[Abstract ] Objective High-mobility group box-1 (HMGB1) is abundantly released in the epileptogenic brain tissue , but few reports are seen about the effect of HMGB 1 on the expression of P-glycoprotein ( P-gp) in the vascular endothelial cells of the epi-leptogenic tissue .This study is to explore whether HMGB 1 can regulate P-gp expression in the brain microvascular endothelial cells of the mouse in vitro . Methods Immortalized brain microvascular endothelial bEnd .3 cells of the mouse were cultured in vitro and al-located to different concentration groups ( treated with culture medium containing 10 , 100 , 500 , and 1000 ng/mL HMGB1 for 8 hours), treatment duration groups (treated with culture medium containing 100 ng/mL HMGB1 for 4, 8, 16, 24, and 32 hours), and a control group ( treated with culture medium without HMGB 1 ) .The mRNA expression of P-gp-encoding gene-multidrug resistance gene 1a (mdr1a) was detected by real-time qPCR, and its protein expression determined by Western blot and immunocytochemistry . Results The results of qPCR manifested that the expressions of mdr 1a mRNA were 1.646 ±0.176, 1.777 ±0.135, 1.617 ±0.043, and 1.398 ±0.182 in the 10, 100, 500, and 1000 ng/mL HMGB1 groups, respectively, significantly higher than 1.030 ±0.284 in the control group (P<0.05), and so were those in the 4, 8, 16, 24 h, and 32 h groups (2.655 ±0.112, 2.168 ±0.212, 1.823 ± 0.232, 1.418 ±0.376, and 1.445 ±0.123) than in the control (1.010 ±0.164) (P <0.05).Western blot showed a significant increase in the P-gp protein expression in all the concentration groups (P<0.05) as well as in the 8 h and 16 h treatment duration groups as compared with the control group (P<0.05).Immunocytochemis-try also revealed a higher P-gp expression in the HMGB1-treated than in the control cells (P<0.01). Conclusion HMGB1 can upregu-late the expressions of mdr1a mRNA and P-gp protein in the brain microvascular endothelial cells of the mouse , which may associated with drug resistance of central nervous system diseases , especially that of epilepsy .

Objective To investigate risk factors related to postoperative death of patients with rectosigmoid junction tumor perforation.Methods The clinical data of 76 cases with rectosigmoid junction tumor perforation confirmed by laparotomy from January 2000 to October 2015 were collected.Results Of the 76 cases,17 patients died postoperatively,the mortality rate was 22％,the single factor analysis showed that age(x2 =4.649,P =0.031),duration of abdominal pain(x2 =8.218,P =0.016),severe heart and lung diseases(x2 =11.996,P =0.007),circulatory and renal function(x2 =10.360,P =0.016),serum albumin(x2 =7.252,P =0.027),white blood cell count(x2 =7.633,P =0.022),Perforation diameter (x2 =9.770,P =0.008),Geroge grade of intraperitoneal contamination (x2 =10.086,P =0.006) were related to postoperative death (P ＜ 0.05).Multivariate analysis showed that complicating severe heart and lung diseases,preexisted circulatory and renal dysfunction,white blood cell count ＜ 4 × 109/L,size ＞ 3 cm,intraperitoneal contamination larger than one quadrant were independent risk factors for postoperative death.Conclusion Risk factors related to postoperative death of rectosigmoid junction tumor perforation were preoperative important organ dysfunction and intraperitoneal infection.

Objective:Expression of protein TSLP and selection of full human anti-TSLP single chain Fv ( scFv).Methods:The cDNA of TSLP was amplified.The amplified target gene and the expression vector pET 101/D-TOPO were ligated , and then transformed into E.coli BL21.The protein was induced to expression by IPTG and purified and identified.The biotinylated TSLP protein was used as antigen to select of human TSLP scFv from a constructed human scFv library by phage display .Results: The size of amplified cDNA of TSLP was about 423 bp,and that of expressed protein was about 26 kD.Dot blot and Western blot results showed that the expressed protein was correct.The constructed human scFv library was enriched for three rounds using biotinylated TSLP as antigen by phage display.ELISA results showed that 35% scFvs had binding activity with TSLP.The scFvs with good binding activity were selected and identified by Western blot and sequencing.Conclusion: The full human scFvs against for TSLP were selected suc-cessfully.

