Objective To observe the influence of serum containing Rhizoma Chuanxiong on the proliferation and differentiation of mouse embryos stem cells(ESCs).Methods Mouse ESCs were co-cultured with serum containing Rhizoma Chuanxiong.The proliferation of ESCs was detected by CCK-8 method.The expression level of specific gene beta-myosin heavy chain (β-MHC) in cardiac myoblasts was detected by reverse transcription-polymerase chain reaction (RT-PCR).Results Compared with the blank rat serum group and the blank fetal bovine serum group,the differences of activities of ESCs in serum containing Rhizoma Chuanxiong group were in significant(P ＞0.05) and the expression level of specific gene β-MHC in cardiac myoblasts was increased (P ＜ 0.05).Conclusion Serum containing Rhizoma Chuanxiong can promote the differentiation of ESCs into cardiac myoblasts.

OBJECTIVETo investigate the effect of ischemic postconditioning on the expression of rat myocardium matris metalloproteinase-2 (MMP-2) induced by ischemia/reperfusion (I/R) and relationship between its expression and interstitium and the effect on left ventricular function.

METHODSTwenty-four rats were randomly divided into 3 groups (n = 8): sham control (SC) group, ischemic/reperfusion (I/R) group and ischemic postconditioning (IPTC) group. The left ventricular peak systolic pressure and its derivate (+/- dp/dt) were calculated; The amount of myocardium collagenous were determined; The vitality of superoxide dismutase (SOD) and content of malondialdehyde (MDA) of plasma were detected; The activity of myocardium MMP-2 was measured by Western blot and RT-PCR.

RESULTSAs compared with I/R group, IPTC could lower the expression of MMP-2, ameliorate left ventricular function and increase the content of myocardium collagenous. In the meantime, the vitality of superoxide dismutase (SOD) of plasma were greatly enhanced and the content of malondialdehyde (MDA) of plasma were reduced in IFC group.

CONCLUSIONProtective effect of IPIC on myocardium may be due to reduce free radical, lower expression of MMP-2 and protect myocardial interstitium. MMPs plays an important role in the myocardial protection provided by IPTC.

Animals ; Collagen ; metabolism ; Ischemic Postconditioning ; Malondialdehyde ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Myocardial Reperfusion Injury ; enzymology ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

OBJECTIVETo study the effect of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2.

METHODSRecombinant plasmids of expressing short hairpin RNA targeting PC-1 mRNA were constructed using DNA recombinant technology and transfected into C4-2 cells via liposome. The positive cell clones were selected by G418. The expression of PC-1 gene was analyzed by RT-PCR and Western blotting technology. MTT and soft agar cloning formation were applied to observe the changes of the growth rate and independent anchor ability of C4-2 cells.

RESULTSPC-1 RNA interference severely affected the expression of PC-1 gene and reduced the growth and colony formation ability of C4-2 cells.

CONCLUSIONRNA interference-mediated PC-1 gene knockdown can decrease the growth and cloning formation ability of C4-2 cells.

Cell Line, Tumor ; Down-Regulation ; Gene Expression ; Humans ; Male ; Phosphoric Diester Hydrolases ; biosynthesis ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Pyrophosphatases ; biosynthesis ; genetics ; RNA Interference ; RNA, Messenger ; genetics ; Transfection

Recently, we have reported a new gfp gene isolated from Aequorea macrodactyla. The protein purified from expressed E. coli exhibited an excitation peak at 476 nm and an emission peak at 496 nm. However, the drawback of only maturing to fluorescence at low temperature limited its applications. In this paper, we further describe twelve mutants of GFPxm. Seven mutants produced enhanced fluorescence when expressed in E. coli at higher temperature (37 degrees C). After six hours of induction at 25 degrees C, 32 degrees C and 37 degrees C respectively, the relative fluorescent intensities of GFPxm16, GFPxm18 and GFPxm19 were higher than that of EGFP, moreover GFPxm16 and GFPxm163 could preserve high fluorescent intensity even expressed at 42 degrees C. Four mutants of the seven could reach high expression level in three kind of mammalian cells. Another 6 mutants had red-shift of excitation-emission maxima, and longest excitation-emission maxima were 514nm and 525nm. Another three mutants had two excitation peaks, and one mutant had only one UV-excitation peak. The most exciting result is the mutant of OFPxm with orange color. The mutant has an excitation peak at 509 nm and an emission peak at 523nm. 523nm is yellowish green but the protein is orange observed by eyes. The mutant could reach high expression level and matured at higher temperature but the fluorescent intensity was comparatively low because of low quantum yield.
Animals ; Electrophoresis, Polyacrylamide Gel ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Mutation ; Temperature

