Objective: To construct a recombinant eukaryotic expression vector, pSilencer 4. l-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 trans-fected 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.%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.
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