[1]黄科,胡宗凯,喻胜鹏,等.Cbfβ人工miRNA干扰载体构建及稳定细胞株系的获得与鉴定[J].陆军军医大学学报(原第三军医大学学报),2014,36(07):682-686.
 Huang Ke,Hu Zongkai,Yu Shengpeng,et al.Construction of artificial microRNA vector interfering mouse Cbfβ gene and establishment and identification of stable cell line[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(07):682-686.
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Cbfβ人工miRNA干扰载体构建及稳定细胞株系的获得与鉴定(/HTML )
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陆军军医大学学报(原第三军医大学学报)[ISSN:1000-5404/CN:51-1095/R]

卷:
36卷
期数:
2014年第07期
页码:
682-686
栏目:
论著
出版日期:
2014-04-15

文章信息/Info

Title:
Construction of artificial microRNA vector interfering mouse Cbfβ gene and establishment and identification of stable cell line
作者:
黄科胡宗凯喻胜鹏傅景曙李伟董世武谢肇
第三军医大学:西南医院骨科,国家地方联合工程实验室,全军矫形外科中心,生物工程学院生物医学材料学教研室
Author(s):
Huang Ke Hu Zongkai  Yu Shengpeng Fu Jingshu Li Wei Dong Shiwu Xie Zhao
Department of Orthopedics, National & Regional United Engineering Laboratory of Tissue Engineering, Center of Orthopaedics, Southwest Hospital, Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Chongqing, 400038, China
关键词:
CbfβamiRNAsamir-Cbfβ间充质干细胞成骨分化
Keywords:
core binding factor β subunitartificial miRNAamir-Cbfβmesenchymal stem cellsosteoblastic differentiation
分类号:
R394-33; R394.2
文献标志码:
A
摘要:
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(qRT-PCR)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。
Abstract:
Objective       To construct and identify miRNA expression vectors interfering mouse core binding factor β subunit (Cbfβ) 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-β-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 osteogenesis marker gene alkaline phosphatase (ALP) in the cells in 3 d after BMP-2 treatment.       Results       Artificial 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 Cbfβ 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.
更新日期/Last Update: 2014-04-03