Objective To investigate the effect of nuclear factor ?B(NF-?B)decoy on the chemokine expression in bladder cancer cell line. Methods Human bladder cancer cell line EJ,NF-?B decoy ODN were used as a NF-?B inhibitor(scrambled NF-?B decoy was used as control).NF-?B DNA binding activity was detected by electrophoretic mobility shift assay (EMSA);and p65 subunit of NF-?B was detected by RT-PCR and Western blot.Chemokines including IL-8,MCP-1,RANTES were detected by RT-PCR. Results EMSA showed that NF-?B decoy inhibited NF-?B activation induced by TNF-?.RT-PCR or Western blot test suggested that p65,IL-8,MCP-1 and RANTES were upregulated by TNF-? and downregulated by NF-?B decoy.However,mutated decoy ODN had no effect on them. Conclusions Chemokines can be detected in bladder cancer.They are activated by TNF-? and inhibited by NF-?B decoy.

Objective To evaluate the application effect of the problem based learning in clinical teaching of orthopedics. Methods We chose sixty five-year program students of Peking univ-ersity health science center as research object. All students were randomly divided into experimental and control groups, with thirty students in each group. Experimental group was given PBL teaching combined with lecture based learning (section-based learning, LBL), while the control group only received the LBL teaching. Two groups were given 8 hours of teaching experiments. After the end of the study, the teaching effect of the two groups was evaluated by the theory course and clinical skills. The questionnaire was distributed to the 2 groups. The scores of both experimental group students and control group students in theory courses and clinical skills were analyzed using SPSS 19.0 software using t-test and x2 test. Result The score of the experimental group was (53.7 ±3.2) in the knowl-edge-based grades exam, while the control group was (52.3±2.2), showing no obvious difference when compared to the control group's grades (P>0.05). The score of the experimental group was (24.0±1.5) in the hands-on technique grades exam, while the control group was (22.3±1.6). The difference in grades showed statistical significance (P<0.05). Feedback survey results showed that the experimental group's teaching satisfaction degree was also significantly higher than the control group, and the dif-ference was statistically significant (P<0.05). Conclusions The application of PBL in clinical teach-ing in orthopedic clinical teaching can improve students' learning interest and self-study ability, and helps to develop students' clinical thinking ability. But the role of PBL teaching in improving students' medical knowledge level is not obvious.

Objective To estimate the sensitivity of malignant cells from ascites of gastric cancer to anticancer agents by in vitro sensitivity test, and to study its value in individualized intraperitoneal chemotherapy. Methods Forty-seven cases of gastric cancers with malignant ascites were selected. The gastric cancer cells were isolated from the ascites of 19 patients with gastric cancers, and in vitro sensitivity tests to carboplatin, taxol, fluorouracil, cisplatin, adriamycin, hydroxycamptothecine, methotrexate, mitomycin and dacarbazine were performed by ATP bioluminescence assay. One of the most sensitive drugs was applied to intraperitoneal chemotherapy. The rates of the patients with complete remission of ascites and with disappearance of intraperitoneal cancer cells in the sensitivity test group were compared to those of 28 patients in control group who all were treated with intraperitoneal cisplatin. Results In sensitivity test group, the sensitive cases to carboplatin, taxol, fluorouracil, cisplatin, adriamycin, hydroxycamptothecine, and mitomycin were 7,6,6,6,5,5 and 5 respectively. No case was sensitive to methotrexate or dacarbazine. The rates of patients with complete remmission of ascites and the rates of patients with disappearance of intraperitoneal cancer cells in the sensitivity test group were significantly higher than those in cisplatin control group(68.4% vs. 32.1%, P

AIM　The purpose is to study the non-isothermal decomposition process and mechanics of vitamin B6. METHOD　The TG technique was used to observe between 30～700℃. RESULTS　The decomposition of vitamin B6 was performed by two stages. Vitamin B6 loses HCl at the first stage together with losing H2O. The kinetic equation obtained was dα/dt=A*e-E/RT*1/2(1-α)3; activation energy obtained was 325.27 kJ/mol; and preexponential factor A obtained was 7.22×1032/s as well. CONCLUSION　Vitamin B6 is rather thermal stable, and it loses HCl together with losing H2O at temperature range of 173℃～271℃.

