Expression Of AC Isoforms And Effection Of AC3 On Primary Cilia Length In Mouse Bone Marrow Mesenchymal Stem Cells

Bone marrow stromal cells(BMSCs) are pluripotent cells that could differentiate into adipocytes, osteoblasts and other cells in bone marrow, and plays an important role in keeping the balance of bone and the composition of bone marrow microenvironment in vivo. Adenylate cyclase(AC) is an important component of the c AMP signaling pathway and plays significant roles in adipogenesis/osteogenesis differentiations of BMSCs. However, the expression of AC isoforms in BMSCs is to be determined. Primary cilia are essential cellular organelles projecting from the cell surface to sense and transduce mechanical and chemical developmental signaling in eukaryotic cells. ACs also play a key role in proliferation and differentiation in BMSCs. AC3 not only plays an important role in mediated the olfactory signal in the main olfactory epithelium of mammals epidermisthe through primary ciliary, also expressed and colocated with primary cilia in BMSCs. It is unclear that whether AC3 affects the proliferation and differentiation of BMSCs via take effect on the cilia length in BMSCs.In this article, to identify the expression of AC isoforms in primary mouse BMSCs, the most suitable culture condition of primary mouse BMSCs is that 4 × 106 nucleated cells is seeded and incubated, and 6 AC isoforms is are founded and identified in primary mouse BMSCs after the total RNA from BMSCs is isolated, reverse transcribed PCR and sequencing, they are AC2, AC3, AC4, AC6, AC7 and AC9. To identify whethere AC3 affects on the length of primary cilia in mouse BMSCs, both of the two cell types-the wild type mice bone marrow mesenchymal stem cells(WT-BMSCs) and AC3 knockout mice bone marrow mesenchymal stem cells(MUT-BMSCs) in vitro, were cultured without fetal bovine serum(FBS) and the length of primary cilia is increased by the treatment with Li Cl. After cell culturing and length of primary cilia analyzed by cell immunofluorescence staining, the results are founded that MUT-BMSCs grow slower than WT-BMSCs,and the length of primary cilia in MUT-BMSCs is shorter than the length of primary cilia in WT-BMSCs,the length of primary cilia in WT-BMSCs is 8.96μm,the length of primary cilia in MUT-BMSCs is 5.61μm. In conclusion,the AC isoforms identified in primary mouse BMSCs are AC2,AC3, AC4, AC6, AC7 and AC9,the length of primary cilia is reduced when AC3 is knock down in mouse BMSCs.