Department of Otolaryngology-Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha 410008.
Otolaryngology Major Disease Research Key Laboratory of Hunan Province, Xiangya Hospital, Central South University, Changsha 410008.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2024 May 28;49(5):655-666. doi: 10.11817/j.issn.1672-7347.2024.230482.
Progressive bone resorption and destruction is one of the most critical clinical features of middle ear cholesteatoma, potentially leading to various intracranial and extracranial complications. However, the mechanisms underlying bone destruction in middle ear cholesteatoma remain unclear. This study aims to explore the role of parathyroid hormone-related protein (PTHrP) in bone destruction associated with middle ear cholesteatoma.
A total of 25 cholesteatoma specimens and 13 normal external auditory canal skin specimens were collected from patients with acquired middle ear cholesteatoma. Immunohistochemical staining was used to detect the expressions of PTHrP, receptor activator for nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in cholesteatoma and normal tissues. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the presence of TRAP positive multi-nucleated macrophages in cholesteatoma and normal tissues. Mono-nuclear macrophage RAW264.7 cells were subjected to interventions, divided into a RANKL intervention group and a PTHrP+ RANKL co-intervention group. TRAP staining was used to detect osteoclast formation in the 2 groups. The mRNA expression levels of osteoclast-related genes, including , cathepsin K (), and nuclear factor of activated T cell cytoplasmic 1 (), were measured using real-time polymerase chain reaction (real-time PCR) after the interventions. Bone resorption function of osteoclasts was assessed using a bone resorption pit analysis.
Immunohistochemical staining showed significantly increased expression of PTHrP and RANKL and decreased expression of OPG in cholesteatoma tissues (all <0.05). PTHrP expression was significantly positively correlated with RANKL, the RANKL/OPG ratio, and negatively correlated with OPG expression (=0.385, =0.417, =-0.316, all <0.05). Additionally, the expression levels of PTHrP and RANKL were significantly positively correlated with the degree of bone destruction in cholesteatoma (=0.413, =0.505, both <0.05). TRAP staining revealed a large number of TRAP-positive cells, including multi-nucleated osteoclasts with three or more nuclei, in the stroma surrounding the cholesteatoma epithelium. After 5 days of RANKL or PTHrP+RANKL co-intervention, the number of osteoclasts was significantly greater in the PTHrP+RANKL co-intervention group than that in the RANKL group (<0.05), with increased mRNA expression levels of , , and (all <0.05). Scanning electron microscopy of bone resorption pits showed that the number (<0.05) and size of bone resorption pits on bone slices were significantly greater in the PTHrP+RANKL co-intervention group compared with the RANKL group.
PTHrP may promote the differentiation of macrophages in the surrounding stroma of cholesteatoma into osteoclasts through RANKL induction, contributing to bone destruction in middle ear cholesteatoma.
进行性骨吸收和破坏是中耳胆脂瘤的最关键临床特征之一，可能导致各种颅内和颅外并发症。然而，中耳胆脂瘤骨破坏的机制仍不清楚。本研究旨在探讨甲状旁腺激素相关蛋白（PTHrP）在中耳胆脂瘤相关骨破坏中的作用。
从患有获得性中耳胆脂瘤的患者中收集了 25 例胆脂瘤标本和 13 例正常外耳道皮肤标本。使用免疫组织化学染色检测 PTHrP、核因子-κB 配体受体激活剂（RANKL）和骨保护素（OPG）在胆脂瘤和正常组织中的表达。使用抗酒石酸酸性磷酸酶（TRAP）染色检测胆脂瘤和正常组织中是否存在 TRAP 阳性多核巨细胞。将单核巨噬细胞 RAW264.7 细胞进行干预，分为 RANKL 干预组和 PTHrP+RANKL 共干预组。使用 TRAP 染色检测两组中的破骨细胞形成情况。干预后，使用实时聚合酶链反应（real-time PCR）测量破骨细胞相关基因（包括组织蛋白酶 K（）和活化 T 细胞核因子细胞质 1（））的 mRNA 表达水平。使用骨吸收陷窝分析评估破骨细胞的骨吸收功能。
免疫组织化学染色显示 PTHrP 和 RANKL 的表达显著增加，而 OPG 的表达显著降低（均<0.05）。PTHrP 表达与 RANKL、RANKL/OPG 比值呈显著正相关，与 OPG 表达呈显著负相关（=0.385，=0.417，=−0.316，均<0.05）。此外，PTHrP 和 RANKL 的表达水平与胆脂瘤骨破坏程度呈显著正相关（=0.413，=0.505，均<0.05）。TRAP 染色显示在胆脂瘤上皮周围的基质中存在大量 TRAP 阳性细胞，包括具有三个或更多核的多核破骨细胞。在 RANKL 或 PTHrP+RANKL 共干预 5 天后，PTHrP+RANKL 共干预组中的破骨细胞数量明显多于 RANKL 组（<0.05），且 、、和 （均<0.05）的 mRNA 表达水平升高。骨吸收陷窝扫描电子显微镜显示，与 RANKL 组相比，PTHrP+RANKL 共干预组骨切片上的骨吸收陷窝数量（<0.05）和大小均显著增大。
PTHrP 可能通过诱导 RANKL 促进胆脂瘤周围基质中的巨噬细胞分化为破骨细胞，从而促进中耳胆脂瘤的骨破坏。
