BACKGROUND: There are various methods to observe and detect early angiogenesis in the process of entochondrostosis, but each holds certain deficiencies.OBJECTIVE: To explore the possibility of tetracycline and alizarin complexone as an indirect marker ofangiogenesis in the process of entochondrostosis.METHODS: New Zealand white rabbit models of bilateral radial bone defects were prepared, followed by β-tricalcium phosphate implantation, and then given the injection of tetracycline and alizarin complexone at 1 and 15 days,respectively. Samples were collected at 28 days, some of which were observed using fluorescence/light microscope after ink perfusion and hard tissue slicing, and the others were decalcified and observed using immunohistochemistry. The uniformity between lumen structures labeled with bone affinity fluorescein and vascular structures marked by immunohistochemistry and ink perfusion was compared.RESULTS AND CONCLUSION: The lumen structure labeled with bone affinity fluorescein was confirmed to be a CD34 positive vascular structure. Under the fluorescence microscope, the bone affinity fluorescein labeled vascular morphology was consistent with ink perfusion-labeled, and black ink lines could be observed in the lumen structures labeled with bone affinity fluorescein after ink perfusion. In addition, the color of the lumen labeled with fluorescein was more gorgeous,three-dimensional structure more vivid, and the vascular evolution process distinguished more easily by different fluorescein colors, exhibiting unique advantages. Therefore, it is available to detect the early angiogenesis in the process of entochondrostosis.

BACKGROUND: Revascularization is a challenge for the tissue-engineered bone carrying cells after implanted into human body. Previous studies have found that tanshinol can improve the functions of endothelial progenitor cells and exert vascular protective effects.OBJECTIVE: To prepare the β-calcium phosphate (β-TCP) scaffold with tanshinol coating, and to observe its cytocompatibility.METHODS: The β-TCP scaffolds coated with 10-7, 10-6 and 10-5 mol of tanshinol were constructed by negative pressure absorption method. The distribution of tanshinol coating on the scaffold was observed using scanning electron microscopy,and the inner ingredients were analyzed by infrared spectrum. Human endothelial progenitor cells (hEPCs) were cultured in the extracts of β-TCP and β-TCP scaffolds with 10-7, 10-6, 10-5 mol of tanshinol coatings, respectively. The cell proliferation was detected at 2, 4, 6, 8 and 10 days of culture; the levels of nitric oxide and vascular endothelial growth factor in the supernatants were detected at 1, 7 and 14 days of culture; the lumen formation on the matrigel was observed after 14-day culture. hEPCs were respectively seeded onto the β-TCP and β-TCP scaffolds with different dosages of tanshinol coating,and then the cell growth was observed under scanning electron microscope at 7 days.RESULTS AND CONCLUSION: The tanshinol coating evenly distributed on the inner surface of the pores, and its crystalline structure became dense with dosage increasing. Infrared spectrum analysis revealed no changes in the characteristic absorption peak of tanshinol and TCP in the scaffold. The β-TCP scaffolds with tanshinol coating could promote the proliferation of hEPCs, especially the scaffolds with 10-6 and 10-5 mol tanshinol coating. Compared with the β-TCP scaffold, the scaffolds with 10-6 and 10-5 mol tanshinol coating significantly upregulated the nitric oxide level at 14 days of culture, and significantly increased the level of vascular endothelial growth factor at 7 and 14 days of culture (P <0.05). Although it could be found in all β-TCP scaffolds with tanshinol coating, the lumen formation was the maturest in the scaffold with 10-5 mol tanshinol coating. These results suggest the β-TCP scaffolds with tanshinol coating can promote the proliferation and endothelial differentiation of hEPCs, and hold a good cytocompatibility.

