Objective; To express and purify HCA518 protein and prepare its monoclonal antibody ( McAb). Methods: The HCA518 protein was expressed with gene recombinant technique in prokaryotic system and purified with nickel chelate nitrilotriacetic acid(Ni-NTA) affinity chromatography column. Hybridoma cell lines that secreted anti-HCA518 McAb were established by cells fusion and screened by enzyme linked immunosorbent assay( ELISA). The specificity of anti-HCA518 McAb wa3 identified by Western blot assay. The HCA518 protein in tumor cells was stained by immunoflourescence assay. Results: Rcombinant HCA518 protein was expressed with a purity of 98%. Two hybridoma cell lines was selected and anti-HCA518 McAb was purified from mice ascites. The titers of anti HCA518 McAb in ascites were 1?10-4 and 5?10-4 respectively. The antibody belonged to IgG2b subtype and IgM. Anti-HCA518 McAb specifically reacted with recombinant HCA518 protein and tumor cells'nuclear protein (P100). The HCA518 protein was mainly located in cell nucleus. Conclusion: Stable hybridoma cell lines that secreted anti-HCA518 McAb have been established and anti HCA518 McAb was prepared with high specificity. It has important significance for detecting HCA518 protein in tumor tissues and determining malignant proliferation status of tumor cells and predicting its prognosis.

Objective:To establish a method for the determination of tanshinoneⅡA and salvianolic acid B in Zhiyou capsules by HPLC. Methods:The chromatographic column was Waters Atlantis@T3(150 mm × 4. 6 mm,3 μm)using acetonitrile and aqueous ammonium acetate solution(adjusting pH to 2. 4 with formic acid)as the mobile phase with gradient elution,the flow rate was 1. 0 ml ·min-1 and the detection wavelength was 260 nm. Results:The contents of salvianolic acid B and tanshinoneⅡA showed good linear relationship within the range of 1. 20-60. 00μg·ml-1(r=0. 999 7)and 0. 40-20. 00 μg·ml-1(r=0. 999 8),respectively. The av-erage recovery was 101. 53%(RSD=1. 14%)and 95. 17%(RSD=4. 33%),respectively. Conclusion:The method is efficient,con-venient and accurate,which can be used in the quality control of Zhiyou capsules.

Objectives To observe the effects of electroacupuncture (EA) treatment of different intensities on learning-memory function and expression of β-amyloid protein 1-40 (Aβ1-40) in hippocampus CA1 region in rats with vascular dementia (VD) and to seek the best intensity of EA treatment.Methods A total of 60 male SD rats (SPF grade) were enrolled.Eight rats were selected as sham operation group with random number table method;others were used to copy the VD model with the modified four-vessel occlusion (4-VO) method.According to the random number table method,the successful model of the rats (n=24) were completely randomly divided into a model group,a 1 am EA group (frequency,2/15 Hz,intensity,1 mA,needle retention time,20 min),and a 3 mA EA group (frequency,2/15 Hz,intensity,3 mA,needle retention time,20 min;n=8 in each group).DU20 (BaiHui) and DU14 (DaZhui) in the EA group were acupunctured once a day for 10 d,and took a rest for 2 d as 1 treatment course.After 2 treatment courses,Morris water maze test was used to detect the ability of learning and memory of rats in each group.Fluorescent quantitative polymerase chain reaction was used to detect the expression level of Aβ1-40 mRNA.Results The mean escape latencies of the water maze test from 2 to 5 days in the sham operation group,model group,1 mA EA group,and 3 mA EA group were 46.8±1.9,40.6±2.3,24.6±1.5,19.4 ±1.2 s;56.3±3.5,51.2±2.6,45.9±2.1,40.8±1.4 s,52.7±1.5,46.0±2.3,31.3±1.2,27.7±1.6 s;and 50.8±3.9,41.5±2.1,29.0±1.1,25.6±1.3 s,respectively;the first time across the original platform were 23.3±1.6,53.9±1.3,30.2±1.4,and 28.1±0.8 s,respectively;the first across the original platform within 120 s were 9.4±0.9,2.6±0.5,6.4±0.7,and 7.2±0.9,respectively;the expression levels of Aβ 1-40 mRNAs in the CA1 regions were 17.3±1.1,40.7±1.1,24.0±1.7,and 22.4±1.8,respectively.There were significant differences among the groups (the F values were 195.88,861.605,103.876,and 380.609,respectively;all P<0.01).Compared with the model group,the mean escape latencies,the first time across the original platform were reduced significantly.Compared with the model group,the times across the original platform within 120 s were increased significantly.Compared with the model group,the gene expression level of Aβ 1-40 mRNA in the center of CA1 region was decreased significantly,and the 3 mA EA group was significantly superior to that of the 1 mA EA group.The difference was statistically significant (all P<0.05).Conclusion EA may improve the learning and memory ability in VD rats and lower the expression level of Aβ 1-40 mRNA in the CA1 region of hippocampus.Effect of 3 mA EA is better than that of the 1 mA EA.