AIM: To obtain a single-chain antibody with high affinity for hepatocellular carcinoma (HCC). METHODS: A second single-chain antibody mutant library was established by using error-prone PCR and DNA shuffling. Single-chain antibodies with high affinity for hepatocellular carcinoma were selected from phage antibody library by using ELISA. RESULTS: The content of the second single-chain antibody mutant library was about 4.5?10~7. Two selected mutants M25 and M36 were obtained after 3 rounds of panning and ELISA. Immunoassay showed that M25 and M36 bound to human HCC cells specifically. The relative affinity of M25 was 2.0 folds higher than that of the original antibody, and M36 was 2.4 folds higher than the original antibody. CONCLUSION: Error-prone PCR combined with DNA shuffling is an effective method to improve affinity of antibodies isolated from phage antibody library.

AIM: To investigate the genetic and epigenetic alterations of p14~ ARF gene and mutation status of p53 gene in human primary colorectal carcinomas and to analyze the relationship between the two gene changes and the role of abrogation of the p14~ ARF -p53 pathway in colorectal carcinogenesis. METHODS: The homozygous deletions, mutations, methylation of 5′ CpG islands, mRNA expression of p14~ ARF gene and mutations of p53 gene were assessed by PCR, direct sequencing, methylation-specific PCR, and RT-PCR in the tumorous and matched adjacent normal colorectal tissues from 56 patients with colorectal carcinoma. RESULTS: ① p14~ ARF alterations were detected in 27% (15/56) of colorectal carcinoma tissues studied, of which 1 case showed homozygous deletion, 14 cases showed 5′ CpG island methylation, and no mutation was found in any tumor. ②15 colorectal carcinomas with p14~ ARF alterations indicated lack of (13 cases) or at low level of expression (2 cases) of p14~ ARF mRNA, while expression of the p14~ ARF transcript was detected in the remaining 41 colorectal carcinomas and any matched adjacent normal colorectal tissues. ③ The mutations of p53 gene were detected in 48% (27/56) of colorectal carcinomas investigated. ④ Of these 56 cases, 12 had p14~ ARF alterations alone, 24 had p53 mutations alone, 3 had both p53 mutations and p14~ ARF methylation, and 17 had neither. 70% (39/56) of the samples had either or both abnormalities of the two genes, and p14~ ARF hypermethylation was related to wildtype p53 (P

AIM: To construct the shuttle plasmid vector for thymidine kinase (tk) and EGFP fusion protein gene driven by IGF - Ⅱ P3 promoter, and investigate the specific killing effect of the HSV - tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro. METHODS: Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening, and identified by restriction digestion and sequencing analysis. Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT -PCR. Cell killing after ganciclovir(GCV) application was determined by MTT. RESULTS: Identification of pDC316 -tkEGFP- P3 by enzyme digestion and sequencing analysis showed that the length, inserted location and direction of the target genes which were inserted into the recombinant were correct. It was found that enhanced green fluorescence protein could only be seen in HepG2 cells, but not in HeLa cells. The results of RT -PCR showed that only two bands could be seen in the samples of pDC316 -tkEGFP- P3 transfected HepG2 cells. The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells. CONCLUSION: The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF - Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.

