Objective To determine the functional motif of the recombinant Ricin B(rRicin B) in Vibrio vulnificus cytolysin (VVC) and understand its molecule pathogenic mechanism.Methods The motif of VVC was predicted through bioinformatics analysis and cloned into a procaryotic expression vector pET28a-rRicin B.The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG to express rRicin B.The expressed protein was further analyzed by SDS-PAGE and purified by Ni2+-NTA agarose.Renaturation of the rRicin B were also carried out for further analysis.ELISA assay and confocal microscope was applied to identify the activity of the rRicin B on human Hela cells.Results Ricin B motif located in the 336-465 amino acids of Vibrio vulnificus cytolysin with a relative molecular weight of 20×103.The result of ELISA showed that the antigenicity of rRicin B was 28.71 U/L after renaturation.FITC labeled rRicin B could bind to the cell membrane and enter the cytoplasm of human Hela cells.Conclusion The Ricin B motif in Vibrio vulnificus cytolysin bearing the similar ability with the natural Ricin B can bind to the cell membrane and enter the cytoplasm.This feature may play an important role in the activity of pore-forming and the cytotoxicity of Vibrio vulnificus cytolysin.

Aim To investigate the effects of chloride channel blockers on the apoptosis of RIN-m? cells of pancreatic islet induced by H2O2.Methods The apoptotic model was made by H2O2 exposed for six hours with a concentration of 500 ?mol?L-1.The chloride channel blockers:DIDS,NPPB and NFA were administered to pretreat the samples respectively.The cell viability,morphological changes,and apoptosis rate were observed.Results Chloride channel blockers alone have no marked effects on the cell viability of RIN-m? cell.However,they elevated the cell viability of RIN-m?cell disposed of by H2O2.Compared to H2O2 group,the groups of DIDS +H2O2,NPPB+ H2O2 and NFA+H2O2 have significant difference in cell viability and apoptosis rate(P

Objective To study the novel methods of VMAT planning based dose volume histogram ( DVH) optimization, evaluated the dosimetry and planning efficiency in VMAT planning for Esophageal Carcinoma. Methods Twelve Esophageal carcinoma patients were enrolled in this study. The conventional VMAT planning as the reference, using multi?criterion optimization DVH ( MCO?DVH ) and overlapping volume histogram prediction DVH ( OVH?DVH ) two different methods to get ideal objectives function for informing VMAT plans, Then evaluate the dosimetric, planning efficiency for all new VMAT plans. The difference between the paired t?test groups. Results The two VMAT plans based DVH objective function can meet the clinical needs. Compared with the conventional VMAT plan, Conformity index ( CI ) and Homogeneity index ( 0. 77 vs. 0. 72, P=0. 017 and 0. 10 vs. 0. 12, P=0. 047 ) is better in DVH informed plans;lung V5 and spinal cord V50 are better in MCO?DVH informed plan (54. 66 vs.60. 23,P=0. 013 and 0. 98 vs.0. 49,P=0. 037).Furthermore,the DVH informed plans had higher planning efficiency (8. 2 vs. 19. 5,P=0. 023) . Conclusions DVH Objective informed VMAT Planning can achieve clinical needs with much uniform dose to target,lower OAR dose and higher planning efficiency.

Objective To observe the protective immunity induced by the nucleic acid vaccine of 21.^7 kDa membrane protein molecule of Schistosoma japonicum Chinese mainland strain (SjC 21.^7) in BALB/c mice. . Methods. A pair of primers (P1 and P2) was synthesized according to the DNA sequence of the SjC21.^7. The ORF sequence of SjC21.^7 was amplified by PCR, and the Kozark sequence was added to the position of initiator. The gene fragment was inserted into the eukaryotic expression plasmid pcDNA3.^1 to form the recombinant plasmid SjC21.^7-pcDNA3.^1. Forty-eight BALB/c mice were divided into three groups: control, test and boost. Each mouse was injected in quadriceps femoris with plasmid pcDNA3.^1 (control) or recombinant plasmid SjC21.^7-pcDNA3.^1 (test, boost); for the boost group, with additional P35-pcDNA3.^1 and P40-pcDNA3.^1. All mice were immunized three times with an interval of 2 weeks, challenged each with 45 cercariae of S.^japonicum at the 30th day after final immunization. At day 45 after challenge,all mice were sacrificed, the numbers of worms and hepatic eggs were counted. Antibody level in the sera of mice before and two weeks after immunization was determined with ELISA. The expression of the target gene in quadriceps femoris was observed with immunohistochemistry. . Results . The immunohistochemistry analysis showed that there were specific antigens expressed in the local tissue of the test group mice. There was specific IgG in the serum of partial mice in test and boost groups. Compared with the control group, the worm reduction rate was 29.^9% and its egg reduction rate 13.^8% in the test group; 31.^9% and 28.^0% respectively in the boost group. The egg reduction rate in the boost group was higher than that of the test group (P

