Kurokawa Atsushi, Kondo Mitsuko, Honda Nahoko, Orimo Mami, Miyoshi Azusa, Kobayashi Fumi, Abe Kazuhiro, Akaba Tomohiro, Tsuji Mayoko, Arimura Ken, Nakatani Kaname, Ikejiri Makoto, Yagi Osamitsu, Takeyama Kiyoshi, Katsura Hideki, Takeuchi Kazuhiko, Tagaya Etsuko
Department of Respiratory Medicine, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan.
Department of Respiratory Medicine, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan.
Respir Investig. 2022 May;60(3):407-417. doi: 10.1016/j.resinv.2022.02.003. Epub 2022 Mar 17.
Primary ciliary dyskinesia (PCD) is diagnosed through multiple methods, including transmission electron microscopy (TEM), a high-speed video microscopy analysis (HSVA), immunofluorescence (IF), and genetic testing. A primary cell culture has been recommended to avoid the misdiagnosis of secondary ciliary dyskinesia derived from infection or inflammation and improve diagnostic accuracy. However, primary cells fail to differentiate into ciliated cells through repeated passages. The conditional reprogramming culture (CRC) method, a combination of a Rho-kinase inhibitor and fibroblast feeder cells, has been applied to cystic fibrosis. The goal of this study was to evaluate the value of CRC in diagnosing PCD in Japanese patients.
Eleven patients clinically suspected of having PCD were included. Airway epithelial cells were obtained from an endobronchial forceps biopsy and cultured at the air-liquid interface (ALI) combined with CRC. Ciliary movement, ultrastructure, and mutated ciliary protein evaluation were performed using HSVA, TEM, and IF, respectively. Genetic testing was performed on some patients.
CRC yielded dense and well-differentiated ciliated cells with a high success rate (∼90%). In patients with PCD, the ciliary ultrastructure phenotype (outer dynein arm defects or normal ultrastructure) and IF findings (absence of the mutated ciliary protein) were confirmed after CRC. In DNAH11-mutant cases with normal ultrastructure by TEM, the HSVA revealed stiff and hyperfrequent ciliary beating with low bending capacity in CRC-expanded cells, thereby supporting the diagnosis.
CRC could be a potential tool for improving diagnostic accuracy and contributing to future clinical and basic research in PCD.
原发性纤毛运动障碍（PCD）通过多种方法进行诊断，包括透射电子显微镜检查（TEM）、高速视频显微镜分析（HSVA）、免疫荧光（IF）和基因检测。推荐进行原代细胞培养以避免误诊由感染或炎症引起的继发性纤毛运动障碍，并提高诊断准确性。然而，原代细胞在反复传代后无法分化为纤毛细胞。条件重编程培养（CRC）方法，即Rho激酶抑制剂与成纤维细胞饲养层细胞的组合，已应用于囊性纤维化。本研究的目的是评估CRC在诊断日本PCD患者中的价值。
纳入了11例临床怀疑患有PCD的患者。通过支气管内钳取活检获取气道上皮细胞，并在气液界面（ALI）结合CRC进行培养。分别使用HSVA、TEM和IF进行纤毛运动、超微结构和突变纤毛蛋白评估。对部分患者进行了基因检测。
CRC产生了密集且分化良好的纤毛细胞，成功率很高（约90%）。在PCD患者中，CRC后证实了纤毛超微结构表型（外动力臂缺陷或正常超微结构）和IF结果（无突变纤毛蛋白）。在TEM显示超微结构正常的DNAH11突变病例中，HSVA显示CRC扩增细胞中的纤毛摆动僵硬且频繁，弯曲能力低，从而支持了诊断。
CRC可能是提高诊断准确性并有助于PCD未来临床和基础研究的潜在工具。