OBJECTIVETurner's syndrome (TS) is characterized by the absence of an X chromosome or the presence of a structurally abnormal X chromosome in a phenotypic female. It was recently reported that autoimmune thyroiditis (AIT) was found in 38% of white patients with TS, and few studies in this aspect have been conducted in China. The purpose of this study was to determine the frequency of AIT among TS patients and risk factors for development of thyroid dysfunction in Chinese children with TS.

METHODSSerum antithyroglobulin antibody (TgAb), thyroperoxidase antibody (TPOAb) and thyroid function (T(3), T(4) and TSH) of 24 children with TS (mean age 12.9 +/- 2.4 years, range 4.8 - 16.8 years) were assessed. Their karyotype distribution was as follows: thirteen patients with 45, XO kayrotype, eight patients with structurally abnormal X chromosome, two with X mosaic kayrotype and one with 46, XX. Techniques including radioimmunoassy and elctro-chemiluminescence immunoassy were used in this study. All TS children were divided into two groups. Group one was thyroid autoantibodies (TAA)-positive group, the levels of TgAb and/or TPOAb in them were higher than the normal levels (TgAb < 30%, TPOAb < 20%), respectively, and the remaining patients were assigned into TAA-negative group.

RESULTSSeven of the 24 (29%) patients had higher levels of TgAb and TPOAb than the normal values (< 30% and < 20%). The level of serum TSH [6.1 (3.6-100.0) mU/L] in TAA-positive group was significantly higher than that [3.9 (1.7-7.9) mU/L] in TAA-negative group (P < 0.05). The frequency of hypothyroidism or subclinical hypothyroidism in TAA-positive group (5/7) was higher than that in TAA-negative group (3/17) (P < 0.05).

CONCLUSIONThe positive rate of serum TAA in children with TS was 29%. About 70% TS children with positive serum TAA developed hypothyroidism or subclinical hypothyroidism. The results have provided the basis for regular follow-up assessment of thyroid autoantibodies and thyroid function in children with TS, and these measures are of importance for timely diagnosis of thyroid dysfunction and application of appropriate treatment.

Adolescent ; Age Determination by Skeleton ; Autoantibodies ; blood ; Child ; Child, Preschool ; Female ; Humans ; Iodide Peroxidase ; immunology ; Thyroid Gland ; physiopathology ; Turner Syndrome ; immunology ; physiopathology

BACKGROUND: The balloon dilatation technique plays an important role in the correction of kyphosis. A balloon catheter can enlarge the spinal cavity in kyphoplasty followed by injection of bone cement under low pressure to lay a foundation for the stability of the spine. OBJECTIVE: To explore the feasibility of balloon dilation with injectable calcium sulfate cement for tibial plateau fractures and to analyze its clinical effect. METHODS: Twenty-four upper tibia specimens of the adults were taken to make the Schatzker Ⅲ collapsed fracture model of the tibial plateau. Then, these specimens were randomized into three groups: the standard group was subjected to poking reduction with autologus bone grafting and screw internal fixation, the bone cement group was inflated with balloon dilatation followed by calcium sulfate cement injection, and the combined group was treated with fixation with cancellous bone screws and balloon dilatation followed by injection of calcium sulfate cement. The general situation of reduction and fixation was observed and the reduction effect was measured. RESULTS AND CONCLUSION: (1) Fixation effect in the model: All three models were well reset, and the average displacement of the standard group, the simple bone cement group and the bone cement screw group was (-0.22±0.62), (-0.23±0.67), and (-0.20±0.69) mm, respectively. There was no significant difference in the displacement between the three groups (P ＞ 0.05). (2) Clinical application: One case of Schatzker type Ⅱ fracture of the left tibial plateau was treated with cancellous bone screw fixation and balloon dilatation followed by injection of calcium sulfate cement. X-ray results showed calcium sulfate cement was visible at 3 postoperative days. At 30 postoperative days, the patient presented with good joint range of motion, and the calcium sulfate was partially absorbed on the X-ray film. At 60 postoperative days, the patient appeared to have no joint extension disorder, and fracture healing and absorption of calcium sulfate as shown by X-rays. To conclude, the balloon dilation with injectable calcium sulfate cement for the treatment of tibial plateau fracture is feasible and has clinical value.

