关键词: 骨纤维异常增殖症, GNAS, 基因突变, 等位基因特异性PCR, 细胞增殖, 成骨分化

Abstract: ObjectiveTo investigate an analytical method of GNAS gene mutations in fibrous dysplasia(FD) of the jaws and toexplore the biological characteristics of bone marrow stromal cells (BMSCs) from FD patients in vitro and lay the foundation for the further study of the pathogenesis of FD.MethodsThe GNAS gene mutations of the 40 cases of FD who visited our hospital between 2005-2016 were analyzed via extracting genomic DNA from paraffin-embedded tissues of the FD patients，allele-specific PCR amplification and sequencing.Fresh FD tissues were immediately obtained from bone lesions for primary cell culture after surgical removal. Informed consent was obtained before volunteers were enrolled in this study. CCK8 was performed for analysis of cell proliferation, the mineralization potential of BMSCs was assessed via Alizarin Red staining, Western blot and real time PCR were used to examine the expression of osteogenesis-associated genes.Results A mutation in the Gsα codon of Arg201 was found in 35 of the 40 (87.5%) cases of FD, with a predilection for Arg-to-His (R201H) substitutions (25 cases, 71.4%) versus Arg-to-Cys (R201C) substitutions (9 cases, 28.6%).No obvious morphological differences between FD BMSCs and normal BMSCs were observed. FD BMSCs exhibited a much stronger proliferation ability relative to BMSCs.Alizarin Red staining indicated that fewer calcium deposits were formed by FD BMSCs. A significant reduction in the expression ofosteogenesis-associated genes was observed in FD BMSCs during osteogenic induction.ConclusionsThere are specific mutations in GNAS gene in BMSCs of FD lesions, allele-specific PCR is a simple and reliable method to diagnose FD.FD BMSCs are characterized by high proliferation activity and weak osteogenic differentiation potential, and the pathogenesis of FD may be associated with osteogenic disorder.

Key words: fibrous dysplasia, GNAS , mutation, allele specific PCR, proliferation, osteogenesis