BACKGROUND: The main role that the osteoprotegedn (OPG) plays in bone tissues is to inhibilate the formation and the activity of osteoclast, while as for the receptor activator of nuclear factor-κB ligand (RANKL), it is to stimulate the differentiation and the activity of osteoclast. Both OPG and RANKL are important for the regulation of osteoclastic function. Recent studies have found the stimulative effects of adiponectin on the proliferation and the differentiation of osteoblast, as well as the coupling between adiponectin and bone metabolism. But effects of adiponectin on osteoclast remain unclear. OBJECTIVE: To investigate the effect of adiponectin on the expression of OPG and RANKL in osteoblast, and to further analyze its effects on the formation of osteoclast. DESIGN, TIME AND SETTING: A contrast observational experiment was conducted in the laboratory of Institute of 2008.MATERIALS: Cancellous bone in anterior superior lilac spine was obtained from adult normal by surgery for cell incubation. Clinical samples were provided by the Xiangya Hospital. METHODS: Human osteoblast was incubated with different doses of adiponcetin (0, 3, 10 and 30 mg/L) for 48 hours, after which OPG and RANKL mRNA and protein expression level was determined. In addition, adiponcetin was added into the co-culture system of osteoblast and peripheral blood monouclear cells for examing its effects on osteoclast formation. MAIN OUTCOME MEASURES: OPG and RANKL mRNA and protein expression in human osteoblast was determined using fluorescent quantitative polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA). Osteoclast was detected by antitartaric acid acid phosphatase staining. RESULTS: Adiponcetin inhibited osteoblast OPG mRNA and protein expression in a dose-dependent way (P < 0.05). Adiponcetin promoted osteoblast RANKL mRNA and protein expression in a dose-dependent way (P < 0.05). Adiponectin induced the osteoclasts formation in the co-culture system of osteoblast and peripheral blood monouclear cells. CONCLUSION: Adiponectin has the effect of inducing osteoclast formation via stimulating RANKL expression and inhibiting OPG

Objective :To establish a rapid molecular identification method for Fallopia multiflora and its adulterants. Methods: Based on psbA-trnH sequences of F. multiflora and its adulterants, the SNP site was searched and the specific primers were designed. The allele-specific PCR amplification of F. multiflora and its adulterants from different producing areas was carried out and the reaction system was optimized. Results: When the annealing temperature was raised to 48 ℃ with 30 cycle number, only the template DNA of F. multiflora could be amplified to obtain the specific 191 bp band whereas the diagnostic PCRs of the other adulterants were all negative. Conclusion: It’s simple and reliable to identify the authenticity of F. multifloa by allele loci specific PCR.

Aged ; Coal Mining ; Humans ; Male ; Middle Aged ; Pneumoconiosis ; complications ; diagnostic imaging ; Pulmonary Heart Disease ; diagnostic imaging ; etiology ; Sensitivity and Specificity ; Tomography, Spiral Computed ; methods

OBJECTIVETo investigate the feasibility of life span extension of transformed human embryonic tendon cells (THETC) by reconstitution of the telomerase activity.

METHODSTHETC were transfected by pGRN145 plasmid containing the human telomerase reverse transcriptase (hTERT) cNDA in vitro by molecular cloning technique. The biological characteristics of transfected cells were detected and compared by morphological observation, plate cloning efficiency, soft agar culture, growth curve of cells cultured in different conditions, immunohistochemistry, telomerase activity assay by telomeric repeat amplification protocol (TRAP).

RESULTSThe THETC transfected by pGRN145 plasmid (telT) could express the telomerase activity with extension of life span. The telT maintained the original characteristics of temperature-dependant and serum-dependant, as well as secretion of type I collagen normally and without tendency of malignant transformation.

CONCLUSIONSThe life span of THETC can be prolonged by reconstitution of telomerase activity, which provides the novel experimental methods to establish the standard cells line.

Cell Line ; Cell Survival ; Embryo, Mammalian ; Humans ; Plasmids ; genetics ; RNA-Directed DNA Polymerase ; Telomerase ; genetics ; metabolism ; Tendons ; cytology ; enzymology ; Transfection