PURPOSE: Rheumatoid arthritis (RA) is an inflammatory joint disorder, the progression of which leads to the destruction of cartilage and bone. Chemokines are involved in RA pathogenesis. In this study, we investigated the chemokine signaling pathway associated with CCL2 in peripheral blood (PB) and synovial tissues (ST) of RA patients based on our previous work about chemokine signaling pathway involved in the activation of CCL2 production in collagen-induced arthritis rat ST. MATERIALS AND METHODS: Total RNA was isolated from PB leukocytes and synovium of the knee joint in both RA patients and control populations. Real-time polymerase chain reaction was used to determine CCL4, CCR5, c-Jun, c-Fos, and CCL2 expressions. Serum level of CCL2 was assessed by enzyme-linked immunosorbent assay, and the production of CCL2 in ST was analyzed immunohistochemically. RESULTS: The expressions of CCL4, CCR5, c-Jun, c-Fos, and CCL2 messenger RNA in RA patients were significantly higher than those in healthy controls, both in ST and on PB leukocyte. Serum CCL2 levels were elevated in RA patients. Histological examination of rheumatoid joints revealed extensive CCL2 expression in RA ST. CONCLUSION: CCL2, CCL4, c-Jun, c-Fos, and CCR5 may play an important role in the recruitment of PB leukocytes into the RA joints. These data provide evidence that the chemokine signaling pathway is involved in CCL2 expression in RA patient tissues, which may contribute to chronic inflammation associated with RA. Targeting this signaling pathway may provide a novel therapeutic avenue in RA.
Adult ; Animals ; Arthritis, Rheumatoid/*blood/metabolism ; Case-Control Studies ; Chemokine CCL2/*blood/metabolism ; Chemokines/metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Gene Expression ; Humans ; Male ; Middle Aged ; RNA, Messenger/genetics/metabolism ; Rats ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Synovial Membrane/*metabolism

OBJECTIVETo investigate contrast-enhanced ultrasound imaging features of diabetic foot ulcers.

METHODSSixteen patients with diabetic foot ulcers underwent conventional and contrast-enhanced ultrasound examinations, and the features of contrast-enhanced ultrasound imaging were analyzed. Pathological examination was also carried out in some cases.

RESULTSContrast-enhanced ultrasound showed slow enhancement in the artery phase in the 16 ulcers after administration of SonoVue. The mean time of initial enhancement was 30.02 ± 2.35 s, and the mean time for the occurrence of peak enhancement was 37.54 ± 4.13 s. In 5 cases a homogeneous enhancement pattern was clearly displayed, and in the other 11 cases, a pattern of homogenous peripheral enhancement with non-enhanced patches within the ulcers was found. Contrast-enhanced ultrasound showed a greater ulcerous area than conventional ultrasound.

CONCLUSIONContrast-enhanced ultrasound is a valuable means for evaluating the ulcerous area and the treatment efficacy for diabetic foot ulcers.

Aged ; Aged, 80 and over ; Contrast Media ; Diabetic Foot ; diagnostic imaging ; Female ; Humans ; Image Enhancement ; methods ; Male ; Middle Aged ; Phospholipids ; Sulfur Hexafluoride ; Ultrasonography

OBJECTIVETo explore the molecular basis of an individual featuring an ABx variant of ABO blood group system.

METHODSSerological assays were used to characterize the erythrocyte phenotypes and salivary ABH secretors. All of the seven exons and flanking introns of ABO glycosyltransferase gene were amplified with polymerase chain reaction (PCR). And the products were sequenced bidirectionally following enzyme digestion. Exons 6 and 7 were also subcloned and analyzed for haplotypes of the ABO gene.

RESULTSErythrocytes of the proband have expressed a strong A antigen and a weak B antigen, which was identified as a rare ABx variant in addition with other serological features. Nine heterozygous sites in exon 6 (297A/G) and exon 7 (467C/T, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 808T/A, 930G/A) of the coding region of the ABO gene were identified. Based on haplotype analysis, one allele was determined as common A102, whilst another was consistent with B101 except for an 808T>A mutation which has resulted in replacement of phenylalanine with isoleucine at position 270 of glycosyltransferase B.

CONCLUSIONThe 808T>A mutation of the glycosyltransferase B gene may decrease the enzymatic activity and result in the Bx variant.

