骨形成发生蛋白9的siRNA筛选及功能检测

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

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,Westernblotting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。

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 cells were infected with the constructed adenovirus simBMP9s,and their BMP9 expression was detected with RT-PCR and Western blotting.C3H10 cells were co-infected with Ad-simBMP9 and Ad-BMP9,and the inhibitory effect on BMP9-induced osteoblasts was evaluated by alkaline phosphatase （ALP） activity.Results GFP expression in the two simBMP9 groups was significantly decreased in HEK293 cells,and the endogenous expression of BMP9 was reduced by 50%-70% by adenovirus-mediated simBMP9 in the fetal liver cells.ALP activity in C3H10 cells was significantly higher in BMP9 group than in the control group （P0.01）,while the activity of the two Ad-simBMP9-infected groups was significantly lower than that in Ad-BMP9-infected group （P0.01）.Conclusion Two specific siRNA targeting mouse BMP9 gene have been obtained,which can effectively inhibit both endogenous and exogenous expressions of BMP9 to facilitate the study of the mechanisms of BMP9 in liver cell differentiation.