We wished to understand the genetic recombination and phylogenetic characteristics of human en- terovirus A71 (EV-A71) and to explore its potential virulence-related sites. Full-length genomes of three EV-A71 strains isolated from patients in Chenzhou City (China) were sequenced and analyzed. Possible re- combination events and crossover sites were analyzed with Recombination Detection Program v4. 1. 6 by comparison with the complete genome sequences of 231 strains of EV-A71. Similarly, plot and bootscanning analyses were undertaken with SimPlot v3. 5. 1. Phylogenetic trees based on the sequences of VP1 regions were constructed with MEGA v5. 2 using the Kimura two-parameter model and neighbor-joining method. Results suggested that recombination events were detected among the three EV-A71 isolates from Chenzhou City. The common main parent sequence was from JF799986 isolated from samples in Guang- zhou City (China) in 2009, and the minor parent sequence was TW/70516/08. Intertypic recombination e- vents were found in the C4b strain (strain SHZH98 isolated in 1998) and C4a strain (Fuyang strain isola- ted in 2008) with the prototype strains of CVA4 and CVA14 in the 3D region. The chi-square test was used to screen-out potential virulence-related sites with nucleotide substitutions of different types of hand, foot, and mouth disease (HFMD) cases using SPSS v19.0. Results suggested that there were no significant nucleotide substitutions between death cases and severe-HFMD cases. Eighteen significant nucleotide substitutions were found between death/severe-HFMD cases and mild-HFMD cases, and all these 18 substitutions were distributed only in P2 and P3 regions. Intertypic recombination among the predominant circulating EV-A71 strains in the Chinese mainland and other EV-A strains probably dates before 1998, and intratypic recombination might have occurred frequently in the HFMD outbreak from 2008 to 2012. Substitutions in the non-capsid region may be correlated with the changes in virulence of EV-A71. These data suggest that researchers should pay more attention to the relationships between substitutions in the noncapsid region and the virulence of the virus.
Enterovirus A, Human ; genetics ; pathogenicity ; Mutation ; Phylogeny ; Recombination, Genetic ; Virulence

Human placental decidua basalis-mesenchymal stem cells (PDB-MSCs) are multipotent cells from the human term placenta, which are ethically conducive, easily accessible and high-yielding source. PDB-MSCs can differentiate into adipogenic, osteogenic and neurogenic cells under appropriate conditions, which may be an attractive and alternative source of seed cells for tissue engineering. To investigate the effect of hypoxia (1% O2) on human PDB-MSCs and the expression of cytokine, PDB-MSCs were isolated from human placenta by density gradient centrifugation and cultured in the Dulbecco's modified Eagle's medium-high glucose (DMEM-HG) containing 10% fetal bovine serum (FBS), and the fifth passage of PDB-MSCs were taken. PDB-MSCs were divided into 4 groups according to the concentrations of O2 and FBS: 20% O2, 10% FBS; 20% O2, 0% FBS; 1% O2, 10% FBS; 1% O2, 0% FBS. The proliferation and apoptosis of PDB-MSCs were detected by MTT and flow cytometric analysis at the time points of 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h, respectively. Vascular endothelial growth factor (VEGF) released from PDB-MSCs was detected by enzyme-linked immunosorbent assay (ELISA) at the same time points. The results showed that hypoxia enhanced the proliferation of PDB-MSCs at 12 h under the condition of 10% FBS, while at 24 h under the condition of 0% FBS (P<0.01, n=3). In normoxia, the cells cultured in 10% FBS displayed a significant proliferation compared to those cultured in 0% FBS. However, in hypoxia, the number of cells cultured in 0% FBS (serum deprivation) increased significantly compared to that cultured in 10% FBS at 24 h and 96 h respectively (P<0.05, P<0.01, n=3). With the flow cytometric analysis of cell apoptosis under the condition of hypoxia and serum deprivation, we found that hypoxia and serum deprivation did not induce PDB-MSCs apoptosis (P>0.05, n=3). This conclusion may relate to the expression of VEGF which needs further research. In conclusion, the results obtained indicate that PDB-MSCs are able to bear hypoxia and serum deprivation, suggesting that PDB-MSCs can be used as seed cells for ischemia related tissue engineering.
Apoptosis ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Decidua ; cytology ; Female ; Humans ; Mesenchymal Stromal Cells ; cytology ; Placenta ; cytology ; Pregnancy ; Tissue Engineering ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the growth and development of brain derived neurophic factor(BDNF)-positive neurons in the frontal lobe of human fetus.

METHODSThe expression of the BDNF-positive neurons in the frontal lobe of human fetus in the 2(nd),3(rd),and 4(th) month of gestation were observed with the streptavidin-biotin-complex/immunoperoxidase(SABC)method.

RESULTSBy the second month of gestation,BDNF-positive neurons were seen in the subventricular layer of the frontal lobe of cerebellum.By the third month of gestation,BDNF-positive neurons in the central layer were in various shapes,with big nucleus,less cytoplasm,and small processes.By the fourth month of gestation,BDNF-positive neurons in the central layer grew larger in size,cytoplasm increased,the BDNF-positive expression was enhanced with deeper dyeing,and the nerve fibers and particles were distributed between neurons;also,the BDNF-positive neurons were seen in the marginal layer of the frontal lobe of cerebrum.