BACKGROUNDOsteochondral lesion repair is a challenging area of orthopedic surgery. Here we aimed to develop an extracellular matrix-derived, integrated, biphasic scaffold and to investigate the regeneration potential of the scaffold loaded with chondrogenically-induced bone marrow-derived mesenchymal stem cells (BMSCs) in the repair of a large, high-load-bearing, osteochondral defect in a canine model.

METHODSThe biphasic scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and characterized by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). Osteochondral constructs were fabricated in vitro using chondrogenically-induced BMSCs and a biphasic scaffold, then assessed by SEM for cell attachment. Osteochondral defects (4.2 mm (diameter) × 6 mm (depth)) were created in canine femoral condyles and treated with a construct of the biphasic scaffold/chondrogenically-induced BMSCs or with a cell-free scaffold (control group). The repaired defects were evaluated for gross morphology and by histological, biochemical, biomechanical and micro-CT analyses at 3 and 6 months post-implantation.

RESULTSThe osteochondral defects of the experimental group showed better repair than those of the control group. Statistical analysis demonstrated that the macroscopic and histologic grading scores of the experimental group were always higher than those of the control group, and that the scores for the experimental group at 6 months were significantly higher than those at 3 months. The cartilage stiffness in the experimental group (6 months) was (6.95 ± 0.79) N/mm, 70.77% of normal cartilage; osteochondral bone stiffness in the experimental group was (158.16± 24.30) N/mm, 74.95% of normal tissue; glycosaminoglycan content of tissue-engineered neocartilage was (218 ± 21.6) µg/mg (dry weight), 84.82% of native cartilage. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at 3 and 6 months, with no significant difference between the experimental and control groups.

CONCLUSIONThe extracellular matrix-derived, integrated, biphasic scaffold shows potential for the repair of large, high-load-bearing osteochondral defects.

Animals ; Bone Marrow Cells ; cytology ; Bone Regeneration ; physiology ; Cartilage, Articular ; surgery ; Dogs ; Extracellular Matrix ; chemistry ; Mesenchymal Stromal Cells ; cytology ; ultrastructure ; Microscopy, Electron, Scanning ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry ; X-Ray Microtomography

BACKGROUNDCartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage. We had previously developed a natural, human cartilage extracellular matrix (ECM)-derived scaffold for in vivo cartilage tissue engineering in nude mice. However, before these scaffolds can be used in clinical applications in vivo, the in vitro effects should be further explored.

METHODSWe produced cartilage in vitro using a natural cartilage ECM-derived scaffold. The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy (SEM), micro-computed tomography (micro-CT), histological staining, cytotoxicity assay, biochemical and biomechanical analysis. After being chondrogenically induced, the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry. The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining. Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.

RESULTSSEM and micro-CT revealed a 3-D interconnected porous structure. The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris, and stained positive for safranin O and collagen II. Viability staining indicated no cytotoxic effects of the scaffold. Biochemical analysis showed that collagen content was (708.2-44.7) µg/mg, with GAG (254.7 ± 25.9) µg/mg. Mechanical testing showed the compression moduli (E) were (1.226 ± 0.288) and (0.052 ± 0.007) MPa in dry and wet conditions, respectively. Isolated canine bone marrow-derived stem cells (BMSCs) were induced down a chondrogenic pathway, labeled with PKH26, and seeded onto the scaffold. Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM. The cell-scaffold constructs contained pink, smooth and translucent cartilage-like tissue after 3 weeks of culture. We observed evenly distributed cartilage ECM proteoglycans and collagen type II around seeded BMSCs on the surface and inside the pores throughout the scaffold.

