Abstract:Objective:To investigate the effect of continuous pulsed-tumor necrosis factor(TNF-α) treatment on nuclear factor-κB(NF-κB) pathway and osteogentic potentials of bone marrow mesenchymal stem cells (BMMSCs). Methods:There were three groups in this study:Group A:negtive control;Group B:BMMSCs treated by TNF-α once;Group C:BMMSCs continuously pulsed-treated by TNF-α for seven days. After the treatment,IκBα and phospho-IκBα (p-IκBα) were detected by Western blot. The mRNA expression of EPHB4,IGF-1,OPG,IL-7 and MMP-1 in BMMSCs were measured by Real-time PCR. Alizarin red staining and alkaline phosphatase(ALP) staining were employed for the measurement of osteogenic differentiation. This experiment also investigated the effect of the addition of NF-κB inhibitor (pyrrolidine dithiocarbamate,PDTC) on TNF-α action. Results:Compared with group A,IκBα protein levels were lower,while phospho-IκBα(p-IκBα) were higher in both group B and C(P < 0.05). Besides,the mRNA expression levels of EPHB4,IGF-1,OPG were up-regulated but IL-7 and MMP-1 down-regulated after TNF-α priming (P < 0.05). The osteogenic potential of TNF-α-primed BMMSCs was enhanced and the pro-osteogenic differentiation effect of TNF-α was reversed in the presence of NF-κB pathway inhibitor PDTC. Conclusion:Our in vitro results indicated that continuously pulsed TNF-α treatment significantly enhances the osteogenic differentiation of BMMSCs and this effect may be mediated at least partially via NF-κB pathway.

Abstract:Objective:To investigate the expression of microRNA-21 (miR-21) in colorectal cancer (CRC) tissues and cells,and investigate the inhibition of miR-21 on protein expression of PI3K/ PTEN/AKT/ mTOR signaling pathway,and look for the target genes which may be regulated by miR-21. Methods:The expressions of miR-21 and PTEN from tissues of CRC were detected by real-time fluorescence quantitative PCR (FQ –PCR) and immunohistochemistry (IHC). The anti-sense oligonucleotide (ASO) technology was performed to inhibit and verify the expression of miR-21 in HCT116 cells. The changes in PTEN,PI3K,Akt,MMP-9 and mTOR protein expressions were detected by Western blotting. The potential target gene prediction was performed by Bioinformatics. Dual luciferase report gene assay was performed to identify the activity of PTEN. Results:The expression level of miR-21 was increased in CRC tissue (P < 0.05),and the expression of PTEN was decreased in CRC tissue (P < 0.05). However,the expression level of miR-21 was negatively associated with PTEN expression (r = -0.396,P < 0.05). The level of miR-21 was the highest in HCT116 cell and lowest in SW480 cell. The expression of miRNA-21 in HCT116 cell lines was significantly inhibited,the expression of PTEN protein was significantly increased and the expressions of PI3K,Akt,MMP-9 and mTOR were down-regulated. PTEN was identified as one of target genes of miR-21 in CRC cell. Conclusion:PTEN,as one of target genes of miR-21 in CRC cell,can regulate the process of CRC. miR-21 expression is increased in CRC,and promotes cancer cell invasion and metastasis by inhibiting PTEN.

Abstract:Objective:To investigate expression of miR-638 and effects of miR-638 on apoptosis of lung adenocarcinoma cell line(SPC-A1). Methods:The expression of miR-638 in lung adenocarcinoma cell line was detected by real-time RT-PCR. Mimics of mir-638 were transiently transfected into SPC-A1 by using lipofectamine method. SPC-A1 cells were transfected with miR-638 mimics or non-special oligonucleotides(as negative control)or nothing(as blank control). After transfection,transfection efficiency was observed by fluorescence microscope. MiR-638 levels were detected by real-time quantitative RT-PCR. Cell apoptosis was detected by flow cytometry. Results:We observed that miR-638 was significantly inhibited in lung adenocarcinoma cells compared with that in normal cells. Cell apoptosis rate was increased significantly in cells transfected with miR-638(P <0.05)compared with negative and blank control cells. Conclusion:MiR-638 was poorly expressed in lung adenocarcinoma cells,and it could dramatically promote apoptosis of lung adenocarcinoma cells,thus provides a new target for the use of miR-638 in lung cancer biotherapy.

