Analysis of the diagnosis of Japanese patients with primary ciliary dyskinesia using a conditional reprogramming culture

BackgroundPrimary 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.MethodsEleven 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.ResultsCRC 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.ConclusionsCRC could be a potential tool for improving diagnostic accuracy and contributing to future clinical and basic research in PCD.     

扁塑藤素调控Notch信号通路诱导人肺癌条件重编程细胞死亡的作用机制研究

目的：应用条件性重编程细胞（conditionally reprogrammed cells，CRCs）技术建立人肺癌细胞体外长期培养体系，研究扁塑藤素对人肺癌CRCs增殖和迁移能力的影响，并探讨相关作用机制。方法：免疫组化法检测Notch 1、HES1和Cyclin D3在肿瘤组织和癌旁组织中的表达水平；使用CRCs技术分离培养非小细胞肺癌原代细胞；使用2，4，8，16 μmol·L^-1的扁塑藤素处理人肺癌CRCs，MTS法检测细胞活力，Annexin V/PI流式细胞术检测细胞凋亡，Transwell法检测细胞迁移能力，Western Blot检测细胞中Notch信号通路相关蛋白Notch 1、HES1和Cyclin D3的表达水平。结果：在非小细胞肺癌患者肿瘤组织中Notch 1、HES1和Cyclin D3的表达水平高于癌旁组织；扁塑藤素能够诱导人肺癌CRCs死亡，并在一定范围内呈现时间和浓度依赖性（P<0.05）；随着扁塑藤素作用浓度的增高，人肺癌CRCs凋亡率明显增高（P<0.05），细胞迁移能力下降（P<0.05）；扁塑藤素能够下调人肺癌CRCs中Notch 1、HES1和Cyclin D3的蛋白表达水平。结论：扁塑藤素可能通过调控Notch信号通路相关蛋白的表达水平，抑制人肺癌CRCs的增殖和迁移，并诱导其凋亡，为肺癌的治疗提供新的实验数据和理论依据。     

Myocardin重组蛋白表达及其在人骨髓间充质干细胞重编程为血管平滑肌细胞中的作用

目的:研究心肌素(myocardin)重组蛋白的表达及分离纯化方法,并探讨myocardin重组蛋白对人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,h BMSCs)向血管平滑肌细胞转分化的作用。方法:构建myocardin原核表达载体,摸索最佳诱导myocardin重组蛋白表达的方法及优化分离纯化方式。使用纳米转导试剂包裹myocardin形成纳米-蛋白复合体,转染h BMSCs,观察蛋白转导效率;蛋白转导成功后,检测血管平滑肌的特异性标志物平滑肌肌球蛋白重链的表达情况,观察h BMSCs向平滑肌细胞转化情况。结果:与传统方法相比较,高浓度法诱导myocardin重组蛋白的表达量更高,新采用的洗脱方式能获取更多的myocardin重组蛋白;纳米-myocardin复合体可成功转染h BMSCs并诱导其向血管平滑肌样细胞转化。结论:Myocardin重组蛋白能被高效转导到细胞内,并促使h BMSCs重编程为血管平滑肌样细胞。     

Protein Kinase C-β Dictates B Cell Fate by Regulating Mitochondrial Remodeling,Metabolic Reprogramming,and Heme Biosynthesis

1. Download high-res image (142KB)
2. Download full-size image

     

卵母细胞发育和受精中的重编程及对雄原核重编程的影响

哺乳动物发育过程中细胞的重编程是当前细胞生物学、转化医学等领域研究的热点之一,因其在体细胞核移植、诱导多能干细胞及再生医学中的应用前景引起了广泛关注。表观遗传学修饰是重编程的主要内容,包括DNA甲基化、组蛋白翻译后修饰、基因组印迹、非编码RNA调控等,这些均可通过不同的机制调控基因组的表达。如卵母细胞发育与成熟过程伴随着基因组的从头甲基化、减数分裂相关基因的去甲基化和组蛋白翻译后的修饰等。此外,受精中,卵母细胞又与进入其中的雄原核发生相互作用,为雄原核基因组的去甲基化等过程提供重编程的环境。总结近年来关于卵母细胞发育和受精过程中重编程机制的研究进展及在胚胎发育中的意义,并探讨自然生殖状况下卵母细胞环境对外源性雄原核重编程的影响。     

Heterogeneity of Osteosarcoma Cell Lines Led to Variable Responses in Reprogramming

Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture.