Objective: To establish a method for isolation and purification of fetal membrane derived adherent cells (FMDACs) , and investigate their biological characteristics. Method: FMDACs were isolated with trypsin inducing and cultured in vitro. FMDACs were induced to differentiate into osteoblasts and adipocytes. FACS and immunocytochemistry technique were used to examine the cell surface antigen. The genetic stability was verified by karyotype analysis. Results: FMDACs were successfully isolated and expanded in vitro. They had strong proliferative ability. FMDACs were positive for CD44 and CD29, but negative for CD34, CD14 and CD45. FMDACs were differentiated into osteoblasts and adipocytes after inducement. The karyotype was stable in the sixth-passaged FMDACs and the tumorigenicity was not found. Conclusion; FMDACs have the possibility of multipotent stem cells, which have strong capacities of self-renewal and multidirectional differentiation. The genetic background of FMDACs is stable. FMDACs may be used as a kind of novel seed cells for tissue engineering.

OBJECTIVETo use a glycemic method to screen hepatocellular carcinoma (HCC) metastasis related aberrant 1-6 fucosylated glycoproteins, and to analyze the metastasis-related alterations of annexin II.

METHODS2-DE coupled with lectin affinity blot, lectin affinity precipitation followed by MALDI-TOF-MS/MS was established to screen glycoproteins related to HCC metastasis. Immunofluorescence analysis, Western blot and real-time PCR were performed on higher and lower metastatic HCC cell lines to detect the protein expression levels and mRNA levels of annexin II.

RESULTSThe Lens culinaris agglutinin (LCA) affinity glycoprotein profiles from MHCC97-L and MHCC97-H cells were differentially displayed when compared with Hep3B. Annexin II was identified by MALDI-TOF-MS/MS and its increased core-fucosylation was associated with HCC metastasis and it was confirmed. In addition, we found that annexin II was distributed in the cytoplasm and it had higher protein and gene expressions in MHCC97-L and MHCC97-H cells than in Hep3B cells.

CONCLUSIONOur results suggest that the increase of annexin II and its expression levels, and the increase of core-fucosylation might all be related to HCC metastatic ability.

Annexin A2 ; analysis ; genetics ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Line, Tumor ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression ; Humans ; Neoplasm Metastasis ; Neoplasm Proteins ; analysis ; genetics

OBJECTIVETo explore the expression of X-linked inhibitor of apoptosis protein (XIAP) in Tca8113 cell, and to investigate its relationship to the chemoresistance. METHODS; The Tca8113 cell line was cultured by IMDM and the concentration of Pingyangmycin (PYM) added to Tca8113 cell line was increased gradually and continually, which was to induce the PYM-resistance in Tca8113 cell line. The sensitivity of Tca8113 cell to PYM and expression of XIAP were measured with methyl thiazolyl tetrazolium (MTT) chromatometry and reverse transcription-polymerase chain raction (RT-PCR). The XIAP level in the cells and its chemoresistance to PYM were analyzed by linear regression.

RESULTSThe IC50 of Tca8113-1-10 group and Tca8113-10-10 group were(12.758 +/- 0.030), (18.986 +/- 0.150) microg x mL(-1) respectively. The IC50 of Tca8113-1-20 group and Tca8113-10-20 group increased to (26.302 +/- 0.072), (35.294 +/- 0.115) microg x mL(-1) respectively. There was a relation between XIAP and the drug-resistance in Tca8113 cell.

CONCLUSIONXIAP may play an important role in the chemoresistance which might serve as a new therapeutic target for oral squamous cell carcinoma.

Apoptosis ; Bleomycin ; analogs & derivatives ; Carcinoma, Squamous Cell ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Humans ; X-Linked Inhibitor of Apoptosis Protein

Cannula ; Catheters ; Drainage ; Duodenal Diseases ; Humans ; Intestinal Fistula/surgery*

Chromatography, High Pressure Liquid ; methods ; Cyclic AMP Response Element-Binding Protein ; genetics ; Female ; Humans ; Male ; Nerve Tissue Proteins ; genetics ; Prenatal Diagnosis ; methods ; RNA-Binding Proteins ; genetics ; SMN Complex Proteins ; Sequence Analysis, DNA ; Spinal Muscular Atrophies of Childhood ; diagnosis ; genetics

OBJECTIVETo construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells.

METHODSThe recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006). These three fragments were ligated to generate a minidystrophin gene. The enhanced green fluorescent protein (EGFP) gene was fused to the C terminal of the minidystrophin gene, and then the pHrnDysG was finally obtained by cloning the fusion gene to pHrneo. Fluorescence microscope and RT-PCR were used to detect the expression of minidystrophin-EGFP fusion gene after the recombinant construct was transfected into Cos-7 cells by lipofectamine.

