Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC, USA.
BMC Biotechnol. 2010 Jan 28;10:5. doi: 10.1186/1472-6750-10-5.
Senescent cells are well-recognized in the aging/degenerating human disc. Senescent cells are viable, cannot divide, remain metabolically active and accumulate within the disc over time. Molecular analysis of senescent cells in tissue offers a special challenge since there are no cell surface markers for senescence which would let one use fluorescence-activated cell sorting as a method for separating out senescent cells.
We employed a novel laser capture microdissection (LCM) design to selectively harvest senescent and non-senescent annulus cells in paraffin-embedded tissue, and compared their gene expression with microarray analysis. LCM was used to separately harvest senescent and non-senescent cells from 11 human annulus specimens.
Microarray analysis revealed significant differences in expression levels in senescent cells vs non-senescent cells: 292 genes were upregulated, and 321 downregulated. Genes with established relationships to senescence were found to be significantly upregulated in senescent cells vs. non-senescent cells: p38 (MPAK14), RB-Associated KRAB zinc finger, Discoidin, CUB and LCCL domain, growth arrest and DNA-damage inducible beta, p28ING5, sphingosine-1-phosphate receptor 2 and somatostatin receptor 3; cyclin-dependent kinase 8 showed significant downregulation in senescent cells. Nitric oxidase synthase 1, and heat shock 70 kDa protein 6, both of which were significantly down-regulated in senescent cells, also showed significant changes. Additional genes related to cytokines, cell proliferation, and other processes were also identified.
Our LCM-microarray analyses identified a set of genes associated with senescence which were significantly upregulated in senescent vs non-senescent cells in the human annulus. These genes include p38 MAP kinase, discoidin, inhibitor of growth family member 5, and growth arrest and DNA-damage-inducible beta. Other genes, including genes associated with cell proliferation, extracellular matrix formation, cell signaling and other cell functions also showed significant modulation in senescent vs non-senescent cells. The aging/degenerating disc undergoes a well-recognized loss of cells; understanding senescent cells is important since their presence further reduces the disc's ability to generate new cells to replace those lost to necrosis or apoptosis.
衰老/退变的人类椎间盘内存在衰老细胞，这是一个公认的事实。衰老细胞虽然存活，但无法分裂，代谢活跃，并随着时间的推移在椎间盘内积累。由于衰老细胞没有表面标记物，因此对组织中衰老细胞的分子分析特别具有挑战性，这使得人们无法使用荧光激活细胞分选作为分离衰老细胞的方法。
我们采用了一种新颖的激光捕获显微切割（LCM）设计，选择性地从石蜡包埋组织中收获衰老和非衰老的环状细胞，并通过微阵列分析比较它们的基因表达。LCM 用于从 11 个人类环状标本中分别收获衰老和非衰老细胞。
微阵列分析显示，衰老细胞与非衰老细胞之间的表达水平存在显著差异：292 个基因上调，321 个基因下调。与衰老相关的基因在衰老细胞中明显上调：p38（MPAK14）、RB 相关 KRAB 锌指、盘状蛋白、CUB 和 LCCL 结构域、生长抑制和 DNA 损伤诱导的β、p28ING5、鞘氨醇-1-磷酸受体 2 和生长抑素受体 3；细胞周期蛋白依赖性激酶 8 在衰老细胞中明显下调。在衰老细胞中显著下调的一氧化氮合酶 1 和热休克 70 kDa 蛋白 6 也显示出显著变化。还确定了其他与细胞因子、细胞增殖和其他过程相关的基因。
我们的 LCM-微阵列分析确定了一组与衰老相关的基因，这些基因在人类环状物的衰老细胞中明显上调，而非衰老细胞中下调。这些基因包括 p38 MAP 激酶、盘状蛋白、生长抑制家族成员 5 和生长抑制和 DNA 损伤诱导的β。其他基因，包括与细胞增殖、细胞外基质形成、细胞信号转导和其他细胞功能相关的基因，在衰老细胞和非衰老细胞中也显示出显著的调节。衰老/退变的椎间盘经历了公认的细胞丢失；了解衰老细胞很重要，因为它们的存在进一步降低了椎间盘产生新细胞以替代坏死或凋亡丢失细胞的能力。