Purpose:To evaluate the effect of oxaliplation (L-OHP)+flurouracil/calcium folinate(5-FU/CF) in combination with concurrent radiotherapy for advanced rectal cancer and to find a more effective way of giving them.Meth- ods:Chemotherapy:L-OHP 130 mg/m~2,iv infusion for 2 hours D_1 combined with calcium folinate 200 mg/m~2 for 2 h ours followed by 5-fluorouracil 300 mg/m~2 for 4h D_1～D_5,repeated every 3 weeks.Radiotherapy:The whole pelvic irradiation DT 50～60Gy/25～30/F(5～6 weeks).Results:30 advanced rectal cancer patients entered the study.One patient reached complete response(CR),15 partial response,13 stable disease,and 1 with progression after treatment,The overall re- sponse rate(CR+PR) was 53.3%,the 1-year survival rate was 70% (21/30).The main adverse acute effects were gas- trointestinitis,anemia,neuro-sensory toxicity and irradiation rectitis,bone marrow suppression was mild.Conclusions: This approach of therapy could improve the therapeutic effect on advanced rectal cancer.

Objective To observe the roles of nuclear factor-κB (NF-κB) and hypoxia-inducible factor-1 o (HIF-1 α) in hippocampal neurodegeneration of status epilepticus (SE) rats, and explore whether HIF-1α activation is regulated by NF-κB.Methods A total of 110 male Sprague-Dawley rats were randomly divided into seven groups : (1) Control group treated with saline (control, n =15), (2) sham group implanted cannula into lateral ventricle and treated with saline (sham, n =15), (3) SE group treated with pilocarpine (SE, n =20), (4) NF-κB activity inhibitor pyrrolidine dithiocarbamate (PDTC) group treated only with PDTC (PDTC, n =15), (5) SE + PDTC group treated with pilocarpine plus PDTC (SE + PDTC, n =15), (6) SE + HIF-1o siRNA group implanted cannula into lateral ventricle and treated with pilocarpine plus HIF-1 α siRNA (SE + HIF-1α siRNA, n =15), (7) SE + control siRNA group implanted cannula into lateral ventricle and treated pilocarpine plus control siRNA (n =15).SE was induced by injecting lithium chloride and pilocarpine.The seizure of rats was observed.The protein expressions of NF-κB and HIF-1 α in hippocampus of rats were examined by Western blotting.The degenerating neurons in hippocampus were detected by Fluoro-Jade C (FJC) staining.Results Twenty-four hours after termination of SE, the nuclear protein expressions of NF-κB and HIF-1α in hippocampus of rats were increased in SE group (0.57 × 0.06, 0.47 ± 0.07) compared with those in control group (0.23 ± 0.03, 0.20 ± 0.03;P ＜0.05);and compared with SE group PDTC significantly decreased the nuclear protein expressions of NF-κB and HIF-1 α in SE + PDTC group (0.23 ± 0.03, 0.14 ± 0.03;P ＜ 0.05);in SE + PDTC group the numbers of FJC positive cells in CA1 area (28.33 ±5.03) were decreased compared with that in SE group (76.67 ± 13.32);HIF-1 o siRNA injected into lateral ventricle of rats significantly decreased the expression of HIF-1α in hippocampus (0.22 ±0.03) and the number of FJC positive cell in CA1 area (27.34 ±7.02) in SE + HIF-1α siRNA group compared with those in SE group (0.39 ±0.06, 76.67 ± 13.32;P ＜0.05).Conclusions These data suggest that SE can result in activation of NF-κB/HIF-1o pathway in brain.Inhibition of the pathway can attenuate hippocampal neurodegeneration caused by SE, which has the brain protective effect.

Objective Construction of human IL‐4 recombinant expression vector and then conduct the expression ,purification and identification of human recombinant IL‐4 .Methods the open reading frame of IL‐4 was amplified by nest PCR with total RNA from PBMC of healthy volunteer .And then the amplified IL‐4 was inserted into pET101/D‐TOPO ,transformed into BL21 ,ex‐pressed ,purified and indentified .Results The size of amplified open reading frame of IL‐4 was about 460 bp and the sequence was correct .After transformed into BL21 ,the IL‐4 clone with higher expression level was selected by selection of different clones insert‐ed with IL‐4 and the size of expressed ,purified IL‐4 was about 28 × 103 .Western blot results showed that the size of single band was identical with the expected protein .Conclusion Human IL‐4 recombinant protein was got successfully .