Abstract:Objective:To investigate effects of ETS1 on early embryo development of Xenopus laevis. Methods:The pCS2+-ETS1 plasmid was produced by ligating pCS2+ plasmid and ETS1 cloned according to PCR. The expressions of the ETS1 during embryogenesis were detected by RT-PCR and Western blot. After micro-injection of ETS1 mRNA into embryo,the expressions of marker genes of different embryonic layers were detected by whole mount in situ hybridization and quantitative RT-PCR. The effect of ETS1 over-expression on apoptosis and proliferation of cells was analyzed by Western blot. Results:The expression of ETS1 started from the period of oosperm,and increased through all periods of organogenesis. The overexpression of ETS1 inhibited differentiation of cells and expression of marker genes of mesoderm and ectoderm in neurulation. The expression of apoptosis-related proteins was increased,whereas the expression of proliferation-related proteins had no significant change. Conclusion:ETS1 is expressed through all periods of embryogenesis of Xenopus laevis. The overexpression of ETS1 inhibits the development of mesoderm and ectoderm,as well as promotes the process of apoptosis,nonetheless no change of proliferation is inspected.

Abstract:Objective:To observe effects of hydrogen sulfide(H2S) intoxication on sodium and water transport function in rat lung and study its mechanism. Methods:SD rats were exposed to H2S gas with semi-lethal concentration(300 ppm) for 3 h. After 6 h,12 h and 24 h exposure to H2S,alveolar fluid clearence(AFC) and wet/dry ratio were measured. HE staining of lung tissues was observed by light microscope,and the change of type Ⅱ alveolar epithelial cells was observed by electron microscope. mRNA expression of α-epithelial sodium channel(ENaC) in the lung tissues was analyzed by real-time PCR. The expression of α-ENaC and ERK1/2 protein in the lung tissue was examined by Western blot . Results:After exposure to H2S, AFC was significantly decreased in SD rats compared to the control group, and reached a lowest level after 6 h and returned to a normal level after 12 h. The lung water content reached a lowest level after exposure for 6 h. Obvious injury changes of the lung tissue of rats were found after exposure for 24 h by light microscope. Dilated mitochondrial cristae and collapsed lamellar bodies were found in typeⅡalveolar epithelial cells of rats after exposure. The expression of α-ENaC mRNA in lung tissue was increased after exposure for 6 h and returned to a normal level after 12 h. Compared to the control group, the protein expression of α-ENaC was significantly decreased after exposure for 6 h. ERK1/2 dephosphorylation in lung tissues was significantly increased after exposure for 6 h. Conclusion:H2S intoxication decreases the AFC of rats and down-regulation of α-ENaC expression may be involved in as a potential mechanism. Furthermore, activated ERK1/2 signaling pathway may participate in the whole damaging process.

