Risbud Makarand V, Di Martino Alberto, Guttapalli Asha, Seghatoleslami Reza, Denaro Vincenzo, Vaccaro Alexander R, Albert Todd J, Shapiro Irving M
Department of Orthopaedic Surgery and Graduate Program in Tissue Engineering and Regenerative Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Spine (Phila Pa 1976). 2006 Apr 15;31(8):884-90. doi: 10.1097/01.brs.0000209335.57767.b5.
Rat lumbar discs comprising nucleus pulposus, annulus fibrosus, and cartilaginous endplates were cultured for 1 week in a specialized media containing either TGF-beta1 or TGF-beta3. Role of TGF-beta isoforms on cell function was evaluated.
To develop an in vitro organ culture of rat intervertebral disc and evaluate effects of TGF-beta3 on disc cell function.
An in vitro model system is of considerable value in understanding the cell biology of the intervertebral disc. Development of a useful organ culture model would enhance understanding of disc function in health and disease.
Rat lumbar intervertebral discs were maintained in organ culture in media supplemented with TGF-beta3 or TGF-beta1 for 1 week. Tissue morphology was studied using routine histologic, histochemical and immunohistochemical techniques. Cell function was assessed by gene expression, sulfate incorporation, and Western blot analysis.
After 1 week in culture with TGF-beta3 and TGF-beta1, the gross morphology and tissue architecture of the disc were preserved. TUNEL analysis indicated that there was no evidence of cell death in the nucleus pulposus or the anulus fibrosus. The level of Alcian blue staining in the nucleus pulposus was similar to that of the freshly isolated disc. However, when compared with TGF-beta1, TGF-beta3 elevated the expression of critical matrix genes, enhanced [S] incorporation into proteoglycans, preserved the expression of TGF-beta receptors, and decreased aggrecan turnover. There was also increased activation (phosphorylation) of ERK, a critical signaling protein. Moreover, inhibition of ERK activity, in the presence TGF-beta3, resulted in suppression of collagen Type II, aggrecan, TGF-beta-RI, TGF-beta-RII and TGF-beta-RIII mRNA expression.
TGF-beta3 maintains the phenotype of disc cells in organ culture. It exerts this effect, in part, by elevating the levels of activated ERK1/2, which in turn regulates the expression of TGF-beta-RI and TGF-beta-RII.
将包含髓核、纤维环和软骨终板的大鼠腰椎间盘在含有转化生长因子β1（TGF-β1）或转化生长因子β3（TGF-β3）的特殊培养基中培养1周。评估TGF-β亚型对细胞功能的作用。
建立大鼠椎间盘的体外器官培养模型，并评估TGF-β3对椎间盘细胞功能的影响。
体外模型系统对于理解椎间盘的细胞生物学具有重要价值。建立一个有用的器官培养模型将增进对椎间盘在健康和疾病状态下功能的理解。
将大鼠腰椎间盘在添加了TGF-β3或TGF-β1的培养基中进行器官培养1周。使用常规组织学、组织化学和免疫组织化学技术研究组织形态。通过基因表达、硫酸盐掺入和蛋白质印迹分析评估细胞功能。
在含有TGF-β3和TGF-β1的培养基中培养1周后，椎间盘的大体形态和组织结构得以保留。TUNEL分析表明，在髓核或纤维环中没有细胞死亡的证据。髓核中阿尔新蓝染色水平与新鲜分离的椎间盘相似。然而，与TGF-β1相比，TGF-β3提高了关键基质基因的表达，增强了硫酸盐掺入蛋白聚糖的能力，保留了TGF-β受体的表达，并降低了聚集蛋白聚糖的更新。关键信号蛋白ERK的激活（磷酸化）也增加。此外，在存在TGF-β3的情况下抑制ERK活性导致II型胶原、聚集蛋白聚糖、TGF-β-RI、TGF-β-RII和TGF-β-RIII mRNA表达受到抑制。
TGF-β3在器官培养中维持椎间盘细胞的表型。它部分通过提高活化的ERK1/2水平来发挥这种作用，而ERK1/2又反过来调节TGF-β-RI和TGF-β-RII的表达。
