Construction of a Luciferase Reporter System to Monitor Osteogenic Differentiation of Mesenchymal Stem Cells by Using a Mammalian Artificial Chromosome Vector
Construction of a Luciferase Reporter System to Monitor Osteogenic Differentiation of Mesenchymal Stem Cells by Using a Mammalian Artificial Chromosome Vector
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发表时间：
2015-03
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通讯作者：
T. Narai;M. Katoh;Toshiaki Inoue;M. Taniguchi;K. Kazuki;Y. Kazuki;Kenzo Sato;Isamu Kodani;K. Ryoke;M. Oshimura
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作者：
T. Narai;M. Katoh;Toshiaki Inoue;M. Taniguchi;K. Kazuki;Y. Kazuki;Kenzo Sato;Isamu Kodani;K. Ryoke;M. Oshimura
间充质干细胞（MSC）有望在成人干细胞介导的骨重建和骨折修复再生医学中应用。地塞米松（DEX）可将体外培养的MSCs定向成骨，但由于地塞米松的副作用如糖皮质激素诱导的骨质疏松症，DEX的应用在临床上并不实用。为了鉴定促进成骨的物质，需要准确且容易地检测MSC的成骨分化阶段的监测系统。方法以人骨钙素（OC）基因为研究对象，利用哺乳动物人工染色体构建了由OC基因增强子/启动子序列驱动的荧光素酶（Luc）报告基因（OC-Luc）。哺乳动物人工染色体是一个合适的装载报告基因构建体的平台，因为它在宿主细胞中稳定的附加型维持，可转移到任何细胞中，并保证长期的生理转基因表达。我们将OC-Luc装载在中国仓鼠卵巢细胞（OC-Luc/MAC）中由小鼠染色体（命名为小鼠人工染色体，MAC）工程化的哺乳动物人工染色体载体上，并通过染色体转移将其转移到人MSC细胞中。结果OC-Luc/MAC在人MSC向成骨细胞分化的过程中，对1 α，25-二羟维生素D3和DEX的正、负刺激均有反应，反映了其生理表达方式。结论OC-Luc/MAC报告基因系统不仅可用于监测MSC的成骨分化阶段，而且可用于筛选新的成骨药物。
BACKGROUND Mesenchymal stem cells (MSCs) hold promise for application in adult stem cell-mediated regenerative medicine in bone remodeling and fracture repair. MSCs in vitro can be directed to osteogenic lineage by dexamethasone (DEX); however, the use of DEX is not practical in clinical settings because of adverse side effects such as glucocorticoid-induced osteoporosis. For identifying substances that facilitate osteogenesis, a monitoring system, which detects the osteogenic differentiation stage of MSCs accurately and easily, is required. METHODS By focusing on the human osteocalcin (OC) gene whose expression profile is described along with osteogenic differentiation, we constructed the luciferase (Luc) reporter gene driven by the enhancer/promoter sequence of the human OC gene (OC-Luc) utilizing a mammalian artificial chromosome. Mammalian artificial chromosome is a suitable platform for loading reporter constructs, because of its stable episomal maintenance in host cells, transferability into any cell and assurance of long-term physiological transgene expression. We loaded the OC-Luc on a mammalian artificial chromosome vector engineered from mouse chromosome (designated as mouse artificial chromosome, MAC) in Chinese hamster ovary cells (OC-Luc/MAC) and transferred this into human MSC cells via chromosome transfer. RESULTS OC-Luc/MAC in human MSC cells are responsive to positive and negative stimulation by 1 alpha,25-dihydroxyvitamin D3 and DEX in differentiation stage of MSCs to osteoblasts, reflecting the manner of physiological expression. CONCLUSION The OC-Luc/MAC reporter system may contribute not only to monitoring the osteogenic differentiation stage from MSC but also to identify novel osteogenic drugs.