BACKGROUND:Bone marrow mesenchymal stem cel s have immunomodulatory properties and have potential applications in immunosuppression. OBJECTIVE:To investigate the isolation and culture of bone marrow mesenchymal stem cel s and the immunoregulation of CD8+T lymphocytes. METHODS:Primary bone marrow mesenchymal stem cel s were isolated from Wistar rats by bone marrow adherence method. The primary cel s were purified by differential adherence and digestion, and col agen type I was added as extracel ular matrix to expand bone marrow mesenchymal stem cel s. Passage 2 bone marrow mesenchymal stem cel s at densities of 0, 1×103, 1×104 and 1×105/wel were co-cultured with CD8+T lymphocytes, fol owed by phytohemagglutinin stimulation for 68 hours. T lymphocyte aggregation and proliferation were detected by staining with staining with 5,6-carboxyfluorescein diacetate succinimidyl ester and MTT, respectively. RESULTS AND CONCLUSION:Different concentrations of bone marrow mesenchymal stem cel s had an inhibitory effect on T lymphocytes, but this effect was weakest for the cel s at the density of 1×103 per wel and strongest for the cel s at the density of 1×105 per wel . Obvious agglomeration was observed in the same media. The proliferation rates of CD8+T lymphocytes were (44.83±4.92)%, (31.94±6.28)%and (15.77±3.98)%, respectively, after co-culture with 1×103, 1×104, 1×105/wel bone marrow mesenchymal stem cel s. There were significant differences between groups (P<0.05). These results showed that primary rat bone marrow mesenchymal stem cel s could be obtained by direct adherence method of the whole bone marrow, and the cel s could be purified by differential adherence combined with digestion and control method. Bone marrow mesenchymal stem cel s could inhibit T lymphocyte proliferation in a concentration-dependent manner.

Objective To evaluate and compare the clinical outcome of coracoclavicular screw and double Endobutton plate in treatment of acromioclavicular dislocation ( Rockwood Ⅲ-Ⅴ ). Methods Twenty-eight patients with Rockwood Ⅲ-Ⅴ acromioclavicular dislocation were subjected to surgical reconstruction from January 2008 to October 2009. The coracoclavicular screw was performed in 14 patients and the double Endobutton plate in the other 14 patients. Clinical evaluation was performed by using Constant score and subject should value (SSV) in both groups, and the preoperative and postoperative radiographs, curative effects and complications were compared. Results The patients in two groups were followed up for a range of 6-25 months (average 12.6 months) , which showed higher postoperative Constant score and SSV score than preoperation in both groups (P＜0.05). But the postoperative Constant sore and postoperative SSV score in the double Endobutton group were (89.8 ±8.3) points and (85.7 ±7. 3) points respectively, significantly better than (78. 0 ± 10. 3) points and (71. 8 ±9. 7) points respectively in the coracoclavicular screw group ( P ＜ 0.05). The radiologic measurement showed no significant difference in regard of the coracoclavicular distance three months after operation in two groups (P＞0.05). Conclusions The double Endobutton plate can attain significantly superior clinical outcomes for Rockwood Ⅲ-Ⅴ acromioclavicular dislocation compared with the coracoclavicular screw. The surgical technique of reconstructing the coracoclavicular ligament through anatomical approach will be the future trend in treatment of the acromioclavicular joint dislocation.

Objective To explore the effects of intestinal trefoil factor ( ITF) on gastric mucosal epithelial cell proliferation and its possible molecular mechanism . Methods The cultured GES-1 cells were treated with ITF in the concentration of 100 ng/mL and 500 ng/mL in vitro, and then were observed using microscope for the morphological analysis .The Cell Counting Kit-8 ( CCK-8) was used to detect the proliferation activity of GES-1.The cultured GES-1 cells were treated with 100 ng/mL ITF and the specific inhibitor of PI3K/Akt signaling pathway LY294002 (15μmol/L) in vitro, and then were observed using microscope for the morphological analysis . The proliferation activity of treated GES-1cells was detected using CCK-8 and the expressions of p-Akt and Akt of PI3K/Akt signaling pathway were determined by Western blot . Results Compared with the control group , the proliferation activity of GES-1 cells in-creased after being treated with ITF and the higher concentration of ITF induced the higher proliferation activity .LY294002 inhibited the increased proliferation activity of GES-1induced by ITF.The data of Western blot indicated that ITF induced the expression of p -Akt and activated the P3IK/Akt signaling pathway to modulate the proliferation activity of GES -1 cells.However, LY294002 inhibited the PI3K/Akt signaling pathway and then decreased the proliferation activity of GES -1 cells. Conclusion ITF could promote the proliferation ac-tivity of GES-1 cells by activating PI3K/Akt signaling pathway .