Objective:To investigate the sensitivity and the specificity of scorpions amplification refractory mutation system ( ARMS) in comparing with that of direct DNA sequencing in the detection of BRAF gene mutations in patients with papillary thyroid microcarcinoma.Methods:Direct sequencing and ARMS were used simultaneously to detect BRAF mutation status in 56 patients with PTMC.Results:BRAF mutations were identified in 46 cases with a mutation rate of 82.9%by ARMS,while in 18 cases with a mutation rate of 32.1%by direct sequencing.Besides,the sensitivity of ARMS was 100%and that of direct sequencing was 39.1%.There were significant differences of both mutation rate and sensitivity between two methods ( P<0.01 ).Conclusion: Compared to direct sequencing,ARMS gains a higher sensitivity in the detection of BRAF mutations in samples with tiny lesions.

0.05). However, in the presence of 16.5 mmol/L glucose, PGF_ 2? increased significantly in insulin secretion (P

Objective To investigate the activity of recombinant Vibrio vulnificus hemolysin (rVvhA) on the apoptosis of J774A.1 cells and the related mechanism. Methods The cytotoxic effect of rVvhA on the growth of J774A.1 cells was identified by MTT, celluar and mitochondrial morphology were observed by transmission electron microscopy, apoptosis or necrosis and mitochondrial membrane potential in J774A.1 cells were measured by flow cytometry, activities of caspase-3 ,-8,-9 were detected by spectrophotometry. Results The viability of J774A.1 cells exposed to rVvhA was inhibited, and it is dependent on dose. Celluar and mitochondrial uhrastructure both occurred to change obviously observed by transmission electron microscopy in J774A.1 treated by 2.0 HU/ml and 3.0 HU/ml rVvhA after 8 hours; and 3.0 HU/ml rVvhA group had a better cytotoxic effect on J774A.1 than that of 3.0 HU/ml rVvhA group. The percentage of apoptosis is (7.80±0.62)%, (12.33±0.12)%, respectively. Besides, the mitochondriai membrane potential also reduced, because the rate of fluorescence which is green increase 1.0% (normal) to 9.8% (2.0 HU/ml rVvhA) and 39.2% (3.0 HU/ml rVvhA). At the same time, the caspase-3, -9 activity increased gradually, but caspase-8 remained unchanging. In J774A.1 cells treated by 3.0 HU/ml rV-vhA + caspase-3 inhibitor(Ac-DEVD-FMK) or caspase-9 inhibitor(Ac-LEHD-FMK), The apoptosis of was reduced to(6.23±3.95)% ,(9.60±3.14)%, and the activity of caspase-3, -9 reduced, too. Conclusion The rVvhA has cytotoxic effect on J774A.1. Mitochondria-mediated apoptosis pathway which is dependent on caspase may be related to apoptosis induced by rVvhA in J774A.1.

