Gruber Helen E, Ingram Jane, Hoelscher Gretchen L, Norton H James, Hanley Edward N
Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC 28232, USA.
Spine (Phila Pa 1976). 2007 May 20;32(12):1287-94. doi: 10.1097/BRS.0b013e31805931d8.
Human intervertebral disc tissue was obtained in a prospective study of cell morphology and gene expression. Experimental studies were approved by the authors' Human Subjects Institutional Review Board. Discs were obtained from surgical specimens or control donors.
To determine if there is morphologic and molecular evidence for polarity in cells of the human anulus.
In many tissues, cells become polarized as they develop functional specializations, which involve cell-cell and cell-extracellular matrix interactions and polarized targeting mechanisms. The highly specialized lamellar organization of the anulus is well recognized and suggests that this structure may be the result of directed secretion of extracellular matrix components by polarized disc cells.
Human disc specimens from donor and surgical patients were examined with light and electron microscopy to assess morphology. Specimens were examined for immunocytochemical localization of PAR3 and claudin-1 and -11, recognized polarity proteins, and additional anulus specimens were examined for expression of polarity-related genes using microarray analysis. In vitro monolayer and 3-dimensional anulus cultures were also studied for gene expression, and additional surgical anulus specimens were used to obtain gene expression data using real time RT-PCR.
At the ultrastructural level, anulus cells showed localization of secretory organelles and directed deposition of extracellular matrix in one portion of the cell, with the nucleus positioned in the opposite side of the cell. Expression of the polarity-related genes claudin-11 and PAR3 and PARD6 was confirmed using RT-PCR and microarray studies and immunocytochemical analyses. The percentage of cells positive for PAR3 immunolocalization was significantly greater in the outer anulus (100%) than in either the inner anulus (43.8%) or nucleus pulposus (22.6%).
At the macroscopic level, the characteristic anular lamellar morphology implies a specialized architectural formation and organization, which is achieved by the tissue-specific function of polarized cells. Morphologic and molecular studies provided evidence for the presence of polarity in cells in the anulus. These findings advance our understanding of anulus disc cell function in production of highly aligned collagen fibrils and macroaggregates of these collagen fibrils into lamellar collagen bundles. Such disc cell activity is important in development and maintenance of the tissue-specific extracellular matrix of the disc.
在一项关于细胞形态和基因表达的前瞻性研究中获取人椎间盘组织。实验研究经作者所在机构的人体受试者机构审查委员会批准。椎间盘取自手术标本或对照供体。
确定人纤维环细胞中是否存在极性的形态学和分子学证据。
在许多组织中，细胞在形成功能特化时会发生极化，这涉及细胞 - 细胞和细胞 - 细胞外基质相互作用以及极化靶向机制。纤维环高度特化的层状结构已得到充分认识，这表明该结构可能是极化的椎间盘细胞定向分泌细胞外基质成分的结果。
对来自供体和手术患者的人椎间盘标本进行光镜和电镜检查以评估形态。检查标本中PAR3、claudin - 1和 - 11（公认的极性蛋白）的免疫细胞化学定位，并用微阵列分析检查其他纤维环标本中极性相关基因的表达。体外单层和三维纤维环培养物也进行基因表达研究，并用实时逆转录聚合酶链反应（RT - PCR）从额外的手术纤维环标本中获取基因表达数据。
在超微结构水平上，纤维环细胞显示分泌细胞器定位以及细胞外基质在细胞的一部分定向沉积，细胞核位于细胞的另一侧。使用RT - PCR、微阵列研究和免疫细胞化学分析证实了极性相关基因claudin - 11、PAR3和PARD6的表达。PAR3免疫定位阳性的细胞百分比在纤维环外层（100%）显著高于纤维环内层（43.8%）或髓核（22.6%）。
在宏观水平上，纤维环特征性的层状形态意味着一种特殊的结构形成和组织，这是通过极化细胞的组织特异性功能实现的。形态学和分子学研究为纤维环细胞中极性的存在提供了证据。这些发现增进了我们对纤维环细胞在产生高度排列的胶原纤维以及将这些胶原纤维聚集成层状胶原束方面功能的理解。这种椎间盘细胞活动在椎间盘组织特异性细胞外基质的发育和维持中很重要。