Objective To compare the successful ratio, efficacy and complications between ultrasound-guided and X-ray-guided endoscopic biliary drainage (EBD). Methods EBD was performed in 62 patients under ultrasound guidance and 54 patients under X-ray guidance. Serum bilirubin, the bile duct diameter and the changes of clinical symptoms were compared before and after the procedure. Results Tube placement was successfully achieved in 54 of 62 patients under ultrasound guidance and 51 of 54 patients under X-ray guidance. The serum direct bilirubin and the common bile duct diameter in patients with ultrasound guidance before and one week after procedure were (205.41±115.27) μmol/L vs. (106.47±82.16) μmol/L and (12.6±7.1) mm vs. (8.5±3.1) mm, respectively, with significant difference (all P values<0.05). Whereas they were (211.14±106.25) μmol/L vs. (110.89±59.47) μmol/L and (13.1±7.0) mm vs. (8.8± 3.2) mm, respectively, in patients with X-ray guidance (P<0.05). No complications such as abdominal pain, fever and elevated amylase were found in patients with ultrasound guidance, while 3 patients (5.9%) with X-ray guidence had above complications. Conclusions X-ray is a most effective method in guidance of EBD. However, ultrasound guidence, which may avoid unfavorable factors such as X-ray radiation and allergic contrast agent, has some advantages including real-time display, mobile convenience and emergency bedside application. It can instead of X-ray in performance of endoscopic nasobiliary drainage and endoscopic retrograde biliary drainage in patients with bile duct stone and mild narrow ducl caused by tumors.

AIM: To construct HBx eukaryotic expression vector pEYFP-C1-X and eukaryotic expression vector pGL3-P4 driven by P4 promoter of human IGF-II gene and to investigate the effect of HBx on the transcription activity of IGF-II gene P4 promoter. METHODS: HBx gene and P4 promoters were cloned into pEYFP-C1 and pGL3-basic vectors respectively by gene recombination techniques to construct recombinant plasmids pEYFP-C1-X and pGL3-P4. HepG2 cells were transfected with pEYFP-C1-X and the resistant cell clones were selected by G418. Then methylated pGL3-P4 was transiently transfected into the above cell clones, and the transcription activity of P4 promoter was determined by dual-luciferase reporter assay system. RESULTS: (1) Aim fragments HBx gene and P4 promoter that were cloned were 465 bp and 1 246 bp, respectively and the DNA sequences were accordant with GenBank data confirmed by restricted enzyme digestion and sequencing. (2) HepG2-EYFP-X cells that expressed HBx protein were obtained. (3) Luciferase activity of methylated P4 promoter in the HepG2-EYFP-X was more than that of control cell HepG2-EYFP (P<0.01). CONCLUSION: HBx may enhance the transcription activity of the P4 promoter.

Objective To investigate the effect of 17β-estradiol on insulin resistance and the expression of insulin receptor-α in skeletal muscle of ovariectomized rats with insulin resistance induced by high fructose.Methods Forty-eight mature female Sprague-Dawley (SD) rats were randomly divided into four groups: the normal control group (NC, n= 12) rats were fed with the normal diet for 8 weeks; the model group (M, n= 12)rats were ovariectomized and fed with the high fructose diet for 8 weeks, meanwhile the physiological-dose of 17βestradiol (30 μg · kg-1 · d-1 ) was injected subcutaneously every day; the vehicle control group (VC, n= 12) rats were ovariectomized and fed with the high fructose diet for eight weeks, meanwhile equivalent alcohol was injected subcutaneously every day. Systolic blood pressure (SBP), fasting blood sugar (FBS), and fasting serum insulin (FSI) were measured and insulin sensitivity index (ISI) was calculated. The expressions of mRNA and protein of insulin receptor-α in quadriceps femoris were measured by RT-PCR and Western-blot. Results Compared with the normal control group, SBP (P<0.05), FBS (P<0.05) and FSI (P<0.01) were increased significantly while ISI was decreased significantly (P < 0. 05) in the model group. The expressions of mRNA and protein of insulin receptor-α and phosphorylated Akt were decreased significantly in quadriceps femoris in the model group (P<0.05), compared with the normal control group. However, these effects were reversed by 17β-estradiol in the 17βestradiol replacement group. Conclusions 17β-Estradiol inhibits insulin resistance, and up-regulates the expression of insulin receptor-α and the level of phosphorylated Akt in ovariectomized rats with insulin resistance induced by high fructose diet.