OBJECTIVETo construct self-assembly fibrocartilage model of goat temporomandibular joint disc and observe the biological characteristics of the self-assembled fibrocartilage constructs, further to provide a basis for tissue engineering of the temporomandibular joint disc and other fibrocartilage.

METHODSCells from temporomandibular joint discs of goats were harvested and cultured. 5.5 x 10(6) cells were seeded in each agarose well with diameter 5 mm x depth 10 mm, daily replace of medium, cultured for 2 weeks.

RESULTSOne day after seeding, goat temporomandibular joint disc cells in agarose wells were gathered and began to self-assemble into a disc-shaped base, then gradually turned into a round shape. When cultured for 2 weeks, hematoxylin-eosin staining was conducted and observed that cells were round and wrapped around by the matrix. Positive Safranin-O/fast green staining for glycosaminoglycans was observed throughout the entire constructs, and picro-sirius red staining was examined and distribution of numerous type I collagen was found. Immunohistochemistry staining demonstrated brown yellow particles in cytoplasm and around extracellular matrix, which showed self-assembly construct can produce type I collagen as native temporomandibular joint disc tissue.

CONCLUSIONProduction of extracellular matrix in self-assembly construct as native temporomandibular joint disc tissue indicates that the use of agarose wells to construct engineered temporomandibular joint disc will be possible and practicable.

Animals ; Cells, Cultured ; Collagen Type I ; Fibrocartilage ; Glycosaminoglycans ; Goats ; Temporomandibular Joint Disc ; Tissue Engineering

OBJECTIVETo investigate the molecular subtypes of 73 strains of Yersinia enterocolitica biotype 1A isolated in Shandong province by PFGE, and thereby to analyze the relationship between PFGE typing and biological characteristics.

METHODSSeventy-three strains of Yersinia enterocolitica biotype 1A were isolated from animal feces and meat products in Gaomi city and Wulian county in Shandong province from 2008 to 2009. Motility test, serum agglutination and virulent genes detection by PCR were used to learn the biological characteristics of the isolated strains. The molecular subtypes were determined by PFGE, whose relationships with motility, serotypes and virulent genotypes were also analyzed.

RESULTSOut of the 73 strains of Yersinia enterocolitica, 5 showed medium-active motility while the other 68 showed well-active motility. The dominated serotypes were O:5(17/73) and O:8(14/73), followed by O:9(5/73) and O:7, 8(1/73), and there was no O:3 serotype found. Meanwhile, 36 strains couldn't be serotyped. All the strains were negative with the gene ail, ystA, yadA and virF, yet the positive rate of ystB gene was 72.6% (53/73). The 73 strains of Yersinia enterocolitica isolated could be subtyped into 54 PFGE patterns (K6GN11SD0001-K6GN11SD0054), most of which only had 1 or 2 isolated strains, and no pattern was dominant. The strains in the same or similar cluster were from different hosts; each serotype and toxic genotype scattered in the clustering trees, without specific correlation with PFGE subtypes. 4 out of 5 strains, which showed medium-active motility, belonged to one branch, with the similarity coefficient at 80.9% - 100.0%; while all the toxic genotype belonged to type B.

CONCLUSIONBiotype 1A Yersinia enterocolitica has many clones, whose PFGE types had relations with motility, but no relations with virulent genotype and host.

Bacterial Typing Techniques ; DNA, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Genes, Bacterial ; Genotype ; Meat Products ; microbiology ; Yersinia enterocolitica ; classification ; genetics ; isolation & purification

OBJECTIVETo know the antibiotic resistance and molecular characteristics of Listeria monocytogenes in Shandong province and to study the relationship between antibiotic resistance phenotypes and genome types.

METHODSFrom 2009 to 2010, a total of 80 Listeria monocytogenes isolates were collected from raw meat, cooked meat, aquatic products and other foods in 6 cities of Shandong province. The antibiotic susceptibility was measured by broth microdilution method, PFGE was performed for molecular typing and the relationship between antimicrobial resistance and PFGE patterns was analyzed.

