Evidence of Enhanced Expression of Osteopontin in Spinal Hyperostosis of the Twy Mouse
Evidence of Enhanced Expression of Osteopontin in Spinal Hyperostosis of the Twy Mouse
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DOI：
10.1097/brs.0b013e3181aa01fc
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发表时间：
2009-07-15
期刊：
影响因子：
3
通讯作者：
Yamazaki, Masashi
中科院分区：
文献类型：
--
作者：
Aiba, Atsuomi;Nakajima, Arata;Yamazaki, Masashi
研究设计.应用原位杂交、免疫组化和北方印迹技术研究骨桥蛋白（osteopontin，OPN）在twy小鼠脊柱骨质增生中的基因表达和蛋白定位.验证骨桥蛋白在小鼠脊柱骨质增生中的作用，并在分子水平上阐明其骨化模式。骨桥蛋白是一种在人类病理条件下与异位钙化一致共定位的分子。twy小鼠显示脊柱韧带异位钙化导致后肢瘫痪，被认为是人类脊柱后纵韧带骨化的模型。在2、4、8、12和16周龄时处死年龄匹配的twy、杂合子和野生型小鼠各28只，并进行组织学和/或分子分析。将切片与OPN的RNA探针杂交，并用抗OPN抗体染色。从2周龄和16周龄的每个基因型的颈胸段脊柱中提取总细胞RNA，通过北方印迹分析定量OPN和COL 10 A1的基因表达。在twy小鼠的脊柱骨质增生病变中观察到OPN mRNA表达增强，特别是在脊柱韧带细胞和纤维环外层的软骨形成细胞中。免疫组化分析也证实了这些趋势。北方印迹分析显示，在2周龄时，所有基因型小鼠都有大量的OPN转录本表达，但只有16周龄的twy小鼠仍有OPN mRNA的强表达。16周龄时，COL 10A 1基因各基因型几乎不表达。骨桥蛋白在twy小鼠骨质增生性脊柱病变中表达过强，骨质增生主要由脊柱韧带异位骨化引起。由于OPN被认为是钙化的抑制剂，因此有必要进行进一步的研究以验证在twy小鼠中过表达的OPN是否具有功能。
Study Design. Gene expression and protein localization of osteopontin (OPN) in spinal hyperostosis of the twy mouse by means of in situ hybridization, immunohistochemistry, and Northern blot analysis.Objective. To verify the involvement of OPN in spinal hyperostosis in the twy mouse and elucidate its ossification pattern at molecular levels.Summary of Background Data. OPN is a molecule that consistently colocalizes with ectopic calcification in human pathologic conditions. The twy mouse, which shows ectopic calcification of the spinal ligament resulting in hind limb paralysis, is considered to be a model for human ossification of the posterior longitudinal ligament of the spine.Methods. Twenty-eight each of age-matched twy, heterozygote, and wild-type mice were killed at 2, 4, 8, 12, and 16 weeks old and subject to histologic and/or molecular analyses. Sections were hybridized with RNA probes for OPN and also stained with anti-OPN antibodies. Total cellular RNA was extracted from the cervicothoracic spine of each genotype at 2- and 16-week-old, and gene expression for OPN and COL10A1 was quantified by Northern blot analysis.Results. Enhanced expression of OPN mRNA was observed in spinal hyperostotic lesions of the twy mouse, specifically in cells of the spinal ligament and chondrogenic cells in the outer layer of the anulus fibrosus. These trends were also confirmed by immunohistochemical analyses. Northern blot analysis showed that a considerable amount of OPN transcripts was detected in all genotypes at 2 weeks old, but the robust expression of OPN mRNA was maintained only in twy mice at 16 weeks old. COL10A1 transcripts were hardly detected regardless of the genotype at 16 weeks old.Conclusion. OPN was overexpressed in the hyperostotic spinal lesions of twy mice, and the hyperostosis was induced mainly by ectopic ossification of the spinal ligament. Because OPN is considered to be an inhibitor of calcification, further studies will be necessary to verify whether OPN overexpressed in the twy mouse is functional.