体外培养人鼻中隔软骨细胞合成硫酸糖胺多糖的检测

背景：软骨细胞的培养及稳定传代是转基因、构建组织工程化软骨组织及异体移植的前提条件。 目的：定量检测体外培养人鼻中隔软骨细胞合成分泌硫酸糖胺多糖的变化情况，评价其对不同代次软骨细胞生物学功能活性。 设计、时间及地点：对比观察的细胞学实验，于2004-01/2005-07在暨南大学组织移植与免疫教育部重点实验室完成。 材料：人鼻中隔软骨细胞来自鼻中隔偏曲患者手术切除的软骨组织，患者对实验内容知情同意。 方法：胶原酶消化分离原代人鼻中隔软骨细胞传代培养至第7代。 主要观察指标：阿尔新蓝比色法测定各代细胞外基质的硫酸糖胺多糖含量，光镜观察甲苯胺蓝染色后细胞形态。 结果：原代软骨细胞具有最高的硫酸糖胺多糖分泌活性，至第4代则出现了非常明显的下降，第4代以后很低下。甲苯胺蓝染色显示原代细胞对甲苯胺蓝呈明显的蓝色异染反应，第3代异染反应明显减弱，第4代以后异染反应微弱，其结果与硫酸糖胺多糖含量变化相一致。 结论：人鼻中隔软骨细胞在体外培养过程中，随传代次数的增加而功能活性逐渐降低，第3代以后的细胞不适于作为人软骨组织工程种子细胞和用于细胞生物学等方面研究

Assay of glycosaminoglycans synthesized by cultivated cartilage in the nasal septum in vitro

BACKGROUND: The culture and passage of chondrocytes is a premise of transgene and contruction for tissue engineering cartilage tissues and allografts. OBJECTIVE: To measure the variation of glycosaminoglycans (GAG) synthesized by cultivated human cartilage of nasal septum in vitro, and to investigate their biological function during different generations. DESIGN, TIME AND SETTING: Controlled observations were performed in the Key Laboratory of Tissue Transplantation and Immunology (Jinan University) by the State Ministry of Education from January 2004 to July 2005. MATERIALS: Human nasal septal chondrocytes were harvested from the patients with deflection of nasal septum, with the informed consents of all patients. METHODS: Primary human nasal septal cartilage was isolated through collagenase enzymatic digestion, and was serial subcultivated to the seventh generation. MAIN OUTCOME MEASURES: The extracellular matrix contents of GAG at each generation were assayed by alcian blue chromatometry, and chondrocytes were stained by toluidine blue for observation under light microscope. RESULTS: Primary cultivated chondrocytes possessed the maximal activity of secreting GAG, but they decreased significantly after the fourth generation. Toluidine blue staining demonstrated primary cultivated cartilage presented apparent reaction of blue staining, but the reaction got weak obviously at the third generation and became feeble after the fourth generation, the results were consistent with the change of GAG. CONCLUSION: During the course of culture in vitro, the biological function of human nasal septal cartilage gradually degrade in accordance with increasing times of passage, chondrocytes after the third generation are not suitable for human cartilage tissue engineering seeds or studying cytobiology.