The dynamic expression of cytokines and phenotypes during the differentiation process of dendritic cell precursors (pre-DCs) to mature dendritic cells (DCs) was investigated,and the effects of inflammatory stimulation with lipopolysaccharide (LPS) on DCs differentiation were understood.The differentiation of bone marrow cells isolated from Balb/c mice was induced to DCs in an 8-day cell culture system with RPMI-1640 complete culture medium containing 10％ FBS,20 ng/mL recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and 10 ng/mL recombinant mouse interleukin-4 (rmIL-4).On the day 3,6 and 7 after culture,DCs were divided into non-LPS group and LPS group,given 500 ng/mL LPS for 24 h stimulation and no stimulation respectively.The expression levels of CD 1 l c+,MHC-Ⅱ +,CD40+,CD80+ and CD86+ were detected by flow cytometry,and those of IL-2,IL-4,IL-10,IL-12 p70 and IFN-γ in the supernatant by ELISA.On the day 3 and 6 after culture,the expression of IL-2,IL-4,IL-10 and IFN-γ in DCs showed no significant differences between non-LPS group and LPS group,whereas the differences were significant at day 7.The expression levels of cytokines (for IL-2,IL-4,IL-10,IFN-γ and IL-12 p70:152.86±6.91,778.33±8.35,44.55±2.54,5.8.26±1.09 and 2423.00±57.21 pg/mL respectively) in LPS group were higher than those in non-LPS group,especially IL-12 p70 increased obviously at day 7.It was concluded that during the differentiation process of pre-DCs to mature DCs,LPS stimulates DCs producing large amounts of IL-12 p70 and Thl-type cytokines as compared with Th2-type cytokines,and day 7 may be a key time-point for DCs polarization.

Objective To observe the influence of congenital heart disease(atrial septal defect,ASD)to intervene closure on the right structure of children(<1 5 years)and adults(1 5-65 years)and to make the safety assessment.Methods Totally 1 1 1 un-derwent interventional treatment of complications in patients with ASD in our hospital from 2010 to 2013 were retrospective ana-lyzed.Closure on changing of right heart structure of child and adult were measured by ultrasonic cardiogram.Closure falls off,shut valve insufficiency,arrhythmia,residual shunt were recorded by ultrasonic cardiogram and electrocardiogram.making statistical a-nalysis.Results The inner diameter of the right atrium(RAD),right ventricle diameter(RVD),pulmonary artery diameter(PA) and right ventricular outflow tract(RVOT)were decreased compared with pre-operation(P < 0.05 ),during the follow-up 1,3,6 month,they was continue decreased in the aged between1 5-65 group(P <0.05),but was stable in less than 1 5 years old age group (P >0.05 ).The complication rate of children and adults were 25.0% and 21.3% respectively,and there were no significantly difference(P >0.05),and was no serious complications.Conclusion Congenital heart disease intervention of atria septal defects can improve heart right structure,which can benefit both children and adult,there is no difference in complication rates.All of these have less serious complications,high safety,curative effect affirmation.

