低表达Cbfa1基因的人牙周膜细胞模型的建立及鉴定

目的:构建针对核心结合因子a1(Cbfa1)的mU6小干扰RNA载体,并将其转染到人牙周膜细胞hPDLCs,观察其干扰效果,以期建立稳定的低表达Cbfa1基因的hPDLCs模型. 方法:设计针对Cbfa1基因编码区的寡核苷酸链,体外退火后克隆入经双酶切线性化的小干扰载体mU6中,对重组质粒进行酶切分析和DNA序列测定. 以脂质体法将mU6空载体和重组质粒分别导人hPDLCs细胞系. 用含G418的培养液筛选,挑取阳性克隆后用RT-PCR技术检测各hPDLCs克隆内Cbfa1 mRNA水平的表达情况. 结果:经酶切鉴定及DNA测序,重组质粒Cbfa1-siRNA中插入的目的基因片段与预计片段完全一致. G418筛选转染细胞4wk后获得稳定转染mU6空载体的细胞克隆株hPDLCs-mU6和稳定转染重组质粒的细胞克隆株hPDLCs-siRNA. 经RT-PCR鉴定,Cbfa1在hPDLCs-siRNA细胞中的表达比对照细胞明显降低. 结论:成功构建了针对Cbfa1的小干扰RNA载体,成功建立了稳定的低表达Cbfa1的hPDLCs模型,为进一步研究Cbfa1的功能奠定了实验基础.

Establishment and identification of human periodontal ligament cells with decreased Cbfa1 expression

AIM: To construct the small interfering RNA (siRNA) expression vector targeting human core binding factor a1 (Cbfa1) gene and to observe its silencing effect in transfected human periodontal ligament cells (hPDLCs) so as to establish stable hPDLCs model with decreased Cbfa1 expression. METHODS: The designed oligos targeting human Cbfa1 gene were cloned into the lineared mU6 vector. The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing. The recombinant vector and the empty vector were respectively transfected by liposome-mediated transfection into hPDLCs. The stable clones were screened by G418 medium and further identified by RT-PCR. RESULTS: The Cbfa1-siRNA expression vector was successfully constructed and confirmed by the enzyme digestion analysis and the DNA sequencing. The stable clones transfected with 2 vectors were respectively constructed. As identified by RT-PCR, the expression of Cbfa1 in hPDLCs-siRNA cells were significantly decreased as compared with that in hPDLCs-mU6 cells. CONCLUSION: The siRNA expression vectors targeting Cbfa1 have been successfully constructed. The expression of Cbfa1 gene is inhibited effectively in hPDLCs cells transfected with Cbfa1-siRNA expression vector, which facilitates the further research on the function of Cbfa1.