Cbfβ人工miRNA干扰载体构建及稳定细胞株系的获得与鉴定

Construction of artificial microRNA vector interfering mouse Cbfβ gene and establishment and identification of stable cell line

目的构建Cbfβ基因干扰载体,获得Cbfβ人工miRNA干扰载体和稳定细胞株系,检测其对间充质干细胞C3H10T1/2细胞成骨分化的影响。方法设计特异性干扰小鼠Cbfβ基因的人工miRNA序列,构建Cbfβ干扰载体Blockit miRNA-cbfβ-miRNA1/miRNA2,将其转入C3H10T1/2细胞获得稳定细胞株;用BMP-2诱导3 d后采用荧光定量PCR(qRTPCR)和Western印迹检测Cbfβ、Runx2和成骨标志基因ALP表达水平。结果成功构建成骨转录因子Cbfβ的干扰载体并获得了稳定细胞株系,成骨标志基因ALP和Cbfβ的mRNA和蛋白表达水平明显下降(P<0.05),而Runx2下降不显著(P>0.05)。结论 miRNA-cbfβ-miRNA1/miRNA2载体可以抑制成骨转录因子Cbfβ的功能,进而降低成骨标志基因ALP蛋白和mRNA的表达,并获得可以影响成骨过程的稳定细胞株系。

Objective To construct and identify miRNA expression vectors interfering mouse core binding factor β subunit （Cbfl3） gene and to determine their effects on osteogenic differentiation of mesenchymal stem cell C3H10T1/2. Methods Artificial microRNA sequences specifically interfering mouse Cbfβ gene were designed and Blockit miRNA-Cbf-13-miRNA1/miRNA2 vectors interfering mouse Cbfβ gene were constructed. The vectors were transferred into C3H10T1/2 cells to get stable cell line. Quantitative RT-PCR and Western blotting were used to detect the expression levels of Cbfβ, Runt-related transcription factor 2 （Runx2） and oste ogenesis marker gene alkaline phosphatase （ALP） in the cells in 3 d after BMP-2 treatment. Results Artifi cial microRNA vectors interfering Cbfβ were successfully constructed and stable cell line was obtained. The expression level of ALP and Cbfβ were obviously reduced （ P 〈 0.05 ） , while Runx2 expression was not notably decreased （P 〉 0.05 ）. Conclusion miRNA expression vectors miRNA-Cbf-β-miRNA1/miRNA2 targeting Cbfl3 gene may inhibit function of osteogenic transcription factor Cbfβ and then decrease protein and mRNA expression of ALP. Stable cell line with influenced osteogenic ability is obtained.