Objective To detect the difference of cerebral gray matter change in children with different karyotype Turner Syndrome (TS) by using voxel-based morphometry (VBM).Methods Nineteen children with 45XO karyotype TS,21 children with heterozygous TS and 20 age-matched control girls were recruited in this study.Wechsler intelligence scale for children was used to obtain their intelligence quotients (IQ).High-resolution magnetic MR imaging was performed in TS children and control girls to collect the whole brain structural data.The data was analyzed by VBM based on SPM8 to compare the volume of gray matter among the monosomy TS children,heterozygous TS children and normal controls by using covariance analysis.Alphasim method in the software of analysis of functional neuroimages(AFNI) was used for clusterlevel multiple comparison.Results The IQ was 89 ± 16 for the monosomy TS children,and it was 91 ± 13 for heterozygous TS children and 109 ± 15 for the controls.Statistical analysis revealed significant difference of IQ among them (F =10.75,P ＜ 0.05).Compared with normal controls,both monosomy TS children and heterozygous TS children showed significantly decreased volume (voxel numbers in clusters were 4117,1392,1085,t =5.75,5.33 and 5.02 for monosomy TS; voxel numbers in clusters were 4501,2437,591,t =5.40,5.11 and 4.95 for heterozygous TS respectively,P ＜ 0.01,FWE-corrected) in the gray matter of bilateral precuneus lobule,postcentral gyrus,and cingulum cortex.However,the volume of the orbitofrontal lobe,parahippocampal gyrus,cerebellum,temporal pole,corpus striatum and posterior midbrain were increased in the monosomy and heterozygous TS children compared to the controls (voxel numbers in clusters were 1444,1188,791,725,695,431,386,t =5.01,5.96,5.67,5.23,4.85,4.43,4.94 for monosomy TS; voxel numbers in clusters were 6988,2709,2510,2380,1987,1709,1185,t =6.50,7.06,7.26,5.27,5.71,6.02,4.56 for heterozygous TS,P ＜ 0.01,FWE-corrected).Compared with monosomy TS,heterozygous TS showed increased gray matter volume in the left parahippocampal gyrus and corpus striatum (voxel numbers were 1014 and 496,t =4.75,4.53,P ＜0.01,FWE-eorreeted),while they had decreased gray matter volume in the right supramarginal gyrus (voxel number was 350,t =4.28,P ＜ 0.01,FWE-corrected).Conclusions Both monosomy and heterozygous TS show brain atrophy in the parietooccipital lobe,indicating similar abnormality of gray matter development.However,heterozygous TS shows more increased gray matter volume in the prefrontal lobes and the cerebellum than monosomy TS,which may be the compensatory mechanism in this condition.

Oxidative stress could induce apoptosis and autophagy process simultaneously, but the role of autophagy is still not clear. Beclin 1, a key gene regulating the preautophagosome formation, is involved in the injury induced by oxidative stress. To observe the role of autophagy in H2O2-induced injury of U251 cells, the recombinant plasmid Psilencer3.1-siRNA-Beclin 1 was transfected into U251 cells by eukaryotic cell transfection technique. Plasmid vector and cell culture medium were used as negative and control groups respectively. The cells were collected 24 h later, and the cell total protein was extracted to detect Beclin 1, Bcl-2 and Bax protein expressions by Western blot. After the Beclin 1-siRNA cells were treated with 1 mmol/L H2O2, the autophagic vacuoles in the cells were stained with monodansylcadaverine (MDC), and the cell apoptotic ratio was determined with PI/Annexin V-FITC staining by flow cytometry analysis. The results showed that the synthetic siRNA decreased the expression of Beclin 1 protein significantly, but had no obvious effect on the levels of Bcl-2 and Bax protein expressions. Compared with those in the control group, the autophagic vacuoles, the level of LC3-II protein expression and the percentage of apoptotic cells increased (P < 0.05) in 1 mmol/L H2O2 group. In Beclin 1-siRNA + H2O2 group, autophagic vacuoles and the levels of LC3-II protein expression decreased obviously, the percentage of apoptotic cells increased significantly compared with that in 1 mmol/L H2O2 group (P < 0.05). H2O2 and autophagy inhibitor 3-methyladenine (3-MA) combination also increased the percentage of apoptotic cells obviously (P < 0.05). These results revealed that the transfection of Psilencer3.1-siRNA-Beclin 1 effectively inhibited the expression of Beclin 1 protein expression, degraded the autophagy level and increased the apoptotic rate in U251 cells under oxidative stress, which was coincident with the effect of autophagy inhibitor 3-MA. This study suggests that autophagy is a cell protective role in oxidative stress process, and the inhibition of autophagy may enhance apoptosis.
Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Autophagy ; physiology ; Beclin-1 ; Brain Neoplasms ; pathology ; Cell Line, Tumor ; Glioma ; pathology ; Humans ; Hydrogen Peroxide ; pharmacology ; Membrane Proteins ; genetics ; metabolism ; Oxidative Stress ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate whether apoptosis induced by low-dose radiation (LDR) is regulated by mitochondrial pathways in testicular cells.

METHODSMale mice were exposed to whole-body LDR, and changes in mitochondrial function and in expression of apoptotic factors were analyzed in the testicular cells as follows. Total nitric-oxide synthase (T-NOS) and Na+/K+ ATPase activities were biochemically assayed. Reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) were determined by flow cytometry using fluorescent probes. Levels of mRNAs encoding cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) were quantified by real-time reverse-transcription PCR (RT-PCR). Expression of Cyt c, AIF, caspase-9, and caspase-3 at the protein level was assessed by western blotting and immunohistochemistry.