RESULTS16.25% (13/80) of the isolates were drug-resistant. Imipenem resistance was the most prevalent (12.50%, 10/80), followed by tetracycline and doxycycline (3.75%, 3/80 and 2.50%, 2/80). A total of the 80 isolates were subtyped into 9 antibiotic resistance patterns and 34 PFGE types which were largely dominated by the type 17 and 29. Antibiotic resistance pattern A corresponded to 79.41% (27/34) of PFGE types.

CONCLUSIONThe antibiotic resistance of Listeria monocytogenes in Shandong province is serious from 2009 to 2010 and there is no correlation between PFGE types and antibiotic resistance patterns.

Anti-Bacterial Agents ; pharmacology ; Bacterial Typing Techniques ; China ; Drug Resistance, Bacterial ; Food Microbiology ; Listeria monocytogenes ; classification ; drug effects ; isolation & purification ; Microbial Sensitivity Tests

The study is aimed to ensure the quality and safety of medicinal plants by using ITS2 DNA barcode technology to identify Corydalis boweri, Meconopsis horridula and their close related species. The DNA of 13 herb samples including C. boweri and M. horridula from Lhasa of Tibet was extracted, ITS PCR were amplified and sequenced. Both assembled and web downloaded 71 ITS2 sequences were removed of 5. 8S and 28S. Multiple sequence alignment was completed and the intraspecific and interspecific genetic distances were calculated by MEGA 5.0, while the neighbor-joining phylogenetic trees were constructed. We also predicted the ITS2 secondary structure of C. boweri, M. horridula and their close related species. The results showed that ITS2 as DNA barcode was able to identify C. boweri, M. horridula as well as well as their close related species effectively. The established based on ITS2 barcode method provides the regular and safe detection technology for identification of C. boweri, M. horridula and their close related species, adulterants and counterfeits, in order to ensure their quality control, safe medication, reasonable development and utilization.
Base Sequence ; China ; Corydalis ; chemistry ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Molecular Sequence Data ; Nucleic Acid Conformation ; Papaveraceae ; chemistry ; classification ; genetics ; Phylogeny ; Plants, Medicinal ; chemistry ; classification ; genetics

OBJECTIVETo investigate the three-dimensional reconstruction methods of the portal vein using 64-slice spiral CT data and the anatomical variation of the portal vein.

METHODSThree-dimensional reconstruction of the portal vein was performed using Mimics software based on the 64-slice spiral CT data of 64 cases. Each model of the portal vein and its branches was evaluated according to the presentation rate, depiction quality and anatomic variation.

RESULTSThe reconstructed model showed a depiction rates of 100% for the 4-grade branches of the portal vein. The stem of the portal vein and the left and right branches of the level III or above were all displayed, but in 2 cases the superior mesenteric vein and in 1 case the spleen vein was displayed only to the level IV. Of the 64 cases, 50 (78.1%) had normal portal vein and 14 (21.9%) showed anatomical variations.

CONCLUSIONThe 3D model vividly mimics the anatomic variations of the portal vein to provide valuable information for surgical plans.

Adult ; Female ; Humans ; Image Processing, Computer-Assisted ; Imaging, Three-Dimensional ; methods ; Male ; Middle Aged ; Portal Vein ; anatomy & histology ; diagnostic imaging ; Tomography, Spiral Computed ; methods ; Young Adult

