Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th St, Pittsburgh, PA 15201, USA.
Biomolecules. 2020 Aug 14;10(8):1182. doi: 10.3390/biom10081182.
: Cilia are actin based cellular protrusions conserved from algae to complex multicellular organisms like Homo sapiens. Respiratory motile cilia line epithelial cells of the tracheobronchial tree, beat in a synchronous, metachronal wave, moving inhaled pollutants and pathogens cephalad. Their role in both congenital disorders like primary ciliary dyskinesia (PCD) to acquired disorders like chronic obstructive pulmonary disease (COPD) continues to evolve. In this current body of work we outline a protocol optimized to reciliate human nasal epithelial cells and mouse tracheal cells in vitro. Using this protocol, we knocked down known cilia genes, as well as use a small molecule inhibitor of Notch, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT), to assess the effect of these on ciliogenesis in order to show the validity of our protocol. : Tracheas were harvested from wild-type, adult C57B6 mice, pronase digested and sloughed off epithelial cells grown to confluence in stationary culture on rat-tail collagen coated wells. Upon reaching confluence, collagen was digested and cells placed suspension culture protocol to reciliate the cells. Using this suspension culture protocol, we employed siRNA gene knockdown to assay gene functions required for airway ciliogenesis. Knock down of Dynein axonemal heavy chain 5 (Dnah5), a ciliary structural protein, was confirmed using immunostaining. Mouse tracheal cells were treated in suspension with varying doses of DAPT, an inhibitor of Notch, with the purpose of evaluating its effect and dose response on ciliogenesis. The optimum dose was then used on reciliating human nasal epithelial cells. : siRNA knockdown of prevented ciliation, consistent with its role as a master regulator of motile cilia. Knockdown of and resulted in immotile cilia, and knockdown, a centrosome protein known to regulate centrosome amplification, inhibited airway ciliogenesis. Dnah5 knockdown was confirmed with significantly decreased immunostaining of cilia for this protein. Inhibiting Notch signaling by inhibiting gamma secretase with DAPT enhanced the percentage of ciliation, and resulted in longer cilia that beat with higher frequency in both mouse and human airway epithelia. : Modifying existing reciliation protocols to suit both human nasal epithelial and mouse tracheal tissue, we have shown that knockdown of known cilia-related genes have the expected effects. Additionally, we have demonstrated the optimal dosage for significantly improving reciliation of airway epithelia using DAPT. Given that cilia length and function are significantly compromised in COPD, these findings open up interesting avenues for further exploration.
纤毛是一种从藻类到人类等复杂多细胞生物中都保守存在的肌动蛋白细胞突起。呼吸道运动纤毛排列在气管支气管树的上皮细胞中，以同步的、顺序的波浪形式跳动，将吸入的污染物和病原体向上移动。它们在先天性疾病（如原发性纤毛运动障碍（PCD））和获得性疾病（如慢性阻塞性肺疾病（COPD））中的作用仍在不断发展。在目前的研究中，我们概述了一种优化的方案，以在体外重新激活人鼻上皮细胞和小鼠气管细胞。使用该方案，我们敲低了已知的纤毛基因，并使用 Notch 的小分子抑制剂 N-[N-(3,5-二氟苯乙酰基)-L-丙氨酰]-S-苯甘氨酸叔丁酯（DAPT），以评估这些对纤毛发生的影响，从而证明我们方案的有效性。：从野生型成年 C57B6 小鼠中收获气管，用蛋白酶消化并脱落上皮细胞，在涂有大鼠尾胶原的培养皿中静止培养至汇合。当达到汇合时，胶原被消化，细胞置于悬浮培养方案中以重新激活细胞。使用这种悬浮培养方案，我们采用 siRNA 基因敲低来检测气道纤毛发生所需的基因功能。使用免疫染色证实了纤毛结构蛋白 Dynein 轴索重链 5（Dnah5）的敲低。用不同剂量的 Notch 抑制剂 DAPT 处理悬浮培养的小鼠气管细胞，以评估其对纤毛发生的作用和剂量反应。然后将最佳剂量用于重新激活人鼻上皮细胞。：siRNA 敲低 阻止了纤毛的发生，这与它作为运动纤毛的主要调节因子的作用一致。 和 的敲低导致无纤毛，中心体蛋白 的敲低已知可调节中心体扩增，抑制气道纤毛发生。用该蛋白的免疫染色明显减少证实了 Dnah5 的敲低。用 DAPT 抑制 γ 分泌酶抑制 Notch 信号通路增强了纤毛的形成百分比，并导致在小鼠和人呼吸道上皮细胞中纤毛更长、跳动频率更高。：通过修改现有的重新激活方案以适应人鼻上皮和小鼠气管组织，我们表明，已知的纤毛相关基因的敲低具有预期的效果。此外，我们还证明了使用 DAPT 显著提高气道上皮细胞重新激活的最佳剂量。由于 COPD 中纤毛长度和功能明显受损，这些发现为进一步探索开辟了有趣的途径。