RESULTSLDR induced an increase in T-NOS activity and ROS levels, and a decrease in Na+/K+ ATPase activity and mitochondrial Δψm, in the testicular cells. The intensity of these effects increased with time after irradiation and with dose. The cells showed remarkable swelling and vacuolization of mitochondria, and displayed a time- and dose-dependent increase in the expression of Cyt c, AIF, procaspase-9, and procaspase-3. Activation of the two procaspases was confirmed by detection of the cleaved caspases. The changes in expression of the four apoptotic factors were mostly limited to spermatogonia and spermatocytes.

CONCLUSIONLDR can induce testicular cell apoptosis through mitochondrial signaling pathways.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspases ; Cytochromes c ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Mitochondria ; drug effects ; Reactive Oxygen Species ; metabolism

OBJECTIVETo observe the effects of signal factors of corticosterone (CS), cAMP, cGMP, Ca2+ andprotein kinase C (PKC) on lymphocyte apoptosis in mouse thymus induced by X-rays of 4 Gy in vitro.

METHODSThe DNA lytic rate for thymocytes was measured by fluorospectrophotometry.

RESULTSThe DNA lyric rate for thymocytes 4-8 hours after irradiation with 2-8 Gy was significantly higher than that in the control (P<0.01). As compared with the control, the DNA lytic rate for thymocytes treated with 0.01 micromol/L CS (P<0.01), 50 ng/mL cAMP (P<0.01), 0.05-0.4 microg/mL ionomycin (Iono, P<0.05 or P<0.01) or 0.05-0.4 ng/mL phorbol myristate acetate (PMA, P<0.05 or P<0.01), respectively, was significantly increased, while the rate for thymocytes treated with 50 ng/mL cGMP was not significantly increased. The DNA lytic rate for thymocytes treated with 0.01 micromol/L CS (P<0.01), 50 ng/mL cAMP (P<0.01), 0.2 and 0.4 microg/mL Iono (P<0.05), and 0.2 and 0.4 ng/mL PMA (P<0.05) plus 4-Gy irradiation, respectively, was significantly higher than that treated with single 4-Gy irradiation, while the rate for thymocytes treated with 50 ng/mL cGMP plus 4-Gy irradiation was not increased. When both 0.4 microg/mL Iono and 0.4 ng/mL PMA acted on the thymocytes, the DNA lytic rate for thymocytes was significantly higher than that in the control (P<0.01), the DNA lytic rate for thymocytes treated with both 0.4 microg/mL Iono and 0.4 ng/mL PMA plus 4-Gy irradiation was significantly higher than that treated with single 4-Gy irradiation (P<0.05), but was not significantly higher than that treated with 0.4 microg/mL Iono plus 4-Gy irradiation or 0.4 ng/mL PMA plus 4-Gy irradiation.

CONCLUSIONCS, cAMP, Ca2+, and PKC signal factors can promote thymocyte apoptosis induced by larger dose X-rays.

Animals ; Apoptosis ; drug effects ; radiation effects ; Calcium ; pharmacology ; Corticosterone ; pharmacology ; Cyclic AMP ; pharmacology ; Cyclic GMP ; pharmacology ; Ionomycin ; pharmacology ; Male ; Mice ; Protein Kinase C ; metabolism ; Spectrometry, Fluorescence ; Tetradecanoylphorbol Acetate ; pharmacology ; Thymus Gland ; cytology ; drug effects ; X-Rays

OBJECTIVETo research the effects of chronic suppurative otitis media on bone conduction threshold in old patients.

METHODSThe files of patients with unilateral chronic otitis media were retrospective analyzed, who were all oder than 60 years, who were inpatient in our department since January 2005 to March 2009. Conventional puretone audiometry test was carried out. Bone conduction thresholds were calculated for frequencies of 0.5, 1, 2, and 4 kHz, with comparison between the ear with chronic otitis media and contralateral ear. Thresholds were examined separately for each frequency.

