Chou Alice I, Bansal Amar, Miller Gregory J, Nicoll Steven B
Department of Bioengineering, The University of Pennsylvania, Philadelphia, PA 1910, USA.
Spine (Phila Pa 1976). 2006 Aug 1;31(17):1875-81. doi: 10.1097/01.brs.0000229222.98051.9a.
Sheep outer and inner anulus fibrosus cells were isolated and analyzed to determine the effect of serial monolayer passaging on their phenotype.
To characterize the effect of sequential serial passage on outer and inner anulus cells to determine at which point passaged cells are significantly different from freshly isolated cells.
Previous studies show that chondrocytic cells lose their differentiated phenotype with sequential monolayer passage. Although intervertebral disc cells are similar, to our knowledge, a complete characterization of passage effects has not been performed.
Sheep outer and inner anulus cells were isolated, serially passaged, and evaluated for changes in cellular morphology, collagen I and II gene expression and protein elaboration, and total protein and deoxyribonucleic acid content.
Outer anulus cells displayed an elongated morphology, while inner anulus cells were initially polygonal and became more fibroblast-like with passage. At low passage, outer anulus cells showed higher collagen I expression, while inner anulus cells indicated higher collagen II expression. At high passage, collagen I expression increased for inner anulus cells and decreased for outer anulus cells, whereas collagen II expression decreased for both cell types. Immunohistochemical staining confirmed gene expression results.
The differences in expression profiles of outer and inner anulus cells support previous findings that zonal differences exist between the cell types. Up to passage 2, both cell types were not significantly different from freshly isolated cells and maintained distinct phenotypic characteristics. However, after 6 sequential passages, outer and inner anulus cells became morphologically indistinguishable, and displayed no significant differences in collagen I gene and protein expression, thus becoming a more homogeneous population. As such, serial monolayer passaging has a marked effect on disc cell behavior, and is an important factor to consider when designing and evaluating in vitro studies and for potential cell-based therapies for disc repair.
分离并分析绵羊的纤维环外层和内层细胞，以确定连续单层传代对其表型的影响。
描述连续传代对纤维环外层和内层细胞的影响，以确定传代细胞在哪个时间点与新鲜分离的细胞有显著差异。
先前的研究表明，软骨细胞在连续单层传代后会失去其分化表型。尽管椎间盘细胞与之相似，但据我们所知，尚未对传代效应进行全面描述。
分离绵羊的纤维环外层和内层细胞，进行连续传代，并评估细胞形态、I型和II型胶原蛋白基因表达及蛋白质合成、总蛋白和脱氧核糖核酸含量的变化。
纤维环外层细胞呈现细长形态，而纤维环内层细胞最初为多边形，传代后变得更像成纤维细胞。传代次数较低时，纤维环外层细胞显示出较高的I型胶原蛋白表达，而纤维环内层细胞显示出较高的II型胶原蛋白表达。传代次数较高时，纤维环内层细胞的I型胶原蛋白表达增加，纤维环外层细胞的I型胶原蛋白表达减少，而两种细胞类型的II型胶原蛋白表达均降低。免疫组织化学染色证实了基因表达结果。
纤维环外层和内层细胞表达谱的差异支持了先前的研究结果，即不同类型细胞之间存在区域差异。传代至第2代时，两种细胞类型与新鲜分离的细胞均无显著差异，并保持了明显的表型特征。然而，连续传代6次后，纤维环外层和内层细胞在形态上无法区分，I型胶原蛋白基因和蛋白质表达也无显著差异，从而成为一个更加同质化的群体。因此，连续单层传代对椎间盘细胞行为有显著影响，在设计和评估体外研究以及潜在的基于细胞的椎间盘修复治疗时，这是一个需要考虑的重要因素。