RESULTSRestrictive enzyme digestion analysis and sequencing confirmed that pHrnDysG vector was constructed successfully. After the recombinant pHrnDysG was transfected to Cos-7 cells, RT-PCR demonstrated that the fusion gene was successfully transcribed, and the green fluorescence was observed at the cell membrane.

CONCLUSIONThe minidystrophin-EGFP fusion gene mediated by pHrneo vector could express in Cos-7 cells and its products' localization in the cell membrane was the same as that of full length dystrophin. These results suggested that the recombinant human source vector pHrnDysG might be potentially used in studies on the gene therapy of Duchenne muscular dystrophy.

Animals ; COS Cells ; Cercopithecus aethiops ; Dystrophin ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Fluorescence ; Models, Genetic ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo identify the pathogenic gene for a non-syndromic hearing loss family.

METHODSMutation analysis was carried out by polymerase chain reaction and direct sequencing of all exons of SLC26A4 (solute carrier family 26, member 4) gene.

RESULTSCompound heterozygous mutations N392Y and S448X were detected in the proband of the family, heterozygous mutation S448X was detected in the father, heterozygous mutation N392Y was detected in the mother.

CONCLUSIONThe proband's hearing loss resulted from the compound heterozygous mutations N392Y and S448X for SLC26A4 gene.

Adult ; Base Sequence ; DNA Mutational Analysis ; Deafness ; diagnostic imaging ; genetics ; pathology ; Family Health ; Female ; Humans ; Male ; Membrane Transport Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; Tomography, X-Ray Computed

OBJECTIVETo study the gene mutation in a patient with multiple exostoses, identify the disease-causing gene mutation.

METHODSPolymerase chain reaction and DNA sequencing were used to screen the EXT1 or EXT2 gene mutation, while mismatch primer amplification and restriction endonuclease digestion were performed to confirm the mutation.

RESULTSBy DNA sequencing, a mutation in the seventh intron was detected and located at 26 bp of 3' splice site upstream in EXT1 gene, which was unreported before. Mismatch primer amplification and restriction fragment length polymorphism analysis suggested that this mutation was not detected in the normal control.

CONCLUSIONThe mutation 1633-26(C-->A) may be the disease-causing mutation in this patient with multiple exostoses.

DNA Mutational Analysis ; Exostoses, Multiple Hereditary ; genetics ; Female ; Humans ; Mutation ; N-Acetylglucosaminyltransferases ; genetics ; Young Adult

Aniridia ; genetics ; Base Sequence ; China ; Codon, Nonsense ; genetics ; DNA Mutational Analysis ; methods ; Eye Proteins ; genetics ; Female ; Frameshift Mutation ; genetics ; Homeodomain Proteins ; genetics ; Humans ; Infant ; PAX6 Transcription Factor ; Paired Box Transcription Factors ; genetics ; Pedigree ; Repressor Proteins ; genetics

OBJECTIVETo screen low molecular weight protein biomarkers relevant to portal vein tumor thrombi (PVTT) in serum of hepatocellular carcinoma (HCC) patients.

METHODSSerum samples were obtained from 12 healthy volunteers, 12 HCC patients without PVTT and 12 HCC patients with PVTT. Using two-dimensional gel electrophoresis (2-DE) in which the second dimension was 16% SDS-PAGE, serum protein images of the 3 groups were analyzed by ImageMaster software. The differential protein spots were further identified by MALDI-TOF MS/MS.

RESULTSComparing the results using 12.5% SDS-PAGE gel, there were more protein bands (between 3 x 10(3) and 20 x 10(3)) and low molecular weight (MW) protein spots (less than 20 x 10(3)) were clearly shown in the 16% SDS-PAGE gel. Fifteen differential protein spots representing 5 proteins were found in the 3 groups by inter-class comparison and they were then identified. Compared with those in the healthy group, apolipoprotein A-I, lipoprotein CIII, transthyretin and DNA topoisomerase II were all down regulated in HCC groups and haptoglobin-2 was over expressed. All 5 proteins decreased more in the PVTT group than in the non-PVTT group.

CONCLUSIONThe expression of low MW serum protein obviously changes in the beginning and in the progressive stage of HCC, and differentially expressed low MW proteins might be potential biomarkers in an early prognostic prediction and surveillance in the treatment for HCC and PVTT.

Adult ; Blood Proteins ; analysis ; Carcinoma, Hepatocellular ; blood ; pathology ; Electrophoresis, Gel, Two-Dimensional ; methods ; Female ; Humans ; Liver Neoplasms ; pathology ; Male ; Middle Aged ; Neoplastic Cells, Circulating ; pathology ; Portal Vein ; pathology ; Proteome ; analysis