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  

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

作　　者：王建浩[1] 徐岚[1,2] 丁庆伟[1] 郑磊[1] 姜智[1] 吴士良[1,2] 

机构地区：[1]苏州大学医学部生物化学与分子生物学系．江苏苏州215123 [2]苏州大学医学部生化工程研究所,江苏苏州215123

出　　处：《中国血液流变学杂志》2011年第3期389-392,396,共5页Chinese Journal of Hemorheology

基　　金：国家自然科学基金资助项目（30670462）;江苏省研究生创新项目（CXIOB-04322）

摘　　要：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插入到RNA干扰载体pGPU6/GFP/Neo中构建成四条干扰载体.②将构建好的BSP siRNA 表达载体pGPU6/GFP/Neo-siBSP-1、pGPU6/GFP/Neo-siBSP-2、pGPU6/GFP/Neo-siBSP-3、pGPU6/GFP/Neo-siBSP-4通过阳离子脂质体将表达质粒分别转染MC3T3-E1 Subclone 14细胞,48h后荧光显微镜下观察不同浓度质粒的转染效率,以RT-PCR 检测BSP和OCN等成骨相关基因mRNA的表达.结果 ①成功构建四条正确的BSP siRNA 表达载体;②成功筛选出一条对BSP有明显抑制作用的siRNA干扰载体,阻断BSP表达后,成骨相关基因cbfa1和OCN的表达均受到明显抑制.结论 构建的BSP siRNA 表达载体可以有效抑制MC3T3-E1 Subclone 14细胞的BSP基因表达,同时抑制相关基因cbfa1和OCN的表达,从而影响该成骨细胞形态与功能,为骨转移等相关疾病的治疗提供了一定的实验基础.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.

关 键 词：BSP SIRNA RNA干扰表达载体 转染 

分 类 号：R73-3[医药卫生—肿瘤]