一氧化氮诱导腰椎软骨终板退变的实验研究  

Experiment study on nitric oxide inducing lumbar cartilage endplate degeneration

作　　者：刘斌[1] 蔡道章[1] 瞿东滨[2] 金大地[2] 

机构地区：[1]中山大学附属第三医院骨科,广州市510630 [2]南方医科大学南方医院脊柱骨病外科

出　　处：《中华显微外科杂志》2007年第1期38-41,I0006,共5页Chinese Journal of Microsurgery

基　　金：国家自然科学基金资助项目（30000168）;广东省科技计划项目（C2003A30212）

摘　　要：目的探讨一氧化氮（nitricoxide，NO）诱导腰椎软骨终板退变的机制。方法取长枫杂种猪腰椎间盘软骨终板细胞，于含20％胎牛血清的DMEM中培养，建立体外软骨终板细胞培养模型，以光镜及苏木精-伊红染色观察其生物学表现，Ⅱ型胶原免疫细胞化学染色对软骨终板细胞进行鉴定，以外源性NO-硝普钠（sodium nitroprusside，SNP）2mmol／l处理软骨终板细胞、^35S掺入法和^3H-脯氨酸掺入法检测蛋白多糖及胶原的合成，放射性免疫测定法测炎性细胞因子IL-1β、IL-6、TNF-α的生成，碘化丙啶荧光染色及DNA片段电泳观察软骨终板细胞凋亡。结果原代细胞生物学性状最接近体内细胞，多次传代后细胞呈现衰老现象，经免疫细胞化学染色证实此细胞具有Ⅱ型胶原表达。施加SNP后，软骨终板细胞蛋白多糖及胶原合成明显减少（P〈0．01），炎性细胞因子含量明显升高（P〈0．01），细胞凋亡率显著增高（P〈0．01）。结论NO抑制软骨终板细胞蛋白多糖及胶原合成，促进炎性细胞因子及细胞凋亡增加，从而促进软骨终板退变。Objective To explore the role of nitric oxide inducing lumbar cartilage endplate degeneration. Methods The chondroeytes were isolated and harvested from the porcine cartilage endopate （CEP）, and the cells were cultured in DMEM containing 20% fetal bovine serum. The biological phenomena was observed by light microscope and HE staining. The biological characteristics was identified by immunocytochemistry for type Ⅱ collagen. After with or without sodium nitroprusside （2 mmol/L） to stimulate the CEP cells, the proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate（ Na2^35 SO4 ）, The incorporation of ^3 H-proline was used to detect synthesis of collagen. The contents of IL-1β, IL-6 and TNF-α were measured by radioimmunoassay. The apoptosis was observed by propidium iodide （PI） fluorescent staining and DNA agarose gel electrophoresis. Results The biological character of primary cells in vitro was similar with that in vivo. The CEP cells became ageing with passage. Type Ⅱ　collagen was expressed in the cells. With 2 mmol/L SNP, the synthetic quantity of proteoglycan or collagen in CEP cells was （ 12682 ± 447 ） cmp, （2626 ± 394） cmp, respectively. The synthetic quantity of proteoglycan or collagen was decreased more significantly than that without SNP（P 〈0. 01 ）. The contents of IL-1β, IL-6 and TNF-α were 0. 68 ±0. 12 ng/ ml, （397. 46 ± 38. 91 ） pg/ml, （ 0. 67 ± 0. 07 ） ng/ml, respectively. The contents of inflammatary cytokines were increased more significantly than those without SNP（ P 〈0. 01 ）. The apoptosis rate of the cells with SNP was significantly increased from （3.68 ±1.37）% to （62.67 ±7.03）% （P〈0.01）. Conclusion NO can increase the apoptosis rate and the contents of IL-1 β, IL-6 and TNF-α in CEP cells, but it decrease the synthetic quantity of proteoglycan or collagen to promote cartilage endplate degeneration.

关 键 词：一氧化氮 软骨终板 细胞培养 细胞凋亡 

分 类 号：R686[医药卫生—骨科学]