Abstract:Objective:To study the effect of adoptive transfer of regulatory T cells(Tregs)on the repair phase of ischemia/reperfusion(IR)induced renal injury in mice. Methods:A total of 24 C57BL/6 mice aged 6-week-old were divided into Sham group,IR group,high-dose transfer group(HT),and low-dose transfer group(LT). Approximately 5 × 106 and 1 × 105 Tregs were injected intravenously into mice by tail vein 1 d before IR injury induced. Left renal pedicle was bluntly dissected and clamped with a microvascular clamp for 45 min and reperfusion 4 d to establish IR renal injury model. Left renal pedicle was only separated without clipping in the Sham group. HE staining was performed to evaluate morphology changes of renal tissues. Immunohistochemical and flow cytometry were performed to detect the infiltrating of Tregs(CD4+CD25+);the expression changes of Ki67,tumor necrosis factor-α(TNF-α),IL-16 and IL-10 were determined by immunohistochemistry,Western blotting and/or ELISA. Results:Compared to the IR group and the LT group,the damage of renal pathological was reduced,cell proliferation,the infiltration of Tregs and the expression of IL-10 were significantly increased(P < 0.05),and the expression of TNF-α and IL-6 were decreased(P < 0.05)in the HT group. Conclusion:Adoptive transfer of Tregs had a dose-dependent protective effect on renal ischemia-reperfusion-induced injury,which is possibly related to inhibition of the proinflammatory cytokines TNF-α,IL-6 and increase of anti-inflammatory cytokine IL-10.

Abstract:Objective:To study the effects of bacterial endotoxin with different concentrations on human gingival fibroblast (HGF) on titanium plate and its product osteoprotegerin(OPG), and to understand the healing function of endotoxin on soft tissue and bone tissue of dental implants. Methods:Tissue block culture method was applied to culture human gingiva and gingival fibroblasts were identified by immunofluorescence staining method. Endotoxin with different concentrations of 0,10 and 100 μg/ml were used to stimulate HGF on the titanium plate and HGF morphology was observed by scanning electron microscope(SEM). ELISA was applied to detect the OPG level. Results:Compare with the normal group,the HGF stimulated by endotoxin of 10 μg/ml had more cellular pseudopods and cellular matrix secretion was increased. However, in the 100 μg/ml endotoxin stimulated group,number of cells and cellular matrix secretion were decreased significantly. ELISA method demonstrated that under the condition of low concentration, OPG increased with the increase of endotoxin concentration. But the OPG level of 100 μg/ml group decreased significantly. Conclusion:Low concentration of bacterial endotoxin can promote growth of HGF and secretion of cellular matrix OPG;whereas high concentration of bacterial endotoxin can inhibit growth of gingival fibroblasts and decrease OPG level.

Abstract:Objective:To synthesize nNOS PDZ structural domain inhibitors and evaluate their neuroprotective effects. Methods:Multiple-series of dicarboxylic acids and their ester compounds were designed and synthesized based on the structure characteristic of multi-basic groups of the nNOS PDZ ligand binding domain. The neuroprotective effects of the compounds were evaluated by glutamate-induced lactate dehydrogenase (LDH) release model of neurons. Results:Eighteen target compounds were synthesized including N-(2-methoxycarbonylacetyl)-D-valine methyl ester(1),N-(2-carboxylacetyl)-D-valine methyl ester(2),N-(2-methoxycarbony-lacetyl)-L-valine methyl ester(7),N-(2-carboxylacetyl)-L-valine(9) showed potent neuroprotective effect. Conclusion:nNOS PDZ structural domain inhibitors are neuroprotective.

