Orthopedic Department of Tangdu Hospital, the Fourth Military Medical University, #1 Xinsi Road, Xi'an, PR China.
Department of Immunology, the fourth Military Medical University, #1 Changle Xi Road, Xi'an, PR China; Transplant Immunology Laboratory, the Fourth Military Medical University, #1 Changle Xi Road, Xi'an, PR China.
Mol Immunol. 2019 Mar;107:123-131. doi: 10.1016/j.molimm.2019.01.013. Epub 2019 Feb 6.
This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N-4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA-binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.
本研究旨在探讨 CD226 对巨核细胞（MKs）分化、激活和多倍体化的影响，并探索其潜在机制。佛波醇 12-肉豆蔻酸 13-乙酸酯诱导 Dami（巨核细胞系）细胞成熟。通过感染 CD226 特异性 shRNA 慢病毒载体沉默 CD226。qRT-PCR 检测 CD226 的 mRNA 水平。通过流式细胞术评估 Dami 细胞表面 CD226、MK 特异性标志物 CD41 和 CD62P 以及 Dami 细胞和 CD226 敲低（KD）细胞中的 DNA 倍性。通过 Western blot 和共聚焦分析测量 CD226 对巨核细胞相关转录因子表达的影响。CD226 shRNA 慢病毒转染显著降低了 Dami 细胞中 CD226 的水平和 CD62P 的表达。沉默 CD226 导致低倍体 MK 形态发生变化和分化延迟。此外，CD226 敲除（KO）小鼠表现出增加的 2N-4N 低倍体 MK 和减少的≥8N 多倍体。有趣的是，巨核细胞中 CD226 的沉默下调了早期转录因子的表达，包括 GATA 结合因子 1（GATA-1）和 Friend 白血病整合 1（FLI-1），但不包括晚期核因子，红细胞 2（NF-E2）。CD226 参与 MKs 的激活和多倍体细胞周期调控。
