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 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 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 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.

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 To investigate modulatory effects of Neb-SNP on inflammatory response and to explore the protection mechanisms of Neb-SNP in newborn piglets with ARDS. Methods Forty-five neonatal swines were randomly divided into five groups:group A (controlled group ,n = 9), group B (physiological saline group,n =9),group C (Neb-SNP 1 mg/ml,0. 9% NaCl, n = 9), group D (Neb-SNP 5 mg/ml,0. 9% NaCl, n = 9) and group E (Neb-SNP 10 mg/ml,0. 9% NaCl, n = 9). The pathological changes and activity of NF-κB in the lung tissue ,TNF-α ,IL-10 and IL-12 concentrations in serum at 30 minutes,60 minutes and 120 minutes after aerosol inhalation were observed. Results Activity of NF-κB and serum concentrations of TNF-α and IL-12 in the Neb-SNP treated group were lower than group B(P ＜0. 05) ,and serum IL-l0 concentration was obviously higher in the Neb-SNP group(P ＜0. 05). With an increase of Neb-SNP concentration,activity of NF-κB and serum concentrations of TNF-α and IL-12 were obviously increased, while serum concentrations of IL-10 was increased in group D and group E than that of group C (P ＜ 0. 05).Conclusion Inhalation of Neb-SNP reduced lung injury induced ARDS through lowering NF-κB activity and inhibiting expression of harmful inflammatory cytokines.

ObjectiveTo compare detection rate of pulmonary nodules and the radiation doses of digital tomosynthesis (DTS) and MSCT chest scanning by using the anthropomorphic chest phantom which containsthermoluminescent dosimeters( TLD ) and simulated pulmonary nodules.Methods The radiation doses of DTS and MSCT scanning were measured by using the anthropomorphic chest phantom which contains 45 TLD and simulated pulmonary nodules.The radiation doses of najor organs were converted into effective dose ( ED ). Three radiologists of different clinical experiences independently reviewed and recorded the density,diameter and position of pulmonary nodules.The sensitivity of nodule detection by DTS and MSCT were compared by Fisher exact test and Chi-square test. The paired t test was conducted to analyze the dose levels of DTS and MSCT.ResultsThe sensitivity of detection nodule by DTS and MSCT were 66.7％ (30/45) and 91.1％ (41/45) respectively.Statistically significant difference between the two examinations existed ( x2 =8.073,P ＜ 0.05).The sensitivity of detection - 650 HU ground glass opacity pulmonary nodule by MSCT and DTS were 93.3％ (14/15) and 73.3％ (11/15) respectively.There was no significant difference between DTS and MSCT ( P ＞ 0.05 ).The sensitivity of detection - 800 HU ground glass opacity nodule and ground glass opacity nodule (d ＜ 8 mm) by DTS were 33.3％ (5/15) and 16.7％ (2/12) respectively,which were lower than those by CT[80.0％ (12/15) and 66.7％ (8/12)].The radiation doses of DTS for various organs in the chest were lower than those of CT. Statistical significant difference between DTS and MSCT existed ( lung t =19.69,thoracic vertebral t =30.01,heart t =16.33,liver t =5.06,breast t =9.43,thyroid gland t =8.05 ;P ＜ 0.05).The effective doses of the DTS and MSCT were 0.65 and 7.71 mSv respectively.ConclusionsThere is no difference between the DTS and MSCT in the detection rate of -650 HU ground glass opacity nodule.For detecting the ground glass opacity nodule ( - 800 HU) and ground glass opacity nodule (d ＜ 8 mm),MSCT is superior to DTS. However,the radiation dosage of DTS is 8.41％ of the MSCT scanning.

BACKGROUND: Inhibiting the degradation of extracellular matrix in the intervertebral disc can delay the degenerative process of intervertebral disc. Matrix metalloproteinases-3 (MMP3) is considered as a key enzyme for degradation of extracelular matrix components such as type II collagen and aggrecan.
OBJECTIVE:To construct the short hairpin RNA lentiviral vector targeting human MMP3 gene and to detect its efficiency of gene silence by infecting human degenerative nucleus pulposus cells.
METHODS:According to the human MMP3 mRNA (NM_002422.4) sequence, four groups of the short hairpin RNA gene sequences targeting MMP3 were designed, synthesized and annealed to form double stranded DNA fragments, which were connected with the LV3 vectors digested by BamHI andEcoRI enzymes, and then transfected into the competent cels. The positive clones were identified by PCR, and analyzed by sequencing. The packaging and titer of lentivirus were determined after transfecting 293T cells. Human degenerative nucleus pulposus cels were infected with lentivirus vector, and the transfection efficiency of each group was observed under inverted fluorescence microscope. The interfering efficiency was detected by real time-PCR and western blot at 72 and 96 hours.
RESULTS AND CONCLUSION:The ds-oligo DNA was successfully inserted into the lentiviral vector as confirmed by electrophoresis and sequence analysis. The recombinant lentivirus was harvested from 293T cels with a viral titer of 1-5 ×108 TU/mL. RNA interference targeting the GCC AGG CTT TCC CAA GCA AAT sequences with the highest interfering efficiency in MMP3 gene at 72 and 96 hours resulted in suppression of MMP3 mRNA expression by 98% and 72%, respectively; and at 96 hours, the interfering efficiency of protein expression was 57.2%. The recombinant lentivirus vector containing RNA interference targeting MMP3 gene is successfuly constructed, which lays a foundation for further studies on the MMP3 function and gene therapy.