摘要
目的研究转化生长因子β1(TGF-β1)在牵张应力刺激促进后纵韧带成纤维细胞骨向分化进程中的作用。方法经颈前路手术获取颈椎后纵韧带骨化症(OPLL)患者的后纵韧带组织,采用组织块培养法进行体外细胞培养及鉴定。通过荧光实时定量PCR检测后纵韧带成纤维细胞在加载牵张应力刺激12 h及24 h后Ⅰ型胶原(COLⅠ)、碱性磷酸酶(ALP)和TGF-β1的mRNA表达变化;并检测在加入外源性TGF-β1或TGF-β1抗体作用后,后纵韧带成纤维细胞在静置或牵张应力刺激下的COLⅠ和ALP的mRNA表达变化。结果 HE及免疫荧光染色证实后纵韧带成纤维细胞培养成功。后纵韧带成纤维细胞经牵张应力刺激12 h及24 h后,细胞COLⅠ、ALP和TGF-β1的mRNA表达与静置相同时间的对照组相比均有升高,且24 h时差异具有统计学意义(P<0.05)。加入0.1、1.0、10.0 ng/mL的外源性TGF-β1并静置24 h后细胞COLⅠ及ALP的mRNA表达量升高,其中10.0 ng/mL浓度组与对照组相比差异具有统计学意义(P<0.05);而加入2.0μg/mL的TGF-β1抗体并加载牵张应力刺激24 h后细胞的COLⅠ及ALP的mRNA表达量的升高程度与对照组相比明显降低,差异具有统计学意义(P<0.05)。结论组织块培养法是进行颈椎OPLL患者后纵韧带成纤维细胞体外培养的有效方法。后纵韧带成纤维细胞在牵张应力刺激下TGF-β1表达上调,且TGF-β1可促进其骨向分化。
Objective To investigate the role of transforming growth factor-β1(TGF-β1) in the ossification of the posterior longitudinal ligament fibroblasts caused by stretch stress. Methods The specimens of posterior longitudinal ligament were collected via anterior cervical surgery for the patients with ossification of posterior longitudinal ligament(OPLL). The fibroblasts were obtained by tissue culture in vitro. The typeⅠcollagen(COLⅠ),alkaline phosphatase(ALP) and TGF-β1 gene expression were detected by real-time RT-PCR at 12 h and 24 h after mechanical stress. The COL Ⅰ and ALP gene expressions at 24 h after being treated with exogenous TGF-β1 or TGF-β1 antibody and mechanical stress was also detected. Results HE and immunofluorescence staining confirmed the posterior longitudinal ligament fibroblast culture was successful. The COLⅠ,ALP and TGF-β1 gene expression of the fibroblasts were up-regulated at 12 h and 24 h after mechanical stress,and the difference was statistically significant at 24 h(P < 0.05). Being treated with different concentrations(0.1,1.0 and 10.0 ng/m L) exogenous TGF-β1,the COLⅠand ALP gene expression were up-regulated at 24 h,and the difference was statistically significant between 10.0 ng/m L and the control group(P < 0.05). Being treated with 2.0 μg/m L TGF-β1 antibody,the rate of COLⅠand ALP gene expression up-regulation at 24 h after mechanical stress were decreased significantly compared to the control group(P < 0.05). Conclusion Tissue culture is an effective method to obtain posterior longitudinal ligament fibroblasts deprived from the OPLL patients. Stretch stress stimulation could promote TGF-β1 gene expression of the posterior longitudinal ligament fibroblasts in OPLL patients,which in turn accelerates the ossification process of the fibroblasts.
出处
《脊柱外科杂志》
2018年第1期41-45,共5页
Journal of Spinal Surgery
基金
上海中医药大学预算内项目(2015YSN043)
关键词
颈椎
骨化
后纵韧带
转化生长因子Β
胶原Ⅰ型
碱性磷酸酶
Cervical vertebrae
Ossification of posterior longitudinal ligament
Transforming growth factor beta
Collagen typeⅠ
Alkaline phosphatase