Abstract:Objective:The aim of this study was to evaluate the potential relationship between single nucleotide polymorphisms/haplotypes of beta-2 adrenergic receptor (ADRB2) gene and asthma in a Han Chinese population. Methods:Six loci (-2 387,-47,46,79,491 and 523 loci) of ADRB2 gene were genotyped by the TaqMan probe assay in 379 asthmatics and 435 healthy controls,and the haplotypes were also analyzed by the SHEsis online platform. Results:There was significant difference in allele frequency distribution of -47 locus between asthmatic patients and control subjects (OR:0.687,95%CI:0.497~0.951,P < 0.05). Compared with TT genotype,the CT and CC + CT genotype both had significant difference (OR were 0.685,0.672,respectively;95%CI were 0.480~0.976,0.474~0.952,respectively;both P < 0.05). There was also significant difference in allele frequency distribution of 79 loci between asthmatic patients and control subjects (OR:0.687,95%CI:0.497~0.951,P < 0.05). Compared with the CC genotype,the CG and CC + CG genotype both had significant difference (OR were 0.672,0.687,respectively;95%CI:0.480~0.976,0.474~0.952,respectively;both P < 0.05). There were no significant differences in genotype or allele frequencies of loci of -2 387,46 and 523 between the two groups (all P > 0.05). The mutant allele at 491 locus was not observed and only the homozygous wild-type was found in both asthmatic patients and control subjects. A strong linkage disequilibrium was found between -47 and 79 loci (D＇ = 0.987,r2 = 0.974) and between 46 and 523 loci (D＇ = 0.996,r2 = 0.593). There was significant difference in the haplotype Ⅲ (CCGGC) distribution frequency between asthmatic patients and control subjects (OR = 0.696,95%CI:0.502~0.966,P < 0.05). Conclusion:The polymorphisms of -47,79 loci and the haplotypeⅢ(CCGGC) of ADRB2 gene may contribute to susceptibility of asthma in Chinese Han population. There were no association between the polymorphisms/haplotypes of -2387,46,491,523 loci and asthma susceptibility.

Abstract:Objective:To determine the extent of inter-observer variation in delineation of heart after and before studying the heart atlas and its impact on dose distribution of the whole heart and every cardiac valve in radical radiotherapy of patients with esophagus cancer in blind modes. Methods:Ten patients with esophagus cancer and without operation were asked to contour the heart in computed tomography transverse section in blind modes by five observers. Inter-observer variability were compared in the delineation of the whole heart and estimated doses for heart and every cardiac valve before and after studying the heart atlas. Results:Compared with the mean heart volume without guidelines,the mean heart volume with guidelines was increased from 635.3 cm3 to 647.6 cm3. Soensen-Dice similarity index(DSI)was increased from 0.84~0.87 to 0.92~0.94. Jaccard similarity index(JSI)was increased from 0.91~0.93 to 0.96~0.97. After learning guidelines,the volume of the heart was increased significantly,I_Tot/U_ Tot increased from 0.75 ± 0.02 to 0.84 ± 0.03. In addition to the heart and the pulmonary valve,Dmean and Dmax of aortic valve,mitral valve,tricuspid valve were increased significantly after learning guidelines. The coefficients of variation(CV)of Dmean and Dmax of hart and valves were increased significantly. Conclusion:There was a significant difference in the delineation of heart for different observers,which could be decreased by the introduction of heart atlas. The delineating inter-observer variability has no dosimetric effects on the whole heart and pulmonic valve,leads to the difference of Dmean and Dmax of the estimated dose on aortic valve,mitral valve,tricuspid valve. Furthermore,the introduction of heart atlas decreases the difference of radiation dose among the above structures.

Abstract:Objective:To establish a rat model of inferior vena cava thrombosis,and to summarize the process of modeling techniques and surgical considerations. Methods:A total of 82 healthy SD rats were divided into the control group and the model group. The model group was adopted a ＂stenosis technology＂,which blocked most of blood flow in the inferior vena cava. At determined time points after ligation,laparotomy was performed and tissues were observed and sampled for pathological examination to evaluate modeling success. Results:Both of the two groups survived during the whole experiment and there were no accidental death,with the survival rate of 100%. The control group had no inferior vena cava thrombosis(0/8);in the Model group,thrombosis(6/8,75%)was found after two hours of surgery,and it was visible after 6 hours(8/8,100%). Between 24 to 48 hours after surgery,a stable thrombus formed with a lumen hyperemia(16/16,100%). After 7 days,organic thrombus was observed,but there was no significant regression(8/8,100%). The dissolving thrombus regression was observed between 14 to 21 days after surgery(16/16,100%). Conclusion:The stenosis technology can be used to block inferior vena cava blood flow successfully to establish the inferior vena cava thrombosis model.