胰岛素样生长因子Ⅰ与透明质酸对人关节软骨细胞的作用

Role of insulin-like growth factor Ⅰ and hyaluronic acid on the articular chondrocytes

目的探讨胰岛素样生长因子Ⅰ（IGF-Ⅰ）和透明质酸（HA）联合培养对人关节软骨细胞增殖及其生物学行为的影响。方法分离培养人关节软骨细胞，分4组，分别为对照组、IGF-Ⅰ组、HA组和IGF-Ⅰ＋HA组。加入不同浓度的IGF-Ⅰ、HA、IGF-Ⅰ和HA，用四氮甲基唑蓝（MTT）法测定不同时间点软骨细胞增殖活性的变化。从第4代开始，用IGF-Ⅰ和HA联合培养，通过电镜观察、甲苯胺蓝染色、Ⅱ型胶原和软骨蛋白聚糖（aggrecan）免疫组化及RT—PCR判断联合培养第8代软骨细胞表型的变化。自然传代的第6代细胞为对照组。结果IGF-Ⅰ在10μg/L浓度时即可明显促进软骨细胞的增殖，当IGF-Ⅰ浓度为50μg/L时，其促增殖作用达最大值。单独应用不同浓度的HA对人关节软骨细胞的促增殖作用不明显。联合应用IGF-Ⅰ与HA对软骨细胞的增殖具有协同效应并使促增殖作用时间后延。IGF-Ⅰ和HA联合培养的第8代细胞胞质内含有丰富的粗面内质网和分泌小泡；RT-PCR显示Ⅱ型胶原mRNA表达阳性而Ⅰ型胶原mRNA表达阴性；甲苯胺蓝染色显示细胞质内有大量的紫色异染颗粒；Ⅱ型胶原和aggrecan免疫组化见92％的细胞染色呈阳性或强阳性，平均灰度分别为85．92±9．71和116．23±16．69。而对照组的平均灰度值分别为105．12±12．20和135．68±10．65，二者均显著低于对照组（P〈0．01）。结论IGF-Ⅰ明显促进软骨细胞的增殖；HA对软骨细胞的增殖无影响；二者联合培养促增殖能力更强，并且能有效地保持软骨细胞表型的稳定。

Objective To investigate the biological role of insulin-like growth factor Ⅰ（ IGF-Ⅰ ） and hyaluronic acid（HA） in human articular chondrocytes. Methods Human articular ehondrocytes were isolated and cultured in DMEM. IGF-Ⅰ , HA as well as both IGF-Ⅰ plus HA were added into the cultured medium respectively. The proliferation of chondrocytes was measured with MTT method. From passage 4, IGF-Ⅰ and HA were added into the cultured media. The electron microscopic observation, toluidine blue staining, human collagen Ⅱ and aggrecan immunohistoehemistry, reverse transcript polymerase chain reaction （ RT- PCR）were conducted to investigate the phenotype of passage 8 chondrocytes treated by IGF-Ⅰ and HA. The passage 6 chondrocytes without any treatment was used as control group. Results The addition of 10 μg/L of IGF-Ⅰ in the culture medium significantly enhanced the proliferation of chondrocytes, and this enhancement was maximal at the concentration of 50μg/L. The chondrocytes proliferation was not obviously with different concentrations of HA. The combination of IGF-Ⅰ and HA was stronger promotion of the proliferation than that of HA or IGF-Ⅰ only, and could prolong the duration of promoting chondrocyte proliferation. There were massive rough surfaced endoplasmic reticulums and secretory vesicles in cytoplasm by electron microscopic observation. RT-PCR revealed positive collagen Ⅱ mRNA and negative collagen Ⅰ. Toluidine blue staining showed metachromia granule. Collagen Ⅱ and aggrccan immunohistochemistry showed 92% positive cell in the passages 8 chondrocytes. Its gray scale displayed distinctively stronger than control group. Conclusions IGF-Ⅰ can promote the proliferation of human articular chondrocyte, but HA can not. When IGF-Ⅰ and HA are combinated together, the ability of promoting the proliferation of chondrocytes is stronger and it can keep the hyaline eondrocyte phenotype more stable.