Objective:To develop non-immunized human phage display library.Methods:The total RNA of lymphocyte cells from peripheral blood of healthy voluntee was isolated and cDNA was synthesized,and the genes of heavy variable chain (VH) and light variable chain (V? and V?) were amplified by direct PCR and half-nested PCR.By overlapping extension PCR,the genes of VH and VL (V? and V?) were linked.The linked genes of single chain Fv fragment (scFv) were ligated with the vector pCANTAB-5E and then cloned into TG1 for the scFv library construction.Results:By direct PCR and half-nested PCR,42 VH fragments,16 V? and 18 V? fragments were obtained.The size of linked scFv library genes was 750 bp and the volume of constructed scFv library was 1.35?108.The results of BstN Ⅰ analysis of scFv genes from the phage library showed that fingerprint map of the selected scFvs was different.Conclusion:The developed phage library is diversity and can be used for selecting humanized scFv.

Objective:Expression and purification of the α-HL of Staphylococcus aureus as antigen for making full human anti-α-HL antibody later ,providing of new treatment for Staphylococcus aureus infection.Methods:The total RNA of Staphylococcus aureus was extracted and the cDNA of α-HL was amplified by RT-PCR.The DNA of α-HL and pCold-TF plasmid was digested and ligated by T4 ligase and then transformed into E.coli TOPO 10.The recombinant plasmid α-HL/pCold-TF which verified by sequencing was trans-formed into E.coli BL21 for expression.The expression products was identified by SDS-PAGE and Western blot.Results: The size of amplified cDNA of α-HL was about 900 bp and the expressed soluble fusion protein of α-HL was about 90 kD(including the molecular chaperone in the vector ) after inducing expression for 24 h at 15℃.The Western blot results showed that the expressed protein was the fusion protein of α-HL.The purified α-HL was injected into BABL/c mice for making antiserum.The results showed that the antiserum had good binding activity with Staphylococcus aureus and the titer was greater than 10 000 times.Conclusion: The α-HL of Staphylococcus aureus was successfully cloned and the soluble fusion protein of α-HL was successfully expressed.

Objective:To single amino acid mutation of the full human scFvs against TSLP to enhance its affinity.Methods: The specific scFvs against TSLP was screened in our previous study and here the three-dimensional structures of TSLP and anti-TSLP scFvs were simulated by Discovery Studio system,then the molecular docking was made.The amino acids of binding epitope were randomly mutated and the mutated amino acids were selected which could remarkably improve the affinity of scFvs.The primers were designed based on the sequence of mutation amino acids and the scFv sequences were mutated by the overlapping extension PCR.The DNA of mutated scFvs was ligated with the expression vector pLZ16 and transformed into E.coli DH5αF′.Then the scFvs were expressed and the scFvs with improved affinity were selected by ELISA and BIAcore.Results: The five scFvs with single amino acid mutation were screened out by DS system,which could elevate the affinity of scFvs.The mutated anti-TSLP-scFvs were amplified by PCR,which size was about 1 000 bp.The mutated scFvs with correct sequence were expressed,and the mutated scFvs with improved affinity were detected by ELISA and BIAcore.The affinity of selected mutated scFv (M4) has been about 10 times higher than the scFv nonmutation.Conclusion: The affinity of anti-TSLP-scFv has been improved successfully.

Objective: To determine the characteristics of postprandial proximal gastric acid pockets (PPGAPs) and their association with gastroesophageal acid reflux in patients with Barrett’s esophagus (BE). Methods: Fifteen patients with BE (defined by columnar lined esophagus of ≥1 cm) and 15 healthy individuals that were matched for age, gender, and body mass index, were recruited. The fasting intragastric pH and the appearance time, length, lowest pH, and mean pH of the PPGAP were determined using a single pH electrode pull-through experiment. For BE patients, a gastroesophageal reflux disease questionnaire (GerdQ) was completed and esophageal 24-h pH monitoring was carried out. Results: The PPGAP was significantly longer (5 (3, 5) cm vs. 2 (1, 2) cm) and the lowest pH (1.1 (0.8, 1.5) vs. 1.6 (1.4, 1.9)) was significantly lower in patients with short-segment BE than in healthy individuals. The PPGAP started to appear proximally from the gastroesophageal pH step-up point to the esophageal lumen. The acidity of the PPGAP was higher in the distal segment than in the proximal segment. In short-segment BE patients, there were significant correlations between the acidity and the appearance time and length of the PPGAP. The length and acidity of the PPGAP were positively associated with gastroesophageal acid reflux episodes. The acidity of the PPGAP was associated with the DeMeester scores, the GerdQ scores, and the fasting intragastric pH. Conclusions: In patients with short-segment BE, a PPGAP is commonly seen. Its length and acidity of PPGAP are associated with gastroesophageal acid reflux, the DeMeester score, and the GerdQ score in patients with short-segment BE.