CONCLUSIONBDNF-positive neurons may participate in the early development of the frontal lobe of cerebrum of human fetus.

Brain-Derived Neurotrophic Factor ; metabolism ; Fetus ; metabolism ; Frontal Lobe ; embryology ; Humans ; Neurons ; cytology ; metabolism

Objective To discuss the clinical features of cerebral schistosomiasis. Methods The clinical data of fourteen patients with cerebral schistosomiasis from March 2010 to March 2016 were collected and analyzed retrospectively. Results The schistosomiasis immunological tests of sera and cerebrospinal fluids from the fourteen patients were all positive. Eosinophils increased in ten cases,and the proportion was 5.1％-60.3％. Schistosoma eggs were found in seven cases by the fecal Kato-Katz method. Fourteen cases were all infected with Schistosome japonicum. Twelve cases were diagnosed as chronic type,and two cas-es as acute type. Thirteen patients received medical treatment,of which twelve were cured,and one improved. One patient re-ceived the surgical resection of the lesion. Conclusion The clinical manifestations of cerebral schistosomiasis mainly include seizure,headache,dizziness and fever. In the enhanced head magnetic resonance imaging(MRI),the lesions are clustered and merged into lumps,which is the characteristic image of cerebral schistosomiasis japonica. The praziquantel treatment can achieve a good prognosis.

OBJECTIVE: To construct two types of Wnt-inducible secreted protein 3(WISP3) gene's mutants(1000T/C,840delT) found in spondyloepiphyseal dysplasia tarda with progressive anthopathy (SEDT-PA) patients, and to observe their expression in COS-7 cells. METHODS: Full-length cDNA of wild type WISP3 gene(WT-WISP3) was amplified from human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes(MUT1000T/C and MUT840delT). The recombined plasmids WT-WISP3/pcDNA3.1(+), MUT1000T/C/pcDNA3.1(+) and MUT840delT/pcDNA3.1(+) were transfected transiently into COS-7 cells by liposome-mediated method, and pcDNA3.1(+) vector was used as a control. The total RNA and protein of the transfected COS-7 cells were extracted after 48 hours of transfection. The expression of WISP3 gene in the transfected COS-7 cells was detected by semi-quantitative RT-PCR and Western blot. RESULTS: By restriction endonuclease analysis and sequencing, the sequence of MUT1000T/C and MUT840delT were consistent with that mutated in SEDT-PA, and the open reading frames matched with the vector sequence. Semi-quantitative RT-PCR and Western blot showed that the recombined plasmids were highly expressed in COS-7 cells. CONCLUSION WISP3 gene's mutants of SEDT-PA are successfully constructed by genetic recombination, and expressed in COS-7 cells, which lays the foundation for the further study on its molecular functions in SEDT-PA.
Animals ; Base Sequence ; CCN Intercellular Signaling Proteins ; COS Cells ; metabolism ; Chlorocebus aethiops ; Humans ; Insulin-Like Growth Factor Binding Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation ; Osteochondrodysplasias ; genetics ; metabolism ; Transfection

OBJECTIVETo explore an effective method to culture oral mucosa epithelial cells (OMECs) of canine in vitro, and to observe the biological characteristics of OMECs growing on small intestinal submucosa (SIS) in order to provide the experimental basis for epithelium tissue engineering.

METHODSThe primary OMECs were cultivated with DKSFM (defined keratinocyte serum free medium) containing 6% fetal bovine serum (FBS). The morphological characteristics and the growth curve of OMECs were observed. The expressions of OMECs marker (CK19) were examined by immunocytochemistry. The 2nd passage of OMECs were seeded on SIS, OMECs co-cultured with SIS were observed by hematoxylin-eosin staining, immunohistochemical staining, and scanning electron microscope (SEM).

RESULTSOMECs were grown well in DKSFM. Immunohistochemical staining of the 2nd passage cultured canine OMECs with broadly reacting anti-cytokeratin anyibodies (CK19) was positive. OMECs formed a single layer on the surface of SIS, and eight days later the cells were polygong and arranged like slabstone.

CONCLUSIONCulture of canine OMECs in DKSFM containing 6% FBS is a simple and feasible method. SIS has good biocompatibility, it is a kind of good bioscafold in the tissue-engineered epithelium.

Animals ; Cattle ; Cell Culture Techniques ; Cells, Cultured ; Coculture Techniques ; Epithelial Cells ; In Vitro Techniques ; Intestine, Small ; Mouth Mucosa ; Tissue Engineering