An Analysis Of Gsα Mutations In Bone Mesenchymal Stem Cells Of Fibrous Dysplasia In Jaw Bones

Objective:Fibrous dysplasia (FD) is a benign fibrous tissue lesion. The gene missense mutation of activated Gsa of bone marrow stromal cells (BMSC) causes leading to FD. The treatment of fibrous dysplasia will depend on a greater understanding of post-natal skeletal stem cell biology and how skeletal stem cells can be manipulated for efficient bone regeneration. Therefore, extraction of BMSC from the specimens of fibrous dysplasia and determination of the Gsa gene encoding has important theoretical and practical value.In this study, based on the results of this study, to investigate a method of isolation and identification of bone mesenchymal stem cells from the patients with fibrous dysplasia of bone in vitro and to explore an analysis method of Gsa protein genes mutations in BMSC in jaw bones of FD, and lays the foundation for the study of the etiology and pathogenesis of fibrous dysplasia.Methods:1. Isolation and identification of human bone mesenchymal stem cells in fibrous dysplasia of bone in vitro. The patients with fibrous dysplasia of bone were confirmed by X-ray and pathological diagnosis. The samples were cut and washed into dish to obtain the bone mesenchymal stem cell (BMSC) In the process of culturing, we observed their morphology.2. The biological characteristics analysis of human bone mesenchymal stem cells in fibrous dysplasia of bone. The cell surface marker of BMSC was detected and a cell growth curve was drawn by MTT.3. The osteogenic Culture and identification of human bone mesenchymal stem cells in fibrous dysplasia of bone. Induced and differentiated BMSC into osteoblasts, their morphology were observed. The activity of alkaline phosphatase (ALP) was measured and mineralized nodule formation was measured by Alizarin Bordeaux staining method.4. Genotype analysis of Gsa of human bone mesenchymal stem cells in fibrous dysplasia of bone. Then DNA was extracted from BMSC. Gene mutation was amplified by nested PCR and restriction enzyme digestion and gene sequences were determined.Results:1. The cells exhibit long spindle-shaped and the cells where are dense exhibit spiral-like. After transfer of culture, the cells all exhibit spiral-like. The cellular form tends to be consistent and assumes long pindle-shaped at last.2. 99% of the cells expressed CD44 and HLA-ABC, but no expressed CD34. After transfer of culture, the incubation period of BMSC is 24 hours, the exponential phase of growth is 3-4 days. On the day 5, it reached plateau phase.3. After induced and differentiated BMSC into osteoblasts, the cells'shapes are osteoblasts-like. The cells exhibit trillion shape or polygon. The cells were positive by alkaline phosphatase staining and calcium nodules after induced.4. The results showed that mutations in the arg201 codon of the Gsa protein subunit in BMSC of patients with FD are Gâ†'A or Câ†'T, which cause the argâ†'his or argâ†'cys substitutions.Conclusions: 1. The acquired BMSC can be successfully obtained from the fresh specimens of FD and can be induced osteogenesis in vitro. The acquired BMSC can satisfy the following experiment's request.2. There are specificity mutations in arg201 codon of the Gsa protein subunit in BMSCs of patients with FD. The gene missense mutation of activated Gsa of BMSC causes leading to FD.3. Nested PCR is a reliable and economical method to diagnose FD.