Effect of Tumor Necrosis Factor to Chondrocytes in Endochondral Ossification

软骨内骨化过程中肿瘤坏死因子对软骨细胞的影响

• 批准号: 12671391
• 负责人: AIZAWA Toshimi
• 金额: $ 1.6万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671391/
• 关键词: fracture healing; endochondral ossification; apotosis; chondrocyte; tumor necrosis factor; サイトカイン

In endochondral ossification, chondrocytes change their morphology and functions and are ultimately removed by the process of programmed cell death (apoptosis). A variety of apoptosis inducing signals and death factors have been characterized including tumor necrosis factor-alpha (TNF-α), Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL). However, there is little known regarding the activity of these factors in the process of fracture healing. Using RNase protection assay (RPA) and immunohistochemistry, this study sought to determine if these factors might participate in endochondral fracture healing. A closed, transverse fracture was made in the right tibiae of 8-10 week old BALB/C mice. Mice were euthanized at 1, 2 and 3 weeks post-fracture. The tibiae were harvested and studied for the expression of caspase-8, a key enzyme of apoptosis. TNF-α, FasL and TRAIL, were assayed using RPA for mRNA and immunostaining for protein. At each time point, caspase-8, FasL and its receptor Fas receptor (FasR), TNF-α and its receptor p55, and TRAIL were detected by RPA. Immunostainings clearly showed that caspase-8, TNF-α, FasL, FasR and TRAIL were expressed in fracture callus chondrocytes. Additionally, the mice in which TNF-α receptors were ablated showed very little expression of caspase-8 by RPA. These findings suggest that cartilaginous callus is replaced by new bone during endochondral fracture healing and chondrocyte removal by apoptosis is controlled by several death factors via autocrine and paracrine mechanisms, particularly TNF-α might play a major role in chondrocyte death.

在软骨内骨化中，软骨细胞改变其形态和功能，并最终通过程序性细胞死亡（凋亡）过程去除。肿瘤坏死因子-α（TNF-α）、Fas配体（FasL）和肿瘤坏死因子相关凋亡诱导配体（TRAIL）等多种凋亡诱导信号和死亡因子已被鉴定。然而，关于这些因素在骨折愈合过程中的活性知之甚少。本研究采用核糖核酸酶保护试验（RPA）和免疫组织化学方法，试图确定这些因素是否可能参与软骨内骨折愈合。在8-10周龄BALB/C小鼠的右胫骨中制造闭合的横向骨折。在骨折后1、2和3周对小鼠实施安乐死。收获细胞并研究细胞凋亡的关键酶caspase-8的表达。TNF-α、FasL和TRAIL分别用RPA检测mRNA和免疫组化染色检测蛋白。RPA法检测各时间点caspase-8、FasL及其受体Fas受体（FasR）、TNF-α及其受体p55、TRAIL的表达。免疫组化结果显示，caspase-8、TNF-α、FasL、FasL和TRAIL在骨折骨痂软骨细胞中均有表达。此外，TNF-α受体被消融的小鼠显示RPA几乎不表达caspase-8。这些结果表明，软骨内骨折愈合过程中，软骨骨痂被新骨取代，软骨细胞通过凋亡被去除，这是由多种死亡因子通过自分泌和旁分泌机制控制的，尤其是TNF-α可能在软骨细胞死亡中起主要作用。