. 62),de-creased significantly at the end of 2nd week(20. 44 ± 9. 03),and decreased at the end of 4th week(2. 19 ± 0. 18)com-pared with the control group;nephrin mRNA expression decreased at the end of 1st week(2. 41 ± 1. 10)and reached to the peak value,decreased at the end of 4th week(0. 52 ± 0. 18);CD2AP mRNA expression did not change significantly in the 1st week(4. 17 ± 0. 79),increased at the end of 2nd week(6. 74 ± 1. 53),reached to the peak value at the end of 4th week(6. 91 ± 1. 13),but did not change significantly at the end of 6th week(4. 04 ± 0. 82);α - actin - 4 mRNA ex-pression did not change significantly at the end of 1st week(1. 75 ± 0. 48),decreased at the end of 2nd week(2. 01 ± 0. 55),reached to the peak value at the end of 4th week(2. 24 ± 0. 81),but did not change significantly at the end of 6th week(1. 39 ± 0. 18). Compared with the control group,the difference had statistical significance( all P ﹤ 0. 05). Conclusion The abnormal expression of podocyte molecules mRNA in ADN rats may be an important molecular mechanism in the development of proteinuria.

Objective To evaluate the effect of Shenfu Decoction pretreatment on the function of intestinal mucosal barrier of endotoxemic rats.Methods Twenty-four adult male Sprague-Dawley rats,aged 4-6 months,weighing 180-220 g,were randomly divided into 3 groups (n =8 each):blank control group (group C),endotoxemia group (group E) and Shenfu Decoction pretreatment group (group S).Endotoxemia was induced by lipopolysaccharide 10 mg/kg injected intravenously in anesthetized rats in groups E and S.Normal saline and Shenfu Decoction 5 ml/kg were injected intraperitoneally at 30 min before the model of endotoxemia was established in groups E and S,respectively.At 2 h after the model of endotoxemia was estabhshed,blood samples were obtained from the abdominal aorta for measurement of the plasma concentrations of tumor necrosis factor-α (TNF-α),interleukin6 (IL-6),intestinal fatty acid binding protein (i-FABP) and diamine oxidase (DAO) by ELISA.The distal ileum was harvested for detection of the expression of tight junctional protein Zonula Occludens-1 (ZO-1) in the intestinal mucosa of epithelial cells by immunohistochemistry.The changes in pathological morphology of the intestinal mucosa were examined after one segment of the distal ileum was stained with haematoxylin and eosin.The pathological changes were assessed and graded according to Chiu's score.Results Compared withgroup C,the plasma TNF-α,IL-6,i-FABP and D-lac concentrations were significantly increased,Chiu's scores were increased,and the expression of ZO-1 was down-regulated in groups E and S (P ＜ 0.05).Compared with group E,the plasma TNF-α and IL-6 concentrations were significantly decreased,the plasma i-FABP and D-lac concentrations were decreased,Chiu's scores were decreased,and the expression of ZO-1 was up-regulated in group S (P ＜ 0.05).Conclusion Shenfu Decoction pretreatment can attenuate the damage to the intestinal mucosa and protect the function of intestinal mucosal barrier in endotoxemic rats and inhibition of systemic inflammatory responses is involved in the mechanism.