ABSTRACT
OBJECTIVE@#To detect the mutations of EXT2 gene in hereditary multiple exostoses (HME) families and to investigate the sensitivity of denaturant gradient gel electrophoresis (DGGE) in screening the mutations in EXT2 gene.@*METHODS@#Five HME families and 3 sporadic patients were screened for the mutation detection in all exons of EXT2 gene covering the coding sequence and the flanking intronic sequence by DGGE, and DNA sequencing was performed for products with abnormal conformation.@*RESULTS@#Among these HME patients, we found 2 disease-causing mutations: A313T (nonsense mutation) and 319 insGT (frameshift mutation).@*CONCLUSION@#Two mutations of EXT2 gene are identified in the sample. DGGE can be an ideal choice for gene diagnoses of HME.
Subject(s)
Adolescent , Adult , Child , Female , Humans , Male , Middle Aged , Base Sequence , DNA Mutational Analysis , Electrophoresis, Polyacrylamide Gel , Methods , Exons , Exostoses, Multiple Hereditary , Diagnosis , Genetics , Genes, Tumor Suppressor , Mutation , N-Acetylglucosaminyltransferases , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the potential of yolk sac mesenchymal stem cells in osteogenic differentiation.</p><p><b>METHODS</b>Murine yolk sacs were harvested on day 8.5 postcoitus, yolk sac cells were obtained after the yolk sacs were digested by 0.1% type I collagenase for 1 hour, the non-adherent cells were removed after being cultured for 1 hour. The adherent cells were cultured in DMEM containing of 5 ng/ml bFGF and 15% FBS, and passaged when they became subconfluent. The morphologic characteristics, and AKP, BMP-2, as well as type I, III collagen of the yolk sac adherent cells were observed and tested. The attached cells were treated with 10(-8) mol/L dexamethasone, 10 mmol/L beta-glycerophosphate, and 50 micrograms/ml vitamin C at passage 4. Alternations of morphological characteristic, AKP activity, collagen of type I, III and mineralization were detected.</p><p><b>RESULTS</b>Pure mesenchymal stem cells which were of spindle shape, uniform in size, positive in type I, III collagen staining and weak positive in AKP activity could be induced to pleomorphism osteoblast-like cells in vitro. The cells were transformed from spindle shape to polygonal cells which were positive in type I collagen, negative in type III collagen, strong positive in BMP-2, and positive in Von Kossa's stain at week 8. The polygonal cells could form nodular structure and their AKP activity was increased. All these were coincidence with the characters of osteoblast.</p><p><b>CONCLUSION</b>Yolk sac mesenchymal stem cell can be purified and induced to osteoblast in vitro.</p>