miR-3960真核表达载体的构建及其对成骨细胞矿化的影响  

Construction of eukaryotic expression vector of miR-3960 and its effect on the mineralization of osteoblast

作　　者：李辉[1] 郭剑[1] 张艳[1] 庞雅玲[1] 李晓燕[1] 吴贵福[1] 

机构地区：[1]陕西省人民医院,西安710068

出　　处：《陕西医学杂志》2012年第6期646-648,668,共4页Shaanxi Medical Journal

基　　金：国家青年科学基金资助项目(81100208);陕西省青年科学基金资助项目(2011JQ4006)

摘　　要：目的:构建miR-3960的真核表达载体,并观察其对成骨细胞矿化的影响。方法:设计引物合成miR-3960的前体序列,将其克隆到真核表达载体pSilencer4.1-CMV puro中,构建成pSilencer4.1-miR-3960重组体。通过酶切及测序证实构建是否成功。随后将miR-3960表达载体转染小鼠基质细胞ST2,Northern blot检测miR-3960表达水平。用BMP-2诱导ST2细胞向成骨细胞分化,通过检测钙沉积量来观察对成骨细胞矿化的影响。结果:pSilencer4.1-miR-3960真核表达载体经酶切及测序鉴定正确。pSilencer4.1-miR-3960转染ST2细胞后,能有效表达miR-3960。经BMP-2诱导分化5d后,稳定转染组钙沉积量较对照组显著增加。结论:成功构建了真核表达载体pSilencer4.1-miR-3960,转染ST2细胞后能有效表达。miR-3960可以促进成骨细胞矿化。Objective：To construct a recombinant eukaryotic expression vector,pSilencer 4.1-miR-3960 and to detect its effect on the mineralization of osteoblast.Methods：The expression vector of miR-3960 was constructed by linked the annealed primers designed according to the precursor sequence of miR-3960 to the pSilencer 4.1CMV puro vector.The expression of miR-3960 was measured by Northern Blot after transfection of ST2 with the expression vector of miR-3960.Osteoblastic differentiation was induced by the addition of 300ng/ml BMP2.the mineralization of osteoblast was quantified with total calcium deposition.Results：The sequences of cloned pre-miR-3960 were correct.Northern blot results indicated that pSilencer 4.1-miR-3960 was effectively expressed in the transfected ST2 cells.Compared with control cells,transfection of miR-3960 promoted calcium accumulation was increased in cells which were transfected of miR-3960.Conclusion：The expression vector of miR-3960 is successfully constructed effectively expresses in ST2 cells.MiR-3960 overexpression promoted osteoblast mineralization of ST2 cells.

关 键 词：微RNAS 成骨细胞 杂合子检测 

分 类 号：R329.23[医药卫生—人体解剖和组织胚胎学]