ABO Blood-Group System ; genetics ; Adult ; Exons ; Female ; Glycosyltransferases ; genetics ; Haplotypes ; Humans ; Mutation

OBJECTIVETo investigate the recombination events between human leukocyte antigen (HLA) loci within two families.

METHODSIdentification of HLA-A, -C, -B, -DRB1 and -DQB1 loci was firstly carried out using polymerase chain reaction-sequence specific oligonucleotide. Then HLA high resolution typing was performed using polymerase chain reaction sequencing-based typing. The recombination between HLA loci was identified by family genetic analysis. The parentage possibility was analyzed by short tandom repeat technique.

RESULTSRecombination between the HLA-A and C loci was identified within two families. One individual inherited a paternal haplotype that was the result of a recombination event between the father's HLA-A and -C loci on his chromosomes. The other individual inherited a maternal haplotype that was the result of a recombination event between the mother's HLA-A and -C loci. The high parentage possibilities were obtained in the family members.

CONCLUSIONThe recombination events of HLA-A and -C have been found in two Chinese families, which may help further study on the mechanism of HLA recombination.

Asian Continental Ancestry Group ; ethnology ; genetics ; China ; ethnology ; Ethnic Groups ; genetics ; Female ; Genetic Loci ; genetics ; HLA-A Antigens ; genetics ; HLA-C Antigens ; genetics ; Haplotypes ; genetics ; Humans ; Male ; Pedigree ; Recombination, Genetic ; genetics

OBJECTIVETo analyze the sequence of the exons 2-4 of human leukocyte antigen (HLA) novel allele HLA-B*15:129.

METHODSDNA of the proband was extracted from whole blood by commercial DNA extraction kit. The amplification for HLA-B exons 2-4 was performed separately by polymerase chain reaction (PCR) with allele group specific primers. The PCR products were digested with enzymes and then directly sequenced for exons 2-4 of HLA-B locus in both directions.

RESULTSSequencing results showed the HLA-B alleles of the proband included B*07:02 and a novel allele. The sequence of the novel allele has been submitted to GenBank (accession no. EF473219) and the allele has been officially named B*15:129 by the WHO Nomenclature Committee. Comparing with the HLA-B*15:01:01:01, the sequence of exons 2-4 of HLA-B*15:129 showed three nucleotide difference in exon 3 at positions 362 and 363 from GG to AT and positions 369 from C to T, which resulted in an amino acid change from Arg to Asn at codon 97.

CONCLUSIONA novel HLA-B allele was identified and has been officially named B15:129 by the WHO Nomenclature Committee.

Alleles ; Base Sequence ; DNA Primers ; Exons ; HLA-B Antigens ; genetics ; Humans ; Male ; Molecular Sequence Data ; Molecular Typing ; Polymerase Chain Reaction

OBJECTIVETo establish the allele specific primer polymerase chain reaction sequence-based typing (PCR-SBT) and investigate the polymorphism of exon 3 of human leukocyte antigen( HLA)-DRB1.

METHODSThe fragment containing exons 2 and 3 of HLA-DRB1 gene was amplified by group specific primers. The amplified products were digested by restriction enzymes and directly sequenced in both directions. The genotype was assigned by using Assign 3.5 SBT software.

RESULTSThe exon 3 sequences of HLA-DRB1*08:09 and HLA-DRB1*12:02:01 were identified for the first time. There were 27 polymorphic sites in exon 3 among the twenty-five HLA-DRB1 alleles, which was 9.56% of all nucleotides of exon 3. The method could discriminate the HLA-DRB1*14:01:01/14:54 ambiguous samples, and the HLA-DRB1*14:01:01 was identified in the Chinese population.

CONCLUSIONThe PCR-SBT method for exon 3 of HLA-DRB1 from the present study was reliable and there were polymorphisms in exon 3 of HLA-DRB1.

