Objective: To reconstruct a substractive complementary deoxyribornucleic acid (cDNA) library of genes sensitive to mechanical stretch in human osteoblast like cells.Methods: Mechanical stretch at 12 cycles per minute was applied to human osteoblast like cells Saos-2 and the deformation of the stretched cells was 12%. Twelve hours after loading, mRNAs were isolated from both stretched and unstretched cells. Substractive cDNA library of the genes sensitive to stretch was constructed with the technique based on polymerase chain reaction (PCR) and substractive hybridization. Primary sequencing of clones in the library was carried out. Results: A substractive cDNA library of genes sensitive to stretch was constructed with a capacity of about 200 clones. According to the results of sequencing, most genes in the library were related to the mechanical stimulation. One novel gene fragment was obtained. Conclusion: The method used in the experiment is effective in cloning genes sensitive to mechanical stretch.

Objective:To investigate the effects of strain force on t he expression of osteoclast differentiation factor(ODF) and osteoclasto-genesis i nhibitory factor(OCIF) in human periodontal ligament cells (HPDLCs). Met hods: HPDLCs were subjected to cyclic strain force for 0, 6, 12 and 24 h ours, mRNA expression of ODF and OCIF were determined by RT-PCR. Result s:After treatment of the cells for 0,6,12 and 24 hours the ODF/?-actio n values were 0.7280?0.0261,0.6831?0.0411,0.5801?0.2230 and 0.4572?0.0373( P0.05) respectively.Conclusion:Strain force may decrease the expression of ODF and increase the expression of OCIF.

Objective:To study the effect of the cyclic-tension force on the expression of matrix metalloproteinase-3 ( MMP-3) mRNA in osteoclasts. Methods: Bone marrow cells were cultured and induced by the combination of IL-6 and 1,25(OH) 2D 3 and identified. The bone marrow cells were seeded onto elastic 6-well culture plate at a density of 2?105 cells/ml in 2ml in each well and cultured for 7 days. Then the cells were subjected to 12% elongation by a strain unit at 6 cycles/min (i.e.5-s elongation and 5-s relaxation) . After 24 hours of stretching, the expression of MMP-3 mRNA in osteoclasts were determined by in situ hybridization analysis. Results:The stretched osteoclasts showed enhanced MMP-3 mRNA expression level. Conclusion: The mechanical stretching may affect the bone-resorbing activity by up-regulated the expression of MMP-3 mRNA in osteoclasts.

Objective To investigate the effect of high mobility group box-1 protein (HMGB1) on immune function of peritoneal macrophages in mice. Methods The peritoneal macrophages were isolated from female BALBc mice in vitro. The macrophages were stimulated with different doses of HMGB1 (1, 10, 100, 1 000ng/ml) for 6 hours, or with 100ng/ml HMGB1 for different duration (0, 6, 12, 24, 48, 72 hours). The phagocytosis ability of macrophages was determined by the neutral red dye uptake assay and optical densities of samples were read at 540nm. The cytotoxicity to L1210 cells was measured with MTT assay and was expressed by absorbance at 570nm. The chemotaxis was assayed by using the Costar Transwell cell culture chamber inserts and the cells on the lower surface of the transwell membrane were fixed and counted. The cells were harvested at the indicated time points, fixed, and stained for indirect immunofluorescence, then the expressions of I-Ak MHC class Ⅱ alloantigen on macrophages were determined with flow cytometry. Results The effects of HMGB1 on phagocytic activities, cytotoxicity, chemotaxis and I-Ak antigen expression of macrophages were enhanced in a dose-dependent manner, starting at doses as low as 1ng/ml and with a maximal response at 100ng/ml (P

Objective:To study the function of transcription factor 4 (TCF4) and to prepare TCF4 polyclonal antibody.Methods:pET-41/TCF4 was transformed into E.coli BL21(DE3) and induced by IPTG.The purified GST-TCF4 fusion protein was applied to immunize rabbit to produce antiserum. The specificity of the affinity of purified anti-TCF4 antibody was examined by Western blotting analysis of the eukaryotic expressed products of TCF4. Dig-labeled probe and antibody against TCF4 were used to examine the expression of TCF4 in Saos-2 cells under mechanical stretch. Results:Western blotting showed that the antibody could bind to TCF4 specifically. The expression of TCF4 mRNA and protein were significantly increased in Saos-2 cells under mechanical stretch. Conclusion: TCF4 antibody has been prepared successfully.

Objective Intranuclear forkhead/winged helix transcription factor p3(Foxp3) plays a key role in T cell-mediated immunosuppression.The present study was performed to investigate the effects of high mobility group box-1 protein(HMGB1) on Foxp3 gene as well as protein expressions in splenic regulatory T cells(Tregs) and their potential regulating mechanisms in mice.Methods CD4+CD25+Tregs isolated from the spleens of male BABL/c mice by magnetic beads were seeded on 96-well(1?105 cells/well) cell culture plates coated with anti-CD3(1 ?g/ml) and soluble anti-CD28(1 ?g/ml),and the cells were stimulated with HMGB1 at various intervals or at different concentrations.After being stimulated,the Foxp3 mRNA/protein expressions in the Tregs were determined.The time-dependent and dose-dependent responses between HMGB1 and intranuclear Foxp3 expression were analyzed by flow cytometry,and the expressions of Foxp3 mRNA of Tregs were analyzed by quantitative PCR of SYBR GREEN.Results After stimulation with HMGB1,the intranuclear Foxp3 protein and mRNA expressions of splenic Tregs in mice were markedly down-regulated in 24 h to 72 h(P

