颈椎后纵韧带骨化患者韧带成纤维细胞中Cx43的表达及其在成骨化中的作用

Expression of Cx43 in the ligament fibroblasts derived from patients presenting ossification of posterior longitudinal ligament and its effect during ossification

目的：观察Connexin43（Cx43）在颈椎后纵韧带骨化患者韧带成纤维细胞中的表达,探讨其在成骨化过程中的作用。方法：选择2013年1月~12月期间在我科行颈前路手术治疗的15例颈椎后纵韧带骨化症患者（骨化组）与15例不伴后纵韧带骨化的颈椎疾病患者（非骨化组）。术中切取两组患者韧带标本并采用组织块培养法进行细胞体外培养,应用免疫细胞化学及免疫荧光技术检测胞浆内波形蛋白进行细胞鉴定。采用RT-PCR方法检测细胞内成骨特异指标骨钙素（OCN）、碱性磷酸酶（ALP）及Ⅰ型胶原（COLⅠ）mRNA表达,Western blot技术检测两组细胞Cx43蛋白表达,评估其成骨活性。采用siRNA技术对骨化组第3代细胞之Cx43抑制72h,检测抑制组与非抑制组OCN、ALP及COLⅠ的表达变化。结果：经鉴定培养出的细胞为成纤维细胞。韧带骨化组细胞OCN、ALP与COLⅠ的mRNA表达量分别为1.36±0.21、0.53±0.18、1.64±0.37,明显高于非骨化组（0.78±0.21、0.29±0.13、1.01±0.26）;骨化组Cx43蛋白表达量（1.00±0.30）亦明显高于非骨化组（0.48±0.18）。对骨化组细胞Cx43抑制72h后,其蛋白表达下调69%（P〈0.01）;非抑制组OCN、ALP及COLⅠ的mRNA表达量为1.54±0.33、1.32±0.36、1.86±0.44,抑制组分别为0.91±0.32、0.47±0.21、0.88±0.29,两组比较差异具有统计学意义（P〈0.05）。结论：颈椎后纵韧带骨化症患者韧带成纤维细胞中存在较高水平的Cx43,并在其成骨化过程中发挥着积极作用。

Objectives： To investigate the expression of connexin43（Cx43） in the ligament fibroblasts derived from patients presenting ossification of posterior longitudinal ligament（OPLL） and its effect during ossification. Methods： Fifteen patients presenting with OPLL and 15 non-OPLL patients underwent anterior decompression between January 2013 and December 2013. Specimens of the posterior longitudinal ligament were collected intraoperatively. Tissue fragment cell culture was performed. The mouse anti-vimentin antibody was used to i- dentify the cultured cells via immunocytochemistry and immunofluoreseence（ICC/IF）. The mRNA expressions of osteoblast-specific genes of osteoealein（OCN）, alkaline phosphatase（ALP） and type I collagen（COL I ） were detected in OPLL and non-OPLL cells by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）. The protein expression of Cx43 was detected via western blotting. And after 72h, when RNA interference against Cx43 was performed in OPLL cells, the expressions of indexes mentioned above were compared again between transfection and non-transfection group. Results： The mRNA expression of OCN, ALP and COL I was 1.36±0.21, 0.53±0.18 and 1.64±0.37. It was significantly higher compared with non-OPLL group, with expression as 0.78±0.21, 0.29±0.13 and 1.01±0.26. The protein expression of Cx43（1.00±0.30） in OPLL group was also obvious higher than the non-OPLL group（0.48±0.18）. Furthermore, after 72h of transfection targeting Cx43, result of western blot showed a down-regulation of Cx43 protein expression by almost 69%（P〈0.01）. The mRNA expression of OCN, ALP and COL I was 1.54±0.33, 1.32±0.36, 1.86±0.44 in non-transfeetion group and 0.91±0.32, 0.47±0.21, 0.88±0.29 in non-transfeetion group. The difference was significant. Conclusions： There is a higher expression of Cx43 in ligament fibroblasts derived from OPLL, which indicates that Cx43 plays an important role during the ossification of the ligament fibroblasts.