ヒト顎関節円板・滑膜病変に対するANK遺伝子を用いた遺伝子診断法の開発

1、ANK基因的人类同源物（ANKH）在大肠杆菌中的表达。人颞下颌关节滑膜和关节盘的研究我们从颞下颌关节紊乱病（TemporomandibularDisorder，T.MMD）患者关节盘中分离滑膜细胞，建立了滑膜细胞的原代培养体系。总RNA为。从五种不同的细胞中提取的使用ANKH特异性引物进行RT-PCR。所有病例均有特异性条带。通过直接测序证实该条带代表ANKH。因此，我们证实了ANKH mRMA在人TMJ滑膜中的表达。2. TMJ内部紊乱中ANKH基因的突变分析人类基因分析-TMD患者于2001年12月9日被京都大学医学伦理委员会批准（#G86）。在获得知情同意书后，我们从TMJ内部的患者中抽取10 ml血液。乱序和关闭锁。目前已收集和储存了80份样品。提取基因组DNA。其中12例扩增并纯化了启动子区和每11个外显子。然后直接在编码区和外显子-内含子边界进行测序。在第2、8外显子和第10内含子中发现了3个新的多态性。此外，我们确定了在启动子区的GGC三重重复的倍增。对于GGC三重重复序列，我们正在进行多病例对照研究。

1, Human homologue of ANK gene (ANKH) expression in. synovial membrane and articular disk of human temporonmandibuiar joint (TMJ)We had established the primary culture system of human synovial cells erived from articuluar disks which were surgically dissected out on temporomandibular disorder (T.MMD) patients. Total RNA was. extracted from 5 different kind of these cells. RT-PCR as performed by using,specific primers for ANKH. Specific band was found in all cases. It was confirmed that this band represented ANKH by direct sequencing. Therefore, we confirmed ANKH mRMA expression in synovial membrane of human TMJ.2, Mutation analysis, of ANKH gene in internal derangement of TMJHuman gene analysis -in TMD patients was approved by the medical ethics committee of Kyoto University on December 9, 2001 (#G86). After informed consent was obtained, we took 10ml of blood from patients which had TMJ internal. derangement and closed lock. 80 samples were collected and stored so far. Genomic DNA was extracted. In 12 cases, promoter region and every 11 exons were amplified and purified. Then they were directly sequenced in coding region and exon-introm boundary. We found 3 novel polymorphism in exon 2, 8 and intron 10. Furthermore, we identified multiplication of GGC triple repeat in Promoter region. As to GGC triple repeat, we are working on case-control study using many-cases.