Objective To demonstrate the effect of tumor necrosis factor-α induced protein 8 like-2 (TIPE2) expression in patients with acute respiratory distress syndrome (ARDS) and its mechanism. Methods A prospective observation was conducted. Thirty-nine patients with ARDS admitted to department of emergency of PLA General Hospital from July 2013 to July 2015 were enrolled, and 35 healthy persons served as control group. The acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score within 24 hours after admission, blood gas analysis, procalcitonin (PCT), and C-reactive protein (CRP) were recorded. The mRNA expressions of TIPE2 in peripheral blood mononuclear cell (PBMC) and myxoma resistance protein 1 (MX1) in plasma were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The correlations were analyzed by Spearman rank correlation analysis. Results The mean of APACHE Ⅱ score in 39 patients with ARDS was 25±3, the mean of PCT was (1.85±0.41) μg/L, and the mean of CRP was (18.0±3.0) mg/L. The TIPE2 mRNA expression in PBMC of ARDS patients was significantly down-regulated as compared with that of healthy control group (2-ΔΔCt: 3.28±0.15 vs. 8.87±0.20, P < 0.001), and the MX-1 mRNA expression in plasma was significantly higher than that of healthy control group (2-ΔΔCt: 39.44±0.46 vs. 20.10±0.32, P < 0.001). It was shown by correlation analysis results that the TIPE2 mRNA expression was negatively correlated with MX1 mRNA expression (r = -0.630, P < 0.001), so as APACHE Ⅱ score (r = -0.781, P < 0.001), but no correlation was found between TIPE2 mRNA and PCT as well as CRP (r value was 0.143 and 0.330, respectively, both P > 0.05). The MX1 mRNA expression was positively correlated with APACHE Ⅱ score (r = 0.893, P < 0.001), but no correlation was found between MX1 mRNA and PCT as well as CRP (r value was 0.230 and 0.210, respectively, both P > 0.05). Conclusion TIPE2 expression was decreased in ARDS patients, which negatively correlate with disease severity, and indicate TIPE2 might be involved in the pathogenic process of ARDS.

BACKGROUND:The regulatory role of extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa B (NF-κB) signal pathways in the osteogenic differentiation of MC3T3-E1 cells subjected to mechanical strain remains unclear.
OBJECTIVE:To investigate the effects of ERK1/2 and NF-kB signal pathway on alkaline phosphatase, type Ⅰcol agen, osteocalcin and interleukin-6 expression in osteoblasts in response to mechanical strain, and to explore the regulatory effects of ERK1/2 and NF-kB signal pathway on osteoblast differentiation.
METHODS:MC3T3-E1 cells cultured in vitro were separately treated with ERK1/2 pathway specific inhibitor PD098059 and NF-kB pathway inhibitor PDTC for 30 minutes, and subjected to12%elongation for 24 hours. Normal cells and cells along loading 12%mechanical strain for 24 hours were considered as controls. Enzyme linked immunosorbent assay and real-time PCR were utilized to detect alkaline phosphatase activities, type Ⅰcol agen, osteocalcin and interleukin-6 mRNA expression before and after cellloading.
RESULTS AND CONCLUSION:Under 12%mechanical strain, alkaline phosphatase, type I col agen, and interleukin-6 expression was regulated by ERK1/2 signal pathway in MC3T3-E1 cells, but osteocalcin gene expression was not affected by ERK1/2 pathway. NF-kB signal pathway inhibitor PDTC significantly suppressed alkaline phosphatase activities in MC3T3-E1 cells under mechanical strain, and inhibited interleukin-6 gene expression. However, type I col agen and osteocalcin gene expression was not affected by NF-kB signal pathway. Results suggested that mechanical strain affected osteogenic differentiation and relevant gene expression in MC3T3-E1 cells by ERK1/2 and NF-kB signal pathway.

Objective To analyze the distribution of image noise in low-dose chest CT scan and optimize the relative scanning parameters.Methods The CT images of the Chinese anthropomorphic chest phantom( CDP-1 C) were simulated into six groups of low-dose images with different noise indexs by using an image noise addition tool.The difference between the preset noise index and analog noise value was compared.The CT images of 20 volunteers were also simulated into nine groups of low dose scans with the tube currents of 10,30,50,80,100,120,150,180 and 240 mA.The noise values of images were recorded and analyzed.Results There was no statistical difference between the analog noise value and the noise index.The image noise of low-dose chest scan was increased with the decrease of tube current.The noise was increased quickly when the current was decreased from 50 to 30 mA ( F =24.09 - 40.79,P ＜ 0.05),but the noise increased slowly when the current decreased from 240 to 80 mA.There was no statistical difference between the noise of 80 mA group and that of 120 mA(P ＞ 0.05).Conclusions The noise addition tool can be used to evaluate the image noise of low-dose chest CT scan.Adoption of 80 mA in chest CT scan would result in low radiation dose without adding image noise.

A valve-free continuous flow method and instrument were established,with only a multi-channel pump for delivering the sample and reagent,and without any injection or solenoid valves and sample loop for selecting and adding the sample or reagent.Nitrate was reduced to nitrite with Cu-Cd reductant column,and then detected with spectrophotometric detector.The proposed method was suitable for determination of nitrate at normal level in most of estuary and coastal seawaters.With the optimum parameters,the linear range and detection limit were 5-180 μmol/L and 0.27 μmol/L,respectively.The samples of 10 and 80 μmol/L nitrate were continually measured for 11 times,and the relative standard deviations were 1.4％ and 1.3％,respectively.The recovery of real samples at different salinity ranged between 99.4％ and 106.1％.There was no significant difference in the analytical results between the proposed method and the flow injection analysis (FIA).In comparison with FIA,the method and instrument were less cost and easy to operate,and was suitable to be applied in general laboratories and field for continuous monitoring.The method was successfully used to measure the nitrate in seawater samples in Xiamen's Western Harbor and monitor nitrate in Jiulongjiang estuary.