髓核细胞分泌的炎症因子对后纵韧带成纤维细胞增殖与成骨能力的影响

Effects of inflammatory cytokines secreted by nucleus pulposus cells on proliferation and osteogenic ability of posterior longitudinal ligament fibroblasts

目的探讨髓核(NP)细胞分泌的炎症因子对后纵韧带成纤维细胞增殖、成骨能力的影响。方法采用组织块培养法对颈椎后纵韧带成纤维细胞进行体外培养,采用酶解法提取NP细胞并进行体外培养,建立OPLL+NP细胞组、OPLL细胞组和NP细胞组。采用ELISA法检测IL-1α、IL-6、TNF-α及PGE2的表达,采用MTT比色法检测OPLL细胞毒性,采用Ed U法检测OPLL细胞增殖,采用RT-PCR法检测OPLL细胞Ⅰ、Ⅺ型胶原及骨钙素mRNA的表达,采用Von Kossa染色法及ALP染色法检测OPLL细胞成骨活性。结果 OPLL+NP细胞组OD490值(0.28±0.09)较OPLL细胞组(0.25±0.06)增高(P<0.05);OPLL+NP细胞组OPLL细胞增殖率明显高于OPLL细胞组;OPLL+NP细胞组ALP染色和Von Kossa染色呈阳性,OPLL细胞组呈阴性;OPLL+NP细胞组Ⅰ、Ⅺ型胶原及骨钙素mRNA表达较OPLL细胞组明显增高(P<0.05);OPLL+NP细胞组炎症因子含量较NP细胞组和OPLL细胞组明显增加(P<0.05)。结论在体外,NP细胞分泌的炎症因子促进后纵韧带成纤维细胞增殖和成骨分化能力。

Objective To investigate effects of inflammatory cytokines secreted by nucleus pulposus cells on proliferation and osteogenic ability of posterior longitudinal ligament fibroblasts. Methods Posterior longitudinal ligamen（ PLL） cells were harvested by tissue fragment culture. Nucleus pulposus（ NP） cells were isolated by enzymatic digestion of nucleus pulposus tissue. The cells were divided into OPLL ＋ NP group,OPLL group and NP group. Secreted cytokines were detected by ELISA. Cell cytotoxicity was measured with MTT assay and cell proliferation was detected with Ed U assay. The expressions of types Ⅰ,Ⅺ collagen and osteocalcin mRNA were determined with real time PCR（ RT-PCR）. The osteogenic activity of OPLL cells was assessed with histochemical stains（ Von Kossa and ALP stain）.Results In the OPLL group,OD490 decreased compared with OPLL ＋ NP group[（ 0. 25 ± 0. 06） vs（ 0. 28 ± 0. 09）,P〈0. 05]. Cell proliferation in OPLL ＋ NP group was significantly higher than that in OPLL group. OPLL ＋ NP group was strong positive for Von Kossa and alkaline phosphatase,while OPLL group was negative. Expressions of types Ⅰand Ⅺ collagen and osteocalcin mRNA in OPLL ＋ NP group were higher then those in OPLL group（ P〈0. 05）. Supernatant from OPLL ＋ NP group showed increased production of IL-1α,TNF-α and PGE2 compared with OPLL groupand NP group（ P〈0. 05）. Conclusion In vitro,inflammatory cytokines secreted by nucleus pulposus cells promote proliferation and osteogenic ability of posterior longitudinal ligament fibroblasts.