Molecular phenotypes of notochordal cells purified from immature nucleus pulposus
Molecular phenotypes of notochordal cells purified from immature nucleus pulposus
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DOI：
10.1007/s00586-006-0088-x
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发表时间：
2006-08-01
影响因子：
2.8
通讯作者：
Setton, Lori A.
中科院分区：
文献类型：
--
作者：
Chen, Jun;Yan, Wei;Setton, Lori A.
未成熟的髓核（NP）由脊索起源的细胞组成，这些细胞较大，含有广泛的细胞骨架网络和许多空泡。随着年龄的增长，这些细胞的消失被认为在调节可能导致年龄相关的椎间盘退变的代谢变化方面很重要。然而，由于在NP中研究混合细胞群体的挑战，这些未成熟NP中的脊索细胞的精确生物学功能仍不清楚。在这项研究中，脊索样细胞纯化未成熟的NP细胞使用新的荧光激活细胞分选（FACS）协议与自体荧光和大小分析。分拣脊索样细胞的独特分子表型的特征在于关键基质蛋白和调节剂的mRNA表达模式，以及细胞基质受体整合素亚基的表达。流式细胞仪分析表明，未成熟的NP包含的大多数细胞比纤维环（AF）细胞大，荧光高于AF细胞。与通过FACS方案分离的小NP细胞相比，分选的脊索样细胞表达较低水平的I型胶原、双糖蛋白聚糖、TIMP 1、HSP 70和c-fos的mRNA，并且通过实时定量RT-PCR不表达可检测水平的核心蛋白聚糖、Lumican、多种MMPs或IL-1 β。与小NP细胞相比，更多的这些脊索样细胞也表达更高水平的α 6、α 1和β 1整联蛋白亚基。总之，我们的研究结果指向一个独特的分子表型，这些脊索样细胞的NP，其特征是缺乏基因表达的特定的小蛋白聚糖和更高的蛋白表达的整合素亚基，调节与胶原蛋白和层粘连蛋白的相互作用。未来的研究将是重要的揭示，如果这种独特的分子配置文件是协调与这些脊索样细胞在NP内的细胞周基质区域和/或Tritegrin介导的细胞-基质相互作用的功能差异。
The immature nucleus pulposus (NP) is populated by cells of notochordal-origin that are larger and contain an extensive cytoskeletal network and numerous vacuoles. The disappearance of these cells with age is believed important in regulating metabolic shifts that may contribute to age-related disc degeneration. The precise biological function of these notochordal cells in the immature NP remains unclear, however, because of challenges in studying the mixed cell population in the NP. In this study, notochordal-like cells were purified from immature NP cells using a new fluorescence-activated cell sorting (FACS) protocol with auto-fluorescence and size analysis. The unique molecular phenotypes of sorted notochordal-like cells were characterized by the mRNA expression pattern for key matrix proteins and modulators, and by the expression of cell matrix receptor integrin subunits. An FACS analysis showed that the immature NP contained a majority of cells that were larger than anulus fibrosus (AF) cells and with fluorescence higher than AF cells. In comparison with the small NP cells separated by the FACS protocol, sorted notochordal-like cells expressed lower mRNA levels of type I collagen, biglycan, TIMP1, HSP70 and c-fos, and did not express detectable mRNA levels of decorin, lumican, multiple MMPs or IL-1 beta via real-time quantitative RT-PCR. A greater number of these notochordal-like cells also expressed the higher levels of alpha 6, alpha 1 and beta 1 integrin subunits as compared to small NP cells. Together, our results point towards a unique molecular phenotype for these notochordal-like cells of NP, characterized by the absence of gene expression for specific small proteoglycans and higher protein expression of integrin subunits that regulate interactions with collagens and laminin. Future studies will be important for revealing if this unique molecular profile is coordinated with functional differences in pericellular matrix regions and/or tritegrin-mediated cell-matrix interactions for these notochordal-like cells within the NP.