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首页> 美国卫生研究院文献>International Journal of Nanomedicine >Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate
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Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

机译：在组织培养板上通过冻干的microRNA反向转染制剂诱导干细胞的成骨分化

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MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 μL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and −20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection which can control stem cell fate and is suitable for loading miRNAs onto various biomaterials.
机译：MicroRNA（miRNA）调节是操纵间充质干细胞命运的一种新颖方法，但是需要一种简单，安全且高效的转染方法。在这项研究中，我们通过冻干组织培养板上的Lipofectamine 2000-miRNA脂质复合物开发了miRNA反向转染制剂。可以将脂质复合物固定在具有完整假球形结构和冻干的组织培养板上，而无需任何冻干保护剂。在这项研究中，反向转染导致细胞对miRNA的高效吸收，并使细胞内靶标miRNA水平得到显着控制。与常规和其他转染方法相比，每孔含Lipofectamine 2000 1μL的反向转染制剂产生更高的转染效率，而没有明显的细胞毒性。此外，反向转染制剂的转染效率在4℃和-20℃下储存90天期间没有恶化。然后，我们评估了miRNA反向转染制剂在促进间充质干细胞成骨分化中的效率。我们发现，用抗miR-138和miR-148b转染可有效增强成骨分化，如成骨相关基因的表达增强，碱性磷酸酶的存在，胶原蛋白的产生和基质矿化所表明。总的来说，这项研究中开发的miRNA逆转染制剂是一种有前途的miRNA转染方法，可以控制干细胞的命运，适合将miRNA加载到各种生物材料上。

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