Segovia-Silvestre Toni, Bonnefond Caroline, Sondergaard Bodil C, Christensen Tjorbjoern, Karsdal Morten A, Bay-Jensen Anne C
Nordic Bioscience A/S, Herlev Hovedgade 207, 2730 Herlev, Denmark.
BMC Res Notes. 2011 Oct 13;4:407. doi: 10.1186/1756-0500-4-407.
Preclinical and clinical studies have shown that salmon calcitonin has cartilage protective effects in joint degenerative diseases, such as osteoarthritis (OA). However, the presence of the calcitonin receptor (CTR) in articular cartilage chondrocytes is yet to be identified. In this study, we sought to further investigate the expression of the CTR in naïve human OA articular chondrocytes to gain further confirmation of the existents of the CTR in articular cartilage.
Total RNA was purified from primary chondrocytes from articular cartilage biopsies from four OA patients undergoing total knee replacement. High quality cDNA was produced using a dedicated reverse transcription polymerase chain reaction (RT-PCR) protocol. From this a nested PCR assay amplifying the full coding region of the CTR mRNA was completed. Western blotting and immunohistochemistry were used to characterize CTR protein on protein level in chondrocytes.
The full coding transcript of the CTR isoform 2 was identified in all four individuals. DNA sequencing revealed a number of allelic variants of the gene including two potentially novel polymorphisms: a frame shift mutation, +473del, producing a shorter form of the receptor protein, and a single nucleotide polymorphism in the 3' non coding region of the transcript, +1443 C>T. A 53 kDa protein band, consistent with non-glycosylated CTR isoform 2, was detected in chondrocytes with a similar size to that expressed in osteoclasts. Moreover the CTR was identified in the plasma membrane and the chondrocyte lacuna of both primary chondrocytes and OA cartilage section.
Human OA articular cartilage chondrocytes do indeed express the CTR, which makes the articular a pharmacological target of salmon calcitonin. In addition, the results support previous findings suggesting that calcitonin has a direct anabolic effect on articular cartilage.
临床前和临床研究表明，鲑鱼降钙素在骨关节炎（OA）等关节退行性疾病中具有软骨保护作用。然而，关节软骨软骨细胞中降钙素受体（CTR）的存在尚未得到证实。在本研究中，我们试图进一步研究CTR在未处理的人OA关节软骨细胞中的表达，以进一步确认关节软骨中CTR的存在。
从4例接受全膝关节置换的OA患者的关节软骨活检组织中分离出原代软骨细胞，提取总RNA。使用专用的逆转录聚合酶链反应（RT-PCR）方案制备高质量的cDNA。由此完成了扩增CTR mRNA完整编码区的巢式PCR检测。采用蛋白质印迹法和免疫组织化学法在蛋白质水平上对软骨细胞中的CTR蛋白进行表征。
在所有4例个体中均鉴定出CTR亚型2的完整编码转录本。DNA测序揭示了该基因的多个等位基因变体，包括两个潜在的新多态性：一个移码突变，+473del，产生一种较短形式的受体蛋白，以及转录本3'非编码区的一个单核苷酸多态性，+1443 C>T。在软骨细胞中检测到一条53 kDa的蛋白条带，与非糖基化的CTR亚型2一致，其大小与破骨细胞中表达的相似。此外，在原代软骨细胞和OA软骨切片的质膜和软骨细胞腔隙中均鉴定出CTR。
人OA关节软骨细胞确实表达CTR，这使得关节成为鲑鱼降钙素的药理学靶点。此外，结果支持了先前的研究结果，表明降钙素对关节软骨有直接的合成代谢作用。
