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 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 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.

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 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.

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 evaluate the effect of NGX6 combined with cisplatin on the inhibition rate of A549 cells and NCI-H1975 cells and antitumor effects in vivo.Methods The NGX6 was loaded in the LPD, and prepare Liposome protamine DNA complexes.A549 cells and NCI-H1975 cells were seperately divided into NGX6 group ( 30 μg/mL NGX6 concentration ) , cisplatin group, NGX6 +cisplatin group, PBS as negative control group.The effect of cytotoxicity of four group on A549 cells and NCI-H1975 cell were evaluated by MTT assay.the clone forming rate and the inhibition rate were determined by Cell colony count.lung transplantation tumor model were successfully established, then nude mice were divided into four groups as abeve, each group of 10, tumor size and survival period were determined tumor cell apoptosis were observed.Results The cell viability of A549 cells and NCI-H1975 cells of (NGX6 +cisplatin) were lower than that of NGX6 group, cisplatin group and saline group, respectively(P<0.01).The cloning efficiency of A549 cells and NCI-H1975 cells of ( NGX6 +cisplatin) were lower than that of NGX6 group, cisplatin group and saline group, respectively(P<0.01).The tumor inhibitory rate was in vivo for (NGX6 +cisplatin) was higher than other group(P<0.01).The median survival of nude mice in (NGX6 +cisplatin), NGX6, cisplatin and saline group were 43,31,29 and 15 days.Conclusion NGX6 combination with cisplatin can inhibit the cell proliferate of lung cancer cells and inhibit the tumor growth and the combination of NGX6 and cisplatin may be a potentially effective treatment for lung cancer.

Objective Using the Chinese anthropomorphic chest phantom to measure the absorbed dose of various tissues and organs under different noise index, and to assess the radiation dose of MSCT chest scanning with the effective dose(ED). Methods The equivalence of the Chinese anthropomorphic chest phantom(CDP-1C) and the adult chest on CT sectional anatomy and X-ray attenuation was demonstrated. The absorbed doses of various tissues and organs under different noise index were measured by laying thermoluminescent dosimeters(TLD) inside the phantom, and the corresponding dose-length products(DLP) were recorded. Both of them were later converted into ED and comparison was conducted to analyze the dose levels of chest CT scanning with automatic tube current modulation (ATCM) under different noise index. Student t-test was applied using SPSS 12.0 statistical software. Results The Phantom was similar to the human body on CT sectional anatomy. The average CT value of phantom are -788.04 HU in lung,45.64 HU in heart,65.84 HU in liver,254.32 HU in spine and the deviations are 0.10%,3.04%, 4.49% and 4.36% respectively compared to humans. The difference of average CT value of liver was statistically significant(t=-8.705,P＜0.05),while the differences of average CT values of lung, heart and spine were not significant(t value were -0.752,-1.219,-1.138,respectively and P＞0.05).As the noise index increased from 8.5 to 22.5, the DLP decreased from 393.57 mGy·cm to 78.75 mGy·cm and the organs dose declined. For example, the average absorbed dose decreased from 22.38 mGy to 3.66 mGy in lung. Compared to ED calculating by absorbed dose, the ED calculating by DLP was lower. The ED values of the two methods were 6.69 mSv and 8.77 mSv when the noise index was set at 8.5. Conclusions Application of the Chinese anthropomorphic chest phantom to carry out CT dose assessment is more accurate. The noise index should be set more than 8.5 during the chest CT scanning based on ATCM technique.