let-7c对大鼠骨髓间充质干细胞增殖及成牙/成骨分化能力的影响

Effects of let-7c on proliferation andodonto/osteogenic differentiation of rat bone marrow mesenchymal stem cells

目的:探讨let-7c在体外对大鼠骨髓间充质干细胞(BMMSCs)增殖及成牙/成骨分化的影响。方法:构建rno-let-7c过/低表达慢病毒载体并且体外转染大鼠BMMSCs,筛选最适转染滴度;倒置荧光显微镜下观察BMMSCs转染后绿色荧光蛋白的表达;实时定量RT-PCR验证rno-let-7c转染效果;CCK-8法和流式细胞术分别检测rno-let-7c过/低表达对细胞增殖和凋亡的影响;Western blot检测胰岛素样生长因子1受体(IGF-1R)及成牙/成骨指标(RUNX2/OSX/OCN/DMP1)的表达。结果:慢病毒载体转染大鼠BMMSCs最佳条件为MOI=3,成功构建rno-let-7c过/低表达的BMMSCs模型。let-7c过/低表达对大鼠BMMSCs增殖和凋亡均无明显影响(P>0.05)。与阴性转染组相比,过表达组IGF-1R表达下调,成牙/成骨指标表达下调,而低表达组IGF-1R表达上调,成牙/成骨指标表达上调(P<0.05)。结论:let-7c对大鼠BMMSCs增殖及凋亡无明显影响,可以抑制其成牙/成骨向分化。

Objective To investigate the effect of let-7c on the proliferation and odonto/osteogenesis differentiation of rat bone mar-row mesenchymal stem cells in vitro. Methods ： Rno-let-7c over/low expression lentiviral vector were constructed and used to transfect rat BMMSCs in vitro, then the optimal transfection titer was screened. The expression of green fluorescent protein was observed by in-verted fluorescence microscope. Real-time quantitative RT-PCR was used to verify the efficiency of rno-let-7c transfection. The effect of rno-let-7c on rBMMSCs proliferation and apoptosis was detected by CCK-8 and Flow Cytometry. The expression of insulin-like growth factor 1 receptor （IGF-1R） and odonto/osteogenic markers （RUNX2/OSX/OCN/DMP1） were detected by western blot. Results： The optimal condition of lentiviral vector to transfect rBMMSCs was MOI = 3. Rno-let-7c over/low expression BMMSCs model was construc-ted successfully. Rno-let-7c over/low expression had no significant effect on proliferation and apoptosis of rat BMMSCs （ P 〉 0 .0 5 ） . Compared with the negative transfection group,the expression of IGF-1R and odonto/osteogenic markers was dow-n-regulated in over ex-pression group,while the expression of IGF-1R and odonto/osteogenic markers was up-regulated in low- expression group （ P 〉 0 .0 5 ） . Conclusions： Let-7c had no obvious effect on the proliferation and apoptosis of rat BMMSCs. Let-7c can inhibit the odonto/osteogenic differentiation of rat BMMSCs.