RESULTSThe bone conduction threshold for the normal side was lower than those for the ear with chronic otitis media. The threshold shift was statistically significant for each frequency (P < 0.01). There were no differences between the groups when analyzed for the presence of cholesteatoma except at 2 kHz frequencies (Z = -1.975, P = 0.048). There were differences between the groups when analyzed for an interruption of the ossicular chain only at 2 kHz frequencies (Z = -2.721, P = 0.007). There were differences between the groups when the duration of middle ear disease was not same at 1 kHz and 2 kHz frequencies (Z value were -2.877, -2.624, P < 0.01, respectively).

CONCLUSIONSThis study shows that chronic otitis media can enhance bone conduction threshold for old patients. All measures for early cure should be considered as early as possible in oder patients with chronic otitis media to prevent advance of sensorineural hearing loss.

Aged ; Audiometry, Pure-Tone ; Auditory Threshold ; Bone Conduction ; Chronic Disease ; Female ; Humans ; Male ; Middle Aged ; Otitis Media, Suppurative ; physiopathology ; Retrospective Studies

AIMTo evaluate the effect of paeonol on the activity of tyrosinase and provide experimental evidence for the treatment of hyperpigmentation disorders.

METHODSTyrosinase activity was estimated by measuring the oxidation rate of L-3,4-dihydroxyphenylalanine (L-Dopa). The inhibitory effects of paeonol on the activity of mushroom tyrosinase and Michaelis-Menten kinetics were deduced from the Lineweaver-Burk plots.

RESULTSThe inhibitory concentration of paeonol leading to 50% enzyme activity lost (IC50) was estimated to be 0.60 mmol x L(-1). The inhibition constants for paeonol binding free enzyme, K(I), and substrate-enzyme, K(IS), are 0.084 and 0.12 mmol x L(-1), respectively.

CONCLUSIONPaeonol is a potential mixed inhibitor of mushroom tyrosinase. The mixed inhibition function may originate from its ability to form a Schiff base with a primary amino group and to chelate copper at the active site of tyrosinase.

Acetophenones ; isolation & purification ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Kinetics ; Levodopa ; metabolism ; Monophenol Monooxygenase ; metabolism ; Paeonia ; chemistry ; Plants, Medicinal ; chemistry

Migration of vascular smooth muscle cells (VSMCs) is involved in vascular development and various vascular diseases; however, the molecular mechanisms of VSMC migration remain unclear. In this study, we established an inverted coverslip migration assay to study the migratory properties of cultured VSMCs on extracellular matrix. Pulmonary arterial smooth muscle cells (PASMCs) from rats were cultured and identified by immunocytochemistry. Each coverslip with a confluent monolayer of PASMCs was inverted to a larger coverslip which was coated with phosphate buffered saline (PBS, as a control), poly-D-lysine hydrobromide (PDL), laminin or Matrigel. After 24 h of migration over the larger coverslip, PASMCs were fixed, and reliably quantified. The roles and mechanisms of extracellular matrix in PASMC migration were further studied by wound-healing assay and immunocytochemistry. The results showed that: (1) The purity of the cultured PASMCs was (97 ± 3)%. (2) The number of PASMCs on laminin or Matrigel migrating out from the inverted coverslip was significantly increased compared with that on PBS or PDL, and the migratory distance of PASMCs on laminin or Matrigel was significantly farther than that on PBS or PDL. (3) The motility of PASMCs on laminin or Matrigel was significantly higher than that on PBS at 8 h, 12 h and 24 h after wounding, respectively. (4) F-actin staining showed that F-actin was congregated along the brim of the migrating cells from the inverted coverslip, and vinculin (a cell marker of focal adhesion) staining showed that the distribution of vinculin in PASMCs plated on laminin or Matrigel was significantly lower than that on PBS or PDL. These results suggest that the inverted coverslip migration assay is suitable to study VSMC migration, and laminin and Matrigel substrates may promote VSMC migration through inhibiting the formation of focal adhesion and regulating the cytoskeletal proteins.
Actins ; chemistry ; Animals ; Cell Adhesion ; Cell Movement ; Cells, Cultured ; Collagen ; chemistry ; Drug Combinations ; Extracellular Matrix ; chemistry ; Laminin ; chemistry ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; Proteoglycans ; chemistry ; Pulmonary Artery ; cytology ; Rats

Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Male ; Osteoarthritis ; epidemiology ; Tibet ; epidemiology