The Differentiation And Formation Of Mouse Osteoclast-like Cell Inhibited By NF-κB Gene Silencing

PurposeOsteopoposis (OP) is a disease with systemic osteopenia, bone microstruc-tural damage and lower bone density, which can increase the risk of bone fracture. Its main pathogenesis is that various bone resorption stimulus factors act on both osteoblast and osteoclast , which promote the proliferation , differentiation and activation of osteoclast and inhibit the apoptosis of osteoclast, consequently result in an excess of the rate of bone absorption over that of bone formation that cause the proportional decrease of organics and mineral. Along with social ageing, it become even more important to further investigate the molecular pathogenesis of osteopoposis accordingly.Osteoblast - osteoclast coupled signal transduction pathways play an important role in both bone resorption and bony remodeling. It is discovered that vitamin D membrane receptor ( VDRm) exists in osteoblast, but its functions is not very clear as yet. We know, 1α,25 (OH)₂ D₃ can up - regulate the expression of osteoclast differentiation factor ( ODF) —that is, receptor activator of nuclear factorκB ligand (RANKL)—and macrophage colony -stimulating factor (M -SCF) , Both of are two essential factors during the differentiation and formation of osteoclast. Moreover, osteoblast - secreted interleukin - 1 ( IL - 1) , inter-leukin - 6 (IL - 6 ) and tumor necrosis factor a ( TNF -α) can promote the differentiation and formation of osteoclast, maintain its survival, inhibit its apoptosis and activate nuclear factor - kappa B (NF - KB ) ; conversely, NF -κB can also promote the expression of IL - 1, IL - 6, TNF -α and M - SCF. In order to futher identify the relations of above - mentioned factors, it is important to research whether 1α,25(OH)₂D₃ can activate NF -κB and NF -κB can regulatethe expression of RANKL or not.RNA interference (RNAi) is first discovered in Arabidopsis, C. elegans in 1998 and demonstrated that it is belong to post - transcriptional level gene silencing mechanism, Endogenous or ectogenic double -stranded RNA (dsRNA) is turned into 21 -23nt small interfering RNA (siRNA) by Dicer, then siRNA combines and activates RNA induced silencing complex ( RISC ). Consequently , homologous single - stranded RNA is specially degraded. This experiment is designed to father investigate the relations of 1 a, 25 ( OH) 2 D3, NF - kB , RANKL and M - CSF on the basis of mouse osteoblast and osteoclast culture in vitro by improved myelomonocyte induction method and RNAi in order to deepen general frame understanding of osteoblast - osteoclast coupled signal transduction pathways.Methods1. Culture and identification of osteoblastOsteoblast were obtained from cranium of newborn mice by successive enzyme digestion ^routinely cultured and then identified by alkaline phosphatase staining.2. Induction by la,25(OH)2D3With first generation osteoblast, experimental gropes were respectively induced by lO'Vol/L la,25(OH)2D3for 6h, 12h, 24h, 36h and 48h; control gropes only added isovolumic 100% alcohol.3. Design, synthesis and transfection of NF - KBp65siRNA 3. 1 Design of NF - KBp65siRNATarget sequence of NF - ?Bp65siRNA, sense siRNA, antisense siRNA and correspondingly mismatching siRNA were respectively obtained on line.3. 2 Synthesis of NF - KBp65siRNAIn accordance with instruction manual in Silencer siRNA Construction Kit, synthesized step by step.3. 3 Transfection of NF - KBp65 siRNAAccording to instruction manual with siPORT lipid Kit. Then gathered cells 2d and 4d after transfection.4. Indirect immumofluorescence staining4. 1 To observe the distribution change of NF - kB in osteoblasts after la, 25(OH)2D3 induction.4. 2 To observe the fluorescence intensity change of NF - kB in osteoblasts 2d after transfection.4. 3 To observe the fluorescence intensity change of RANKL and M - CSF in osteoblasts 4d after transfection.5. Culture, transfection and Giemsa staining of osteoclast -like cell 5. 1 Culture and transfection of osteoclast - like cellBy improved myelomonocyte revulsion, mice osteoblast and myelomonocyte were co -cultured for 6d under la,25(OH)2D3 induction, trypsinizated, inoculated on sterile coverslips; 20 minutes later, remove non - adherent cells, transfected after full spread of cells, then continued to culture for 4d under 1 a, 25(OH)2D3 induction.5-. 2 Giemsa stainingBy Giemsa staining, to observe the change of myelomonocyte before transfection and 4d after transfection.Results1. Alkaline phosphatase stainingIt shows dark positive particles are distributed in mature osteoblast cytoplasm to various degrees, but immature osteoblast is not.2. The distribution change of NF - kB in osteoblasts after 1 a, 25 ( OH ) 2 D3 induction.Indirect immumofluorescence staining shows NF - kB is only distributed in osteoblast cytoplasm in control group ( non - induction) , while in experiment groups it transfers from cytoplasm to nucleus, its distribution start to change at 6h after induction, reach to peak at 36h, and decrease at 48h.3. The fluorescence intensity change of NF - kB in osteoblasts 2d after gene silencingThe fluorescence intensity of NF - kB obviously weaken in transfected osteoblast cytoplasm in experiment groups, but there is not obvious difference be-...