Alleles ; Base Sequence ; DNA Primers ; genetics ; Evolution, Molecular ; Exons ; genetics ; Genotype ; HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Humans ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; methods ; Polymorphism, Genetic ; genetics

OBJECTIVETo analyze the molecular basis for an individual with ABx09 phenotype of ABO subtype.

METHODSThe ABO group antigens on red blood cells of the proband were identified by monoclonal antibodies, and the ABO antibody in serum was detected by standard A, B, O cells. The exons 1 to 7 of ABO gene were amplified by polymerase chain reaction (PCR) respectively and the PCR products were sequenced directly. The amplified products for exons 5 to 7 were also cloned by TOPO TA cloning sequencing kit to split the two alleles apart, selected colonies were sequenced bidirectionally for exons 5 to 7 of the ABO gene. The samples of the proband's parents were collected, then serological test of the blood group and sequence analysis for exons 6 and 7 of ABO gene were preformed.

RESULTSBoth A and B antigens were detected on red blood cells of the proband and there was anti-B antibody in the serum. There was no G deletion at position 261, while 297AG in exon 6, 467CT, 526CG, 657CT, 703GA, 796CA, 803GC, 889GA and 930GA heterozygote in exon 7 were detected by direct DNA sequencing, which can be assigned for A102Bx09 genotype. After cloning and sequencing, two alleles A102 and Bx09 were obtained. The sequence of Bx09 had one nucleotide changes (G to A) at position 889 compared with that of B101, which resulted in an amino acid change of Glu to Lys at 297 position. The Bx09 in the proband was inherited from her mother by family investigation.

CONCLUSIONG to A at nt889 of alpha-1,3 galactosyltransferasegene can result in Bx09 phenotype, with the presence of anti-B antibody in serum.

ABO Blood-Group System ; genetics ; metabolism ; Adolescent ; Alleles ; Base Sequence ; Blood Grouping and Crossmatching ; Female ; Gene Frequency ; Genotype ; Humans ; Molecular Sequence Data ; Pedigree ; Phenotype

OBJECTIVETo establish the eukaryotic cell expression system for the alpha-1,2 fucosyltransferase gene FUT1 293C to T and 658C to T mutations and explore the mechanism of FUT1 mutations resulting in the reduced expression of H antigen.

METHODSGenomic DNAs were extracted from individuals with para-Bombay phenotype and full coding region of FUT1 was amplified. The amplification fragments were ligated with pcDNA3.1 plasmid to construct the recombinant expression vectors. The recombinant plasmids were transfected into the COS-7 cells using lipofectamine transfection reagent and stable expression screening was performed. FUT1 mRNA expression was determined by real-time quantitative PCR. The activity of enzyme was measured by high performance liquid chromatography. Expressed protein was identified by SDS-PAGE and Western blotting.

RESULTSpcDNA3.1-FUT1 wildtype, pcDNA3.1-FUT1 293C to T and pcDNA3.1-FUT1 658C to T recombinant vectors were constructed, respectively. COS-7 cells with stable expression of FUT1 were obtained through recombinant plasmid transfection and screening with G418. The FUT1 mRNA level of transfected cells with 293C to T and 658C to T recombinant vectors reached 97.10% and 104.74% of the wildtype FUT1 transfected cell. A specific protein band with about 46 000 was confirmed in the transfected cell lysates by SDS-PAGE electrophoresis and Western blotting with 6×His Tag antibody. The wildtype FUT1 transfected cell lysates can catalyze the enzymatic reaction, while the enzyme activity of cell lysates from 293C to T and 658C to T were abolished.

CONCLUSIONThe results suggested that the 293C to T and 658C to T mutations of FUT1 gene did not affect the RNA and protein expression levels, but the enzyme activity of cells with FUT1 mutations was significantly decreased which resulted in the reduced expressin of H antigen.

Animals ; COS Cells ; Cercopithecus aethiops ; Fucosyltransferases ; genetics ; metabolism ; Gene Expression Regulation ; Genetic Vectors ; genetics ; Mutant Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics