摘要
目的探讨颌骨骨纤维异常增殖症(fibrous dysplasia,FD)GNAS基因突变的分析方法;细胞学实验比较正常颌骨与FD患者颌骨来源骨髓间充质干细胞(bone marrow stromal cells,BMSCs)的生物学特征及其差异,为进一步研究FD的病因及发病机制奠定基础。方法对我院2006—2016年间40例FD患者的石蜡标本切取组织切片并提取DNA,通过等位基因特异性PCR及测序,分析GNAS基因突变特征。在患者知情同意前提下,收集FD病灶切除术中取得的病灶区颌骨组织,贴壁培养法获得BMSCs;CCK8检测细胞增殖能力;茜素红染色检测矿化结节形成能力;Western blot及real time PCR检测RUNX2等成骨分化相关基因的表达。结果本组40例FD患者中35例GNAS基因发生突变,突变率为87.5%,其中R201H占71.4%,R201C占28.6%。与正常颌骨BMSCs相比,FD BMSCs细胞形态无明显差异;但FD BMSCs具有更强的增殖能力;在成骨诱导液的作用下,FD BMSCs具有更弱的成骨分化能力,表现为成骨分化相关基因的低表达及较弱的矿化结节形成能力。结论FD患者GNAS基因存在特异性突变,等位基因特异性PCR是检测该突变的简便、可靠方法之一;细胞学研究表明FD BMSCs不仅表现为高增殖活性,且其成骨分化能力显著下降,提示FD的发病机制可能与成骨分化过程障碍有关,对进一步认识FD的发病机制具有重要意义。
Objective To investigate an analytical method of GNAS gene mutations in fibrous dysplasia( FD) of the jaws and to explore 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. Methods The GNAS gene mutations of the 40 cases of FD who visited our hospital between2005-2016 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( R201 H) substitutions( 25 cases,71.4%) versus Arg-to-Cys( R201 C) 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 of osteogenesis-associated genes was observed in FD BMSCs during osteogenic induction. Conclusions There 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.
作者
肖涛
傅瑜
朱伟文
徐玲
张平
江宏兵
XIAO Tao;FU Yu;ZHU Weiwen;XU Ling;ZHANG Ping;JIANG Hongbing(Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University,Nanjing 210029, China)
出处
《口腔医学》
CAS
2018年第4期289-294,共6页
Stomatology
基金
国家自然科学基金(8150030656)
江苏高校优势学科建设工程资助项目(2014-37)