CONCLUSIONThis study suggests that a cartilage ECM scaffold holds much promise for in vitro cartilage tissue engineering.

Animals ; Biomechanical Phenomena ; Cartilage ; cytology ; Cell Survival ; Cells, Cultured ; Dogs ; Extracellular Matrix ; physiology ; Humans ; Immunohistochemistry ; Male ; Mesenchymal Stromal Cells ; cytology ; Tissue Engineering ; methods ; Tissue Scaffolds

BACKGROUNDThe genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) includes sequences encoding the putative protein X4 (ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix, indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection.

METHODSThe prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV-infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over-expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles.

RESULTSWe expressed and purified soluble recombinant protein X4 from E.coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6 cells. It was also detected in the lung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 3T3 cells as determined by cell counting and MTT assays.

CONCLUSIONThe results provide the evidence of protein X4 expression following SARS-CoV infection, and may facilitate further investigation of the immunopathological mechanism of SARS.

Amino Acid Sequence ; Animals ; BALB 3T3 Cells ; Cercopithecus aethiops ; Growth Inhibitors ; analysis ; physiology ; HeLa Cells ; Humans ; Immunohistochemistry ; Lung ; chemistry ; Mice ; Molecular Sequence Data ; SARS Virus ; chemistry ; Severe Acute Respiratory Syndrome ; metabolism ; Vero Cells ; Viral Structural Proteins ; analysis ; physiology

BACKGROUNDEchinococcosis, coenurosis and cysticercosis are debilitating diseases which prevail in China. Immunological diagnosis of metacestodosis is important in disease control. The 8-kDa glycoproteins from taeniid cestodes have successfully been used for diagnosis of human cysticercosis in immunological assays. The aim of the present study was to investigate genetic variations and phylogenetic relationships of the 8-kDa proteins for evaluating the possibility of utilizing these proteins as diagnostic antigens for other metacestode infections.

METHODSThe genes and complementary DNAs (cDNAs) encoding the 8-kDa proteins from Echinococcus (E.) granulosus, Taenia (T.) multiceps and T. hydatigena were amplified using PCR method. Their amplicons were cloned into the vector pMD18 and the positive clones were sequenced. Sequence data were analyzed with the SeqMan program, and sequence homology searches were performed using the BLAST program. Alignments were conducted using the ClustalX program, and the phylogenetic analyses were performed with the Protein Sequences Program and the Puzzle Program using the Neighbor-joining method.

RESULTSFifteen, 18 and 22 different genomic DNA sequences were identified as members of the 8-kDa protein gene family from E. granulosus, T. multiceps and T. hydatigena, respectively. Eight, four and six different cDNA clones respectively from E. granulosus, T. multiceps and T. hydatigena were characterized. Analysis of these sequences revealed 54 unique 8-kDa protein sequences. Phylogenetic trees demonstrated that the taeniid 8-kDa proteins are clustered into eight clades at least: Ts18, Ts14, TsRS1, TsRS2, T8kDa-1, T8kDa-2, T8kDa-3 and T8kDa-4.

CONCLUSIONWe found that the gene family encoding for the taeniid 8-kDa antigens is comprised of many members with high diversity, which will provide molecular evidence for cross-reaction or specific reaction among metacestode infections and may contribute to the development of promising immunological methods for diagnosis of metacestodosis.

Amino Acid Sequence ; Animals ; DNA, Helminth ; genetics ; Echinococcus granulosus ; genetics ; metabolism ; Genetic Variation ; genetics ; Glycoproteins ; chemistry ; classification ; genetics ; Helminth Proteins ; chemistry ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Sequence Homology, Amino Acid ; Taenia ; genetics ; metabolism