慢性鼻窦炎病人鼻腔呼吸上皮基底细胞的分化和自我更新功能研究

Although the pathophysiology of chronic rhinosinusitis(CRS) remains unclear, aberrant epithelial remodeling is one of main characteristics of nasal mucosal inflammation. Recent studies have revealed that nasal respiratory epithelium basal cells (NREBCs) may play important roles in the process of tissue remodeling and repair in CRS. Emerging evidence has shown that NREBCs can self-renew and differentiate into other epithelial cells, however, it is still not fully clear whether or not the functions of NREBCs in CRS are dysregulated. We recently found that the number of p63 positive NREBCs were remarkedly decreased in the ethmoid sinus tissue of patients with CRS. Our results indicate that dysfunction of NREBCs may result in abnormal epithelium remodeling and repair in CRS. In this project, we plan to isolate the NREBCs and unravel genes profiles of NREBCs; sphere with a pseudostratified respiratory epithelium from NREBCs will be formed after cell culture, and ability of differentiation of NREBCs after p63 gene was blocked by RNA interference will be also compared. Meanwhile, we aim to observe the functions of self-renewal and differentiation of NREBCs and explore the role of NREBCs in the animal nasal mucosa injury model. In conclusion, this project will reveal the differentiation of NREBCs in CRS and its roles in the process of mucosa repair. This project will thus provide new insights in the pathogenesis of CRS.

慢性鼻窦炎(CRS)发病机制尚未明确，异常的上皮重塑是鼻黏膜炎症的重要特征之一。近来发现鼻腔呼吸上皮基底细胞 (Basal cells, BCs) 在CRS鼻腔黏膜重塑、修复中起着重要作用。有证据表明鼻腔BCs具有自我更新和分化成其他上皮细胞的能力，但在CRS鼻腔BCs是否存在这些功能异常尚未明。我们前期工作发现CRS筛窦黏膜p63阳性BCs数量明显下降，这提示，BCs的功能异常可能导致CRS的异常上皮重塑和修复过程。本研究拟分离CRS鼻腔呼吸上皮BCs，揭示BCs基因表达特征; 通过培养单个BCs，使其分化成球形假复层纤毛柱状上皮层，并利用RNA干扰沉默p63基因，并比较观察其分化能力；建立动物鼻腔黏膜损伤模型，观察BCs自我更新和分化的能力，揭示其在黏膜损伤后修复中的作用。本课题有助于揭示CRS鼻腔呼吸上皮BCs的分化功能及其在黏膜损伤修复中的作用，为阐明CRS发病机制提供新的思路。

背景：.慢性鼻窦炎发病机制尚未明确，异常上皮重塑是鼻黏膜炎症的重要特征之一。研究发现鼻腔黏膜上皮基底细胞是一种多能干细胞，基底细胞不仅维持上皮稳态，还促进疾病的发生。基底细胞高表达p63 (tumor protein63)基因，其发育依赖于p63基因。我们研究发现慢性鼻窦炎鼻黏膜上皮p63+基底细胞分化和增殖异常。这提示p63异常表达的基底细胞致鼻腔炎症性疾病的发生。.研究方法：.本课题采取免疫荧光三标方法p63/CK5 (Cytokeratin 5,角化蛋白5)+、p63+/CK14+ ，揭示人类鼻腔黏膜及成年小鼠鼻腔黏膜上皮基底细胞分布特征; 选择雌性小鼠C57BL/6 (9-10周)，同35%三氯醋酸鼻腔给药，建立动物鼻腔黏膜化学损伤模型，观察p63+基底细胞分化和再生能力。获取鼻腔黏膜上皮，通过培养液方法，分离鼻腔基底细胞，观察基底细胞分化能力。.主要结果：.我们研究结果揭示人类鼻腔黏膜呼吸上皮存在p63+、p63+/CK5+、p63+/CK14+基底细胞。通过35%三氯醋酸进行单侧鼻腔(左侧)单次注射，成功造成鼻腔黏膜上皮的化学损伤，对侧鼻腔(右侧)作为对照 。三氯醋酸可以造成鼻腔黏膜水肿、纤毛脱落、上皮细胞水肿、细胞变性等病理变化。通过这个黏膜损伤炎症模型，观察鼻腔黏膜上皮细胞的损伤和修复过程。在黏膜上皮损伤后一周左右，鼻腔基底细胞开始再生、增殖，最终修复黏膜上皮细胞。通过基因芯片筛选出172个与鼻腔上皮细胞修复和再生相关的差异表达基因。我们成功完成鼻腔黏膜上皮细胞培养和分离，目前RNA干预仍在进行中。.我们研究揭示人类鼻腔黏膜呼吸上皮存在p63+、p63+/CK5+、p63+/CK14+基底细胞。我们发现慢性鼻窦炎病人鼻腔呼吸黏膜上皮细胞数量显著较对照组增多。然而，慢性鼻窦炎病人鼻窦区域中p63+细胞明显较下鼻甲黏膜中p63+细胞减少。 .结论：.人类、小鼠鼻腔黏膜呼吸上皮均存在p63+、p63+/CK5+、p63+/CK14+基底细胞。我们的研究结果揭示p63+鼻腔基底细胞在鼻腔黏膜损伤修复中、再生和重塑中有重要的作用。慢性鼻窦炎病人鼻腔黏膜上皮细胞增生活跃，但鼻腔p63+基底细胞分化和增殖异常。结果提示，鼻腔黏膜上皮p63+基底细胞分化和增殖异常导致鼻腔慢性炎症的发生或迁延不愈。