体外持续传代引起小鼠透明软骨细胞表型和细胞外基质平衡变化的研究

Effects of in vitro continuous passaging on the phenotype of mouse hyaline chondrocytes and the balance of the extracellular matrix

目的研究体外持续传代对透明软骨细胞形态表型、分化特性及细胞外基质(ECM)平衡状态的影响。方法酶消化法分离培养小鼠透明软骨细胞,连续传代至第5代。采用苏木精-伊红染色观察软骨细胞的形态改变,采用半定量聚合酶链式反应分析软骨细胞特异性基因、常规基因、基质金属蛋白酶家族(MMPs)和基质金属蛋白酶组织抑制剂家族(TIMPs)m RNA水平的变化,采用明胶酶谱分析鉴定软骨细胞明胶酶活性的改变。结果随着传代次数增加,软骨细胞形态由圆形或多边形变为长梭形,其特异性基因的表达量明显下降(P<0.05),至第5代已基本丧失。相比而言,常规基因的下调并不明显。MMPs和TIMPs均有下调(P<0.05),仅MMP-1和TIMP-1改变的差异无统计学意义(P>0.05);MMPs/TIMPs比值随传代发生紊乱。在蛋白质水平,随传代次数增加,明胶酶的活性下降,P4和P5代细胞下调显著(P<0.05)。结论软骨细胞在体外培养过程中,其特异性表型特征会随传代次数增加而逐渐丧失,软骨细胞发生反分化而表现出纤维化趋势。同时,ECM的平衡状态被打破,合成与分解代谢紊乱。在利用软骨细胞进行相关疾病的研究或软骨组织工程学实验时,应选用前3代的软骨细胞。

Objective This study aimed to investigate the effects of in vitro continuous passaging on the morphological phenotype and differentiation characteristics of mouse hyaline chondrocytes, as well as on the balance of the extracellular matrix（ECM）. Methods Enzymatic digestion was conducted to isolate mouse hyaline chondrocytes, which expanded over five passages in vitro. Hematoxylin-eosin stain was used to show the changes in chondrocyte morphology. Semi-quantitative polymerase chain reaction was performed to analyze the m RNA changes in the marker genes, routine genes, matrix metalloproteinases（MMPs）, and tissue inhibitors of MMPs（TIMPs） in chondrocytes. Zymography was carried out to elucidate changes in gelatinase activities. Results After continuous expansion in vitro, the morphology of round or polygonal chondrocytes changed to elongated and spindled shape. The expression of marker genes significantly decreased（P〈0.05）, and it was almost negatively expressed by P5 chondrocytes. By contrast, the down regulation of routine genes was insignificant. The gene expression levels of MMPs and TIMPs both decreased（P〈0.05）, but the change in MMP-1 and TIMP-1 was not statistically significant（P〉0.05）. Meanwhile, the ratio of MMPs/TIMPs was altered. At the protein level, the activities of gelatinases decreased after passaging, especially for P4 and P5 chondrocytes（P0.05）. Conclusion Serially passaged chondrocytes dedifferentiated and lost specific phenotypic characteristics during in vitro expansion culture. Simultaneously, the anabolism and catabolism of the cartilage ECM became uncontrollable and led to the imbalance of ECM homeostasis. When hyaline chondrocytes are applied in research on relevant diseases or cartilage tissue engineering, P0–P2 chondrocytes should be used.