Construction of a eukaryotic expression vector pEGFP-C1-BMP-2 and its effect on cell migration
Construction of a eukaryotic expression vector pEGFP-C1-BMP-2 and its effect on cell migration
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真核表达载体pEGFP-C1-BMP-2的构建及其对细胞迁移的影响
DOI：
10.1631/jzus.b1100386
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发表时间：
2012-05-01
影响因子：
5.1
通讯作者：
Ding, Yue-min
中科院分区：
文献类型：
--
作者：
Wang, Xiao-ying;Chen, Zhong-hua;Ding, Yue-min
众所周知，骨形态发生蛋白（BMP）在骨和软骨发育中发挥着重要作用。最近的研究表明BMPs可能诱导肿瘤发生并促进肿瘤扩散，但其分子机制尚未阐明。本研究旨在探讨BMP-2在COS-7细胞迁移中的调控功能及其机制。扩增人BMP-2基因片段并导入pEGFP-C1载体。经XhoI和BamHI酶切分析及DNA测序确认后，将重组pEGFP-C1-BMP-2质粒转染COS-7细胞。通过细胞划痕实验和Western blotting检测BMP-2对细胞迁移和丝切蛋白活性的影响。重组pEGFP-C1-BMP-2在COS-7细胞中有效表达。 BMP-2过表达细胞中LIMK1和cofilin磷酸化增加，细胞迁移能力增强。成功构建重组pEGFP-C1-BMP-2载体，BMP-2过表达调节Rho GTPase信号通路下游分子的活性，可能有助于细胞迁移能力的增强。
Bone morphogenetic proteins (BMPs) are known to play an important role in bone and cartilage development. Recent research has shown that BMPs may induce tumorigenesis and promote tumor to spread, but the molecular mechanisms have not been elucidated. The aim of the present study was to investigate the regulatory function of BMP-2 in the migration of COS-7 cells and the underlying mechanisms.Human BMP-2 genetic fragment was amplified and introduced into the pEGFP-C1 vector. After being confirmed by XhoI and BamHI digestion analyses and DNA sequencing, the recombinant pEGFP-C1-BMP-2 plasmid was transfected into COS-7 cells. The influence of BMP-2 on cell migration and cofilin activity was detected by cell scratch assay and Western blotting.The recombinant pEGFP-C1-BMP-2 was effectively expressed in COS-7 cells. An increased phosphorylation of both LIMK1 and cofilin and an enhancement of cell migration were observed in cells with overexpression of BMP-2.A recombinant pEGFP-C1-BMP-2 vector was successfully constructed and overexpression of BMP-2 regulated the activities of the downstream molecules of the Rho GTPase signaling pathway, which might contribute to the enhancement of cell migration.