颈椎后纵韧带骨化细胞体外培养及成骨活性检测

In vitro cell culture and osteogenecity of cells derived from cervical ossificaitonl of posterior longitudinal ligament

目的探讨体外培养颈椎后纵韧带骨化（ossification of posterior longitudinal ligament,OPLL）患者韧带细胞的方法并检测其成骨活性。方法 2013年1~12月颈椎外伤与后纵韧带骨化患者各15例行颈前路手术治疗。术中切取韧带标本,采用组织块培养法进行体外培养。取第3代细胞进行细胞鉴定,逆转录聚合酶链反应方法测量2组细胞骨钙素、碱性磷酸酶与Ⅰ型胶原mRNA表达差异。结果组织块培养法培养7~14 d见细胞萌出,细胞呈梭形、纺锤形及多角的星形,细胞核大、卵圆形、细胞边界不清。免疫细胞化学及免疫荧光检测显示波形蛋白阳性表达,证实细胞培养成功。逆转录聚合酶链反应结果提示韧带骨化组细胞骨钙素、碱性磷酸酶与Ⅰ型胶原mRNA表达明显高于外伤组。结论组织块培养法可成功培养颈椎后纵韧带组织细胞,形态学表现为成纤维细胞特点。体外培养的OPLL患者的韧带细胞具有明显的成骨活性。

Objective To discuss the method of in vitro cell culture and osteogenecity of cells derived from cervical ossifi- cation of posterior longitudinal ligament（OPLL）. Methods Fifteen patients with OPLL and 15 patients with cervical injury but no OPLL were performed anterior decompression from January 2013 to December 2013. Intraoperative specimen of posteri- or longitudinal ligament was derived and cultured with method of tissue fragment culture. The mouse anti-vimentin antibody was used to identify the third passage cultured cells via immunocytochemistry and immunofluorescence （ICC/IF）. The mRNA expression of osteoblast-specific genes of osteocalcin （OCN）, alkaline phosphatase （ALP） and type I collagen （COL I ） were detected by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） in the cells derived from 2 groups. Results The cultivated cells were found 7-14 d after tissue fragment culture, which showing fusiform and multi-angu- lar star morphologies, large and elliptical cell nucleus and ill-defined cell appearances. ICC/IF exhibited positive results of vimentin staining. This was accordance with fibroblast. The mRNA expression of OCN, ALP and COL I from OPLL ligament fibroblasts were greater than those from non-OPLL ceils, the difference was significant. Conclusion The method of tissue fragment culture can successful culture cervical OPLL cells. The morphology of cultured cells was in fact fibroblasts. Cells from OPLL patients had better osteogenecity than non-OPLL patients.