1 Bone Metabolism Lab, School of Life Sciences, Northwestern Polytechnical University , Xi'an, Shaanxi, China.
2 Shenzhen Research Institute of Northwestern Polytechnical University , Shenzhen, Guangdong, China.
Hum Gene Ther. 2018 Feb;29(2):259-270. doi: 10.1089/hum.2017.153.
Microtubule actin crosslinking factor 1 (MACF1) is a large spectraplakin protein known to have crucial roles in regulating cytoskeletal dynamics, cell migration, growth, and differentiation. However, its role and action mechanism in bone remain unclear. The present study investigated optimal conditions for effective transfection of the large plasmid PEGFP-C1A-ACF7 (∼21 kbp) containing full-length human MACF1 cDNA, as well as the potential role of MACF1 in bone formation. To enhance MACF1 expression, the plasmid was transfected into osteogenic cells by electroporation in vitro and into mouse calvaria with nanoparticles. Then, transfection efficiency, osteogenic marker expression, calvarial thickness, and bone formation were analyzed. Notably, MACF1 overexpression triggered a drastic increase in osteogenic gene expression, alkaline phosphatase activity, and matrix mineralization in vitro. Mouse calvarial thickness, mineral apposition rate, and osteogenic marker protein expression were significantly enhanced by local transfection. In addition, MACF1 overexpression promoted β-catenin expression and signaling. In conclusion, MACF1 overexpression by transfecting the large plasmid containing full-length MACF1 cDNA promotes osteoblast differentiation and bone formation via β-catenin signaling. Current data will provide useful experimental parameters for the transfection of large plasmids and a novel strategy based on promoting bone formation for prevention and therapy of bone disorders.
微管肌动蛋白交联因子 1（MACF1）是一种大型 spectrin 蛋白，已知在调节细胞骨架动力学、细胞迁移、生长和分化方面具有关键作用。然而，其在骨骼中的作用和作用机制尚不清楚。本研究旨在探讨转染包含全长人 MACF1 cDNA 的大质粒 PEGFP-C1A-ACF7（约 21 kbp）的有效条件，以及 MACF1 对骨形成的潜在作用。为了增强 MACF1 的表达，通过电穿孔将质粒转染到体外成骨细胞中，并通过纳米颗粒转染到小鼠颅骨中。然后，分析了转染效率、成骨标志物表达、颅骨厚度和骨形成。值得注意的是，MACF1 的过表达在体外显著增加了成骨基因的表达、碱性磷酸酶活性和基质矿化。局部转染显著增强了小鼠颅骨厚度、矿化速率和成骨标志物蛋白的表达。此外，MACF1 的过表达促进了β-连环蛋白的表达和信号转导。综上所述，通过转染包含全长 MACF1 cDNA 的大质粒过表达 MACF1 通过 β-连环蛋白信号促进成骨细胞分化和骨形成。本研究提供了转染大质粒的有用实验参数，以及一种基于促进骨形成的新策略，用于预防和治疗骨疾病。