This study was aimed to explore the frequency of PTPN11 mutation in children with leukemia and its clinical significance. Genomic DNAs were extracted from peripheral leukocytes of 131 patients with leukemia, including 101 cases of ALL, 26 cases of AML, 3 cases of CML and 1 case of juvenil myelomonocytic leukemia (JMML). The sequences of PTPN11 exons 3, 8, 13 were amplified by polymerase chain reaction (PCR), and the clinical characteristics of positive patients were analyzed. The results indicated that the PTPN11 mutation was found in 10 cases (9.9%) from newly diagnosed 101 cases of ALL. Grouping the newly diagnosed ALL children by various clinical features, it was found that the PTPN11 mutation did not show associations with sex, age, white blood cell (WBC) count, prednisone test sensitivity, clinical risk and disease recurrences at the first visit (P > 0.05). PTPN11 mutations were found in 2 cases out of 26 AML patients, including one AML-M(2) and one AML-M(4). No PTPN11 mutation in 3 CML patients was found. Exon 13 mutation of PTPN11 gene was found in 1 case of JMML. It is concluded that the E76 of exon 3 is the hot spot of PTPN11 mutation in children leukemia. The novel G503E (1508G > A) mutation is detected in one JMML patient. The PTPN11 mutation does not associate with the sex, age, WBC count, prednisone sensitive test and early recurrence.
Adolescent ; Base Sequence ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Leukemia ; genetics ; Male ; Mutation ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; genetics

OBJECTIVETo develop a TaqMan real-time PCR for the detection of Aeromonas hydrophila.

METHODSThe conserved region of major adhesion gene of Aeromonas hydrophila (aha) was used to design primers and TaqMan probe. A total of six concentration gradients for forward and reverse primers ranging from 200 -700 nmol/L were chosen, and four concentration gradients for probe ranging from 100 - 400 nmol/L were chosen. And then the concentration of primers and probe were optimized by ANOVA of completely randomized design respectively. The specificity of the established method was evaluated by using bacteria as contrast, including 45 strains Vibrio cholerae, 20 strains Vibrio parahaemolyticus, 10 strains Vibrio fluvialis, 4 strains Vibrio mimicus, 5 strains Vibrio vulnificus, 1 strain Vibrio alginolyticus, 1 strain Vibrio furnissii, 5 strains Salmonella, 10 strains Shigella and 2 strains Plesiomonas shigelloides. The sensitivity, bacterial sensitivity and DNA sensitivity included,were evaluated. The stool of healthy people was contaminated by Aeromonas hydrophila artificially, and the ability of the established TaqMan real-time PCR system for detection of Aeromonas hydrophila was also evaluated.

RESULTSThe cycle threshold (Ct) value deserved from 6 groups of primers concentration gradient was (x +/- s):20.69 +/- 0.33, 20.72 +/- 0.21, 20.81 +/- 0.12, 20.74 +/- 0.12, 20.51 +/- 0.16 and 20.69 +/- 0.11, respectively, and the concentration of forward primer and reverse primer was determined to be 200 nmol/L (F = 1.33, P = 0.28). The Ct value deserved from 4 groups of probe concentration gradient was (x +/- s):20.56 +/- 0.08, 20.82 +/- 0.05, 20.82 +/- 0.11 and 20.93 +/- 0.09, respectively, and the concentration of probe was determined to be 100 nmol/L (F = 5.26, P = 0.01). The bacterial sensitivity and DNA sensitivity were 80 CFU/ml and 100 fg/microl respectively, and the sensitivity to detect Aeromonas hydrophila from stool was 8 x 10(3) CFU/ml, which might be 8 CFU/ml after 8 hours' enrichment. No amplification was observed in the templates of other bacterial.

CONCLUSIONThe TaqMan real-time PCR method targeting the aha gene of Aeromonas hydrophila had a high sensitivity and specificity and might be used to detect Aeromonas hydrophila from pure bacterial and stool rapidly.

Aeromonas hydrophila ; genetics ; DNA Primers ; Genes, Bacterial ; Molecular Probes ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Species Specificity

OBJECTIVETo examine the effects of high and low concentrations of transforming growth factor (TGF) β(1) and insulin-like growth factor-I (IGF-I) on the extracelluar matrix synthesis of the self-assembled constructs of temporomandibular joint (TMJ) disc.

