体外培养骺板、关节及肋软骨细胞外基质中糖胺多糖含量的检测和比较

Quantitative detection and comparison of sulfate glycosaminoglycans content in extracellular matrix of in vitro cultured epiphyseal, articular and rib chondrocytes

目的 建立定量检测体外培养软骨细胞外基质中糖胺多糖 (GAG)含量的方法 ,以评价体外培养骺板、关节及肋软骨细胞的生物学功能活性。 方法 改进的二甲基亚甲蓝 (DMB)比色法测定细胞外基质的糖胺多糖含量 ,细胞 -玻片甲苯胺蓝 (TB)染色光镜观察。 结果 原代软骨细胞具有最高的GAG分泌活性 ,骺板 (70 .12± 7.72 ) μg cm2 ,关节 (92 .0 0± 10 .15 ) μg cm2 ,肋软骨(80 .61± 11.40 ) μg cm2 ,至第三代则出现了非常明显的下降 ,骺板 (5 3 .2 7± 9.5 0 ) μg cm2 ,关节 (63 .88± 11.92 ) μg cm2 ,肋软骨 (5 8.94± 8.2 1) μg cm2 ;TB染色结果与GAG含量变化相一致。三种软骨细胞间比较差异无显著性意义。 结论 在体外培养过程中 ,软骨细胞随传代次数的增加而其功能活性逐渐降低 ,第三代以后的软骨细胞不适于作为组织工程种子细胞。DMB比色法准确、灵敏 ,可作为一种用于评价体外培养软骨细胞功能活性的有效分析方法。

Objective To establish a method for quantitative detection of the sulfate glycosaminoglycans (GAG) content in extracellular matrix of in vitro cultured chondrocytes so as to evaluate the biological characteristics of epiphyseal, articular and rib chondrocytes. Methods Sulfate GAG content in extracellular matrix of three chondrocytes was measured by the modified dimethylmethylene blue (DMB) method. The changes of the toluidine blue (TB) stain of chondrocytes were observed by light microscope. Results Primary chondrocytes had the highest content of sulfate GAG in the extracellular matrix, ie, epiphyseal chondrocytes reached (70.12±7.72) μg/cm 2, articular chondrocytes (92.00±10.15) μg/cm 2 and rib chondrocytes (80.61±11.40) μg/cm 2, respectively. On the third passage chondrocytes, epiphyceal chondrocytes decreased to (53.27±9.50) μg/cm 2, articular chondrocytes to (63.88±11.92) μg/cm 2 and rib chondrocytes to (58.94±8.21) μg/cm 2, respectively. The change of TB in every passage chondrocyte was consistent with the sulfate GAG content. There was no significant difference between three chondrocytes. Conclusions In in vitro culture, the biological properties of chondrocytes gradually decrease with passaging. After the third passage, chondrocytes are not fit for cartilage tissue engineering. DMB method is precise, accurate and helpful in evaluating the biological properties of chondrocytes in vitro cultured.