Nakoshi Yutaka, Hasegawa Masahiro, Akeda Koji, Iino Takahiro, Sudo Akihiro, Yoshida Toshimichi, Uchida Atsumasa
Department of Orthopedic Surgery, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu City, Mie 514-8507, Japan.
J Orthop Sci. 2010 Sep;15(5):666-73. doi: 10.1007/s00776-010-1513-x. Epub 2010 Oct 16.
Tenascin-C (TN-C) is expressed in the cartilage of osteoarthritis (OA). We examined whether TN-C was involved in cartilage repair of the diseased joints. Human articular cartilage samples were obtained from patients with OA and those with normal joints.
Immunohistochemistry testing of TN-C, chondroitin sulfate (CS), and proliferating cell nuclear antigen (PCNA) was performed. Chondrocytes were isolated from human cartilage and cultured. After treatment with TN-C, chondrocyte proliferation s was analyzed by bromodeoxyuridine (BrdU) incorporation assay using an enzyme-linked immunosorbent assay kit. Glycosaminoglycan content was determined by dimethylmethylene blue (DMMB) assay. The mRNA expression of aggrecan was also analyzed, by quantitative real-time polymerase chain reaction (PCR).
In osteoarthritic cartilage, increased TN-C staining was observed with the degeneration of articular cartilage in comparison with normal cartilage. TN-C staining was shown in the cartilage surface overlying CS-positive areas. In addition, the expression of PCNA in the positive areas for TN-C was significantly higher than that in the negative areas. Treatment of human articular chondrocytes with 10 μg/ml TN-C accelerated chondrocyte proliferation, increased the proteoglycan amount in culture, and increased the expression of aggrecan mRNA.
Our findings indicate that the distribution of TN-C is related to CS production and chondrocyte proliferation in osteoarthritic cartilage and that TN-C has effects on DNA synthesis, proteoglycan content, and aggrecan mRNA expression in vitro. TN-C may be responsible for repair in human osteoarthritic cartilage.
骨桥蛋白-C(TN-C)在骨关节炎(OA)的软骨中表达。我们研究了TN-C是否参与患病关节的软骨修复。从骨关节炎患者和关节正常的患者获取人关节软骨样本。
进行TN-C、硫酸软骨素(CS)和增殖细胞核抗原(PCNA)的免疫组织化学检测。从人软骨中分离软骨细胞并进行培养。用TN-C处理后,使用酶联免疫吸附测定试剂盒通过溴脱氧尿苷(BrdU)掺入试验分析软骨细胞增殖。通过二甲基亚甲基蓝(DMMB)测定法测定糖胺聚糖含量。还通过定量实时聚合酶链反应(PCR)分析聚集蛋白聚糖的mRNA表达。
与正常软骨相比,在骨关节炎软骨中,随着关节软骨退变观察到TN-C染色增加。在覆盖CS阳性区域的软骨表面显示出TN-C染色。此外,TN-C阳性区域中PCNA的表达明显高于阴性区域。用10μg/ml TN-C处理人关节软骨细胞可加速软骨细胞增殖,增加培养物中蛋白聚糖的量,并增加聚集蛋白聚糖mRNA的表达。
我们的研究结果表明,TN-C的分布与骨关节炎软骨中CS的产生和软骨细胞增殖有关,并且TN-C在体外对DNA合成、蛋白聚糖含量和聚集蛋白聚糖mRNA表达有影响。TN-C可能参与人类骨关节炎软骨的修复。