Objective To evaluate the relationship of preoperative neutrophil-lymphocyte ratio (NLR) with clinicopathological features and prognosis of colorectal cancer in middle-aged and elderly patients.Methods A retrospective analysis was performed in 212 patients with colorectal cancer in the First Affiliated Hospital of Nanjing Medical University from January 2011 to June 2013.All patients were divided into middle-aged group (46-65 year old,n=130) and old-aged group (66-89 year old,n=82),The optimal cut-off point of NLR was identified by the area under receiver operating characteristic curve,while NLR ＞ 3.13 and NLR≤3.13 were classified as high and low NLR group.The clinicopathological features and prognosis between the two groups were compared.Results There was no difference in gender,tumor growth site,depth of invasion,tumor embolus,lymphatic metastasis,distant metastasis,TNM stage between low and high NLR group (allP＞ 0.05).However,the difference between high NLR group and low NLR group in old-aged group with diabetes mellitus was statistically significant (P＜0.05).The 1-,2-,and 3-year survival rate of the overall 212 patients were 96.2％ (204/212),87.7％ (186/212) and 74.5％ (158/212) In middle-aged group,the 1-,2-,and 3-year survival rates were 98.8％ (85/86),90.7％ (78/86) and 84.9％ (73/86) respectively in low NLR group,but 95.5％ (42/44),84.1％ (37/44) and 72.7％ (32/44) respectively in high NLR group,(allP＜0.05).In old-aged group,the 1-,2-,and 3-year survival rates were 95.7％ (44/46),89.1％ (41/46) and 73.9％ (34/46) respectively in low NLR group,but 91.7％ (33/ 36),83.3％ (30/36) and 52.8％ (19/36) respectively in high NLR group (all P＜0.05).Cox regression showed that TNM stage and NLR were independent risk factors for the prognosis of the middle-aged and elderly patients with colorectal cancer (P＜0.05 or P＜0.01).Conclusions Preoperative NLR ＞ 3.13 suggest that the prognosis is poor in middle-aged and elderly patients with colorectal cancer.

Objective To observe the effects of CoC12 treatment on the expression of Hypoxia-inducible factor-1(HIF-1α) in mice hippocampus at different time point.Methods Balb/c mice were injected with CoCl2 and the change of HIF-1 α was detected by western blot and immunofluorescence and confocal laser scanning microscope at different time point(0h,1h,2h,3h,4h,5h and 6h) after injection.Results The relative protein level of HIF-1α was 0.135 ±0.01,0.572 ±0.01,0.595 ±0.03,1.09 ±0.03,1.30 +0.04,1.275 ±0.03,0.947 ±0.03respectively at different time point after the injection.The HIF-1α protein level reached its peak value at 4 h and decreased at 5h and 6h.Fluorescence intensity of HIF-1α was 13.33 ± 3.42,30.95 ± 7.86,46.50 ± 9.65,61.50± 10.02,88.30 + 15.69,71.39 ± 11.28,67.41 ± 10.78 respectively at different time point after the injection.The HIF-1α fluorescence intensity also reached its peak value at 4 h and decreased at 5h and 6h.Conclusion Time dependent HIF-1α accumulation was in close correlation with the CoCl2.

Objective To compare temperature curve and ablation zone between 915 MHz and 2450 MHz cooled shaft microwave antenna in ex vivo porcine livers.Methods The 915 MHz and 2450 MHz microwave ablation and thermal monitor system were used in this study.A total of 56 ablation zones and 280 temperature data were obtained in ex vivo porcine livers.The output powers were 50,60,70,and 80W and the setting time was 600s.The temperature curve of every temperature spot,the short- and long-axis diameters of the coagulation zones were recorded and measured.Results At all four power output settings,the peak temperatures of every temperature spot had a tendency to increase accordingly as the output power was increased,and except for 5 mm away from the antenna,the peak temperatures for the 915 MHz cooledshaft antenna were significantly higher than those for the 2450 MHz cooled-shaft antenna (P ＜0.05).Meanwhile,the short- and long-axis diameters for the 915 MHz cooled-shaft antenna were significantly larger than those for the 2450 MHz cooled-shaft antenna ( P ＜0.05).Conclusions The 915 MHz cooledshaft antenna can yield a significantly larger ablation zone and achieve higher temperature in ablation zone than 2450 MHz cooled-shaft antenna in ex vivo porcine livers.

Objective To identify the association strength of the prevalence of HBeAg, cccDNA with the occurrence of HBV related hepatocellular carcinoma (HCC) in high risk male cohort in Qidong area in China. Methods A cohort of 377 middle aged HBV infected men in Qidong was followed from 1989 for 13. 25 years. HCC cases were registered. A matched case-controlled study was conducted on 32 pairs of inherent HCC cases with non-HCC controls. Serum HBeAg was measured by ELISA. cccDNA was detected by semi-nested PCR and verified by DNA sequencing. Standard statistical comparison between the prevalence of each HBV marker in HCC versus control group provided the odds ratio and P-value was used to evaluate its association strength with HCC occurrence. Results Serum HBeAg prevalence was 53. 1% (17/32) in HCC group versus 15. 6% (5/32) in controls, odds ratio (OR) =6. 12, P