Objective To compare the errors of double-center and single-center setup, and to study the role of both on reducing the rotational setup errors for the patients with esophageal carcinoma depend on rigid registration errors between online kV-cone-beam computed tomography (kV-CBCT) images and plans for CT images. Methods 20 patients with middle esophageal carcinoma received image scanning before treatment every week by using double-center setup and CBCT, and single-center setup images of 20 patients were taken from the X volume image (XVI) system. Then the images of both setup types, registration errors of CT image and rotational setup errors were compared respectively. Every patient received kV-CBCT scanning analysis before treatment every week, and 6 times in total. 240 group of kV-CBCT images from all of the patients were off-line matched with plans for CT images to calculate the errors of X-axis, Y-axis, Z-axis. Then the data of linear errors and rotational setup errors from patients were collected, aiming at putting the error data into the patients treatment program and analyzing the significances. Results The standard registration of double-center setup was as follows: T (X) (0.28 ±0.19) cm, T (Y) (0.27 ±0.19) cm, T (Z) (0.33 ±0.12) cm, R (X) (0.40 ±0.19)° , R (Y) (0.30 ±0.18)° , R (Z) (0.30 ±0.19)° . The standard registration of single-center setup was as follows:T(X) (0.32±0.20) cm, T(Y) (0.29±0.25)cm, T(Z) (0.31±0.16) cm, R(X) (2.2±0.68)°, R(Y) (0.5±0.32)°, R(Z) (2.10±0.60)°. There were statistical differences between linear errors in T(X) and rotational setup errors in R(X), R(Y) or R(Z) (P< 0.05). Conclusion Double-center position can reduce the rotational setup errors, especially in X-axis, Y-axis errors, and may provide more help for the radiation oncology departments without on-board CBCT.

Objective To clone and express a high mobility group box 1(HMGB1)protein of Schistosoma japonicum(Main-land strain)and analyze its function. Methods The DNA fragment of open reading frame encoding Sj HMGB1 protein was ampli-fied by RT-PCR from the mRNA of S. japonicum worms,then it was subcloned into the expression vector pET28a(+)to form the recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21(DE3),and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombi-nant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding ca- pacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT-PCR. Female ICR mice were immunized with the recombinant SjHMGB1 pro-tein and infected with 45±2 cercariae of S. japonicum after three immunizations. Forty-two days post-infection,the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue,respectively. The worm and egg reduction rates were calculat-ed respectively. Results A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT-PCR,which was the open reading frame(ORF)encoding SjHMGB1protein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1-pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a(+). The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG, and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA,and the recombinant protein immu-nized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum-infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT-PCR and Western blot results showed that SjHMGB1 was abun-dantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effec-tive immune protection against S. japonicum. Conclusion The gene encoding HMGB1 from S. japonicum and the soluble recombi-nant SjHMGB1 protein with natural functional activity are obtained,and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.

OBJECTIVETo observe the effect of Huzhang on the progress of pulmonary fibrosis in rats, evaluate the role of Huzhang in this process and explore its mechanism.

METHODWistar male rats were randomized into 7 groups (normal control group, model group, positive control group, prophylactic group, 3rd day treatment group, 7th day treatment group and 14th day treatment group). Bleomycin was administered by intratracheal injection to produce pulmonary fibrosis groups except the normal control group. The positive control group began to be given DXM (4 mL x kg(-1) x d(-1)) on the day of the model-making. The normal control group and model group were given NS (4 mL x kg(-1) x d(-1)) on the day of the model-making. The prophylactic group was given reagent (4 mL x kg(-1) x d(-1)) 2 days ahead of the model-making, whereas the 3rd day treatment group, the 7th day treatment group and the 14th day treatment group given the same dose respectively on the third day, the seventh day and the fourth day behind of the model-making. Lung tissue was stained with hematoxylin-eosin (HE) and Masson's trichrome to determine the pathological grading. The lung fibroblast (LF) was cultured in vitro by way of pancreatic enzyme digestion, which was used to detect the contents of the expression of MMP-2 and TIMP-1mRNA with RT-PCR method.

RESULTCompared with those in the model group, the alveolitis, pulmonary fibrosis and collagen accumulation were significantly alleviated in the positive control group, Huzhang prophylactic group and each treatment groups. In the positive control group, Huzhang prophylactic group, the 3rd day treatment group, the 7th day treatment group and the 14th day treatment group, the expression of MMP-2 mRNA was weaker significantly than that in the BLM model group (P < 0.05 or P < 0.01) except that on the 42nd day. The expression of TIMP-1mRNA was also weaker significantly than that in the BLM model group at all set times in all treatment groups (P < 0.05 or P < 0.01). The inhibition of TIMP-1 lasted until the 42nd day.

CONCLUSIONHuzhang inhibited the expression of MMP-2mRNA and TIMP-1mRNA of lung fibroblast in different periods to reduce the alveolitis and pulmonary fibrosis, which was probably one of the anti-fulmonary fiborsis mechanisms of Huzhang.

Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; therapeutic use ; Fibroblasts ; drug effects ; metabolism ; Humans ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Pulmonary Fibrosis ; drug therapy ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism