伴肌动蛋白相关锚定蛋白（N-RAP）在颈椎后纵韧带骨化中的表达及意义

Expression and significance of Nebulin related anchoring protein(N-RAP) in the ossified posterior longitudinal ligament

中文关键词:  后纵韧带骨化症  伴肌动蛋白相关锚定蛋白  实时定量PCR

英文关键词:Ossification of the posterior longitudinal ligament  Nebulin related anchoring protein  RT-PCR

中文摘要:

  【摘要】 目的：从蛋白质及核酸水平研究伴肌动蛋白相关锚定蛋白（N-RAP）在颈椎后纵韧带骨化中的表达，探讨其存在的临床意义。方法：选择2006～2010年间行手术治疗的20例颈椎后纵韧带骨化症患者（OPLL组）及10例因外伤行颈椎手术患者（对照组）的颈椎后纵韧带标本。所有标本福尔马林固定、石蜡包埋，以N-RAP兔抗人多克隆抗体作为Ⅰ抗免疫组化SP法制片，DAB显色。选择染色满意的标本切片，观察表达N-RAP的细胞（棕黄染色）在两组样本中的数量及分布特点。5例骨化样本及5例对照样本冰浴匀浆、提取总RNA，行实时定量PCR检测。观测后纵韧带骨化症患者及对照病例后纵韧带组织中N-RAP mRNA的表达情况。结果：20例骨化韧带标本中，16例染色满意；10例对照组后纵韧带标本中，6例染色满意。免疫组化结果显示N-RAP表达于韧带组织中细胞的胞浆中，呈棕黄色染色。在OPLL组的后纵韧带组织中，可见多个黄染细胞及少量蓝色无N-RAP表达的细胞。而在对照组后纵韧带中，仅可见蓝色无N-RAP表达的细胞。实时定量PCR检测发现N-RAP在对照组标本中平均表达量为14.29±4.70，在OPLL组标本中为161.29±60.14，两组间差异有统计学意义（P＜0.05）。结论：颈椎OPLL韧带组织中存在较高水平的N-RAP，其可能参与了韧带骨化的发生、发展过程。

英文摘要:

  【Abstract】 Objectives: To investigate the expression of Nebulin related anchoring protein(N-RAP) in the ossified posterior longitudinal ligament, and to explore its clinical significance. Methods: Ligament samples were harvested from the patients undergoing anterior cervical surgeries. 20 were from patients with OPLL and 10 from patients suffering from trauma were served as control group. All samples were fixed with formalin and embedded with paraffin. N-RAP polyclonal rabbit anti-human antibody, immunohistochemical SP method tablets and DAB color were used for immunohistochemical detection. Slices with satisfying staining were submitted for observation of cells expressing N-RAP(Brown-yellow staining). Samples from 5 ossified ligaments and 5 normal controls were submitted for RT-PCR. Samples were homogenizered on ice and total RNA was extracted. Real-time quantitative PCR was used to verify the mRNA expression of N-RAP in two groups. Results: 16 samples from ossified ligaments and 6 samples from normal controls got satisfying immunohistochemical staining. Immunohistochemistry showed that N-RAP expressed in cytoplasm in cellular components of ligament tissue. Cells expressing N-RAP were brown-yellow, while cells with no N-RAP were in blue. In ossified ligament from patients with OPLL, most cells were brown-yellow and a samll amount of blue cells without expressing N-RAP were observed. While in control samples, only blue cells with no N-RAP expression could be seen. Under real-time quantitative PCR detection，the average expression level of N-RAP was 14.29±4.70 in control groups, while 161.29±60.14 in ossified ligaments, which showed significant difference between two groups(P＜0.05). Conclusions: There is a higher expression of N-RAP in ligament tissue from patients with OPLL, which indicates that N-RAP may be involved in the onset or development of OPLL.