骨形成发生蛋白9的siRNA筛选及功能检测

目的 筛选特异性沉默小鼠BMP9基因的siRNA序列,在BNLCL.2胚胎肝细胞及C3H10细胞进行功能鉴定.方法 体外退火获得4组simBMP9双链DNA序列,克隆至含有BMP9靶基因的pSOS-BMP9质粒中获得pSOS-simBMP9质粒,脂质体转染HEK293细胞,GFP检测筛选有功能的simBMP9片段,将筛选出的simBMP9构建腺病毒,感染BNLCL.2胚胎肝细胞,通过RT-PCR,Western blotting检测BMP9的表达,simBMP9与BMP9腺病毒共感染C3H10细胞, 碱性磷酸酶活性检测simBMP9对BMP9成骨诱导的抑制作用.结果 在HEK293细胞中,四组simBMP9中有两组的GFP表达明显降低,同时,腺病毒介导的这两对simBMP9均能抑制胚胎肝细胞中内源性的BMP9表达,抑制率为50%～70%,BMP9能诱导C3H10细胞的成骨分化,碱性磷酸酶的活性明显高于对照组(P<0.01),而两组simBMP9与BMP9共感染组C3H10细胞的ALP活性均明显的低于BMP9组(P<0.01).结论 成功筛选出两对特异性沉默BMP9基因的siRNA片段,能有效抑制内源性和外源性的BMP9表达,为研究BMP9在肝细胞成熟分化中的作用及具体机制提供重要的分子手段.

Screening and identification of effective small interfering RNAs against bone morphogenetic protein-9

Objective To screen specific small interfering RNA (siRNA) targeting mouse BMP9 gene and identify its function in BNLCL.2 fetal liver cells and C3H10 cells.Methods Four pairs of double-stranded DNA fragments for silencing mouse BMP9 were annealed in vitro and cloned into pSOS-BMP9 vector with BMP9 gene to construct pSOS-simBMP9 plasmid.The 4 pSOS-simBMP9 plasmids were separately transfected in HEK293 cells via Lipofectamine,and the gene silencing efficiency was assessed by GFP detection.BNLCL.2 fetal liver ...