BSP siRNA 的构建及其对成骨样细胞株MC3T3-E1相关基因表达的影响

目的 构建BSP干扰表达载体,有效沉默小鼠成骨样细胞MC3T3-E1 Subclone 14细胞的BSP基因表达,研究其对MC3T3-E1 Subclone 14增殖分化过程中OCN等成骨相关基因表达的影响.方法 ①利用互联网资源针对BSP基因mRNA序列设计四条可能的小干扰RNA(siRNA),将这四条小干扰RNA插...

Construction,Screening of BSP Effective siRNA Expression Vector and the In.uence on the Related Gene Expression of MC3T3-E1 with BSP Expression Blocked by RNA Interference

Objective To construct a vector of RNA interference（RNAi） on BSP and to effectively silence the gene expression of BSP in mice pre-osteoblast-like cell line MC3T3-EI Subclone 14,study it＇s influence on the bone-related gene expression such as BSP during the proliferation and differentiation of MC3T3-E1 Subclone 14.Methods ①Four possible small RNAi were designed for BSP gene mRNA through the use of Interact resources.These four small RNAi were recombined with the vector pGPU6/GFP/Neo,and four vectors of RNA interference were built.②MC3T3-E1 Subclone 14 were transfected with the constructed vector pGPU6/ GFP/Neo-siBSP- 1,pGPU6/GFP/Neo-siBSP-2,pGPU6/GFP/Neo-siBSP-3,pGPU6/GFP/Neo-siBSP-4 plasmid. The transfection efficiency could be observed by the fluorescence microscope those transfected with different concentrations of plasmid.The expression variation of bone-related gene such as BSP and OCN were detected by RT-PCR.Results ①Four correct BSP expression vector of RNAi were constructed successfully.②The siRNAexpression vector that could block the BSP effectively was screened out.The bone-related gend cbfal and OCN mRNA were all inhibited distinctly after the gene BSP was blocked.Conclusion The constructed BSP siRNA expression vector could effectively inhibit the BSP gene expression of MC3T3-EI Subclone 14 cell,could also inhibit the expression of gene cbfal and OCN,and could influence the bone morphology and function.The I3SP study has laid a experimental foundation for the therapy of bone metastasis and other related diseases.