METHODSThe experimental groups of self-assembled constructs were exposed to IGF-I (10, 100 µg/L) and TGF-β(1) (5, 50 µg/L), the control groups were not added with any growth factors. All groups were examined at 3 and 6 weeks for gross morphological, histological, and biochemical changes. Safranin-O/fast green staining was used to examine glycosaminoglycan (GAG) distribution, picrosirius red and immunohistochemical staining to observe type I collagen distribution. Type I collagen contents were tested by ELISA assay kit, GAG contents were measured by Blyscan GAG assay kit, and the cell numbers were quantified with a Picogreen reagent kit.

RESULTSThe growth factor groups all upregulated the matrix synthesis of the self-assembled constructs compared with control groups. TGF-β(1) (5 µg/L) and IGF-I (10 µg/L) were the two most potent concentration in increasing type I collagen and GAG synthesis and cells proliferation. IGF-I group (10 µg/L) produced nearly 2 times (109.16 ± 5.12 µg) as much type I collagen as the control group (69.13 ± 5.94 µg) at 3 weeks. The matrix contents and the number of the proliferated cells in control group and all GF groups at 6 weeks were more than those at 3 weeks.

CONCLUSIONSIGF-I (10 µg/L) is the most beneficial growth factor and can be applied in tissue-engineering stratigies of the temporomandibular joint disc. At the same time, the exposure time of growth factors is another key factor that affects matrix synthesis of TMJ disc constructs.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Extracellular Matrix ; metabolism ; Glycosaminoglycans ; biosynthesis ; Goats ; Insulin-Like Growth Factor I ; pharmacology ; Temporomandibular Joint Disc ; cytology ; metabolism ; Tissue Engineering ; methods ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo construct the cloning vector of glycoprotein G2 gene of hantavirus (HV), to analyze the sequence of G2 gene by the phylogenetic tree, and to study the differences among glycoprotein G2 genes from the world around.

METHODSEnvelope glycoprotein G2 gene was amplified from four specimens of Shandong province by RT-PCR, and the product recombined into the PMD-18T vector. The clones that carry the G2 gene were identified. After sequencing, the gene sequence was handled with the software DNASTAR, compared with 24 strains worldwide and the phylogenetic tree was drawn.

RESULTSHV G2 gene was amplified by RT-PCR from 4 specimens, named GM04-38.G2, ZB8.G2, JUN5-14.G2, RCH5.G2, respectively. The map of the phylogenetic tree showed that all the 4 strains belonged to SEO-type hantavirus. The analysis of the sequence showed that all the four HV strains had the highest rates of homology with Z37 strain. The sequence homology of SEO-type HV strains was from 82.3% to 99.8%.

CONCLUSIONThe four cloning vectors containing the glycoprotein G2 genes were successfully constructed. Envelope glycoprotein G2 gene of four specimens from Shandong province had high homology rates.

Animals ; China ; Cloning, Molecular ; Hantavirus ; genetics ; Mice ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Viral Envelope Proteins ; genetics

Objective To study the characteristics and epidemic trend of Shigella in Shandong province through the analysis of serotype, virulence genes, molecular typing and drug sensitivity. Methods The serotype was classified using the method of slide agglutination. Polymerase chain reaction (PCR) was used to amplify the related virulence genes. The molecular typing was carried out by pulsed field gel electrophoresis (PFGE), and the antibiotic sensitivity of the strains was determined by micro-broth dilution method. Results The main serogroups of 44 Shigella strains were Shigella flexneri (54.55%) and Shigella sonnei (43.18%). The carrying rates of ipaH, Set1, Sen and ial were 100%, 43.18%, 56.82% and 50.00%, respectively. By PFGE typing, the strains of Shigella flexneri were divided into 18 patterns with a low similarity. The strains of Shigella sonnei were divided into 14 patterns, and the similarity of 89.47% of the strains was more than 90%. 44 strains of Shigella had different levels of resistance to 14 of the 15 antibiotics. 93.18% of the strains were multidrug resistant. Conclusion The Shigella in Shandong province is dominated by serogroups of Shigella flexneri and Shigella sonnei, with high virulence gene carrying rate, clustering distribution and severe antibiotic resistance. It is necessary to strengthen the monitoring on serotype, traceability and antibiotic resistance of Shigella in Shandong province.