传代对残耳软骨细胞体内软骨形成能力的影响

Effects of Continuous Passage on the Cartilage Forming Ability of Human Remnant Ear Chondrocytes in Vivo

目的：研究传代对残耳软骨细胞体内软骨形成能力的影响。方法分离培养人残耳软骨细胞，将第3-8代细胞分别复合聚羟基乙酸/聚乳酸支架，构建组织工程化软骨；体外培养4周后植入裸鼠体内观察8周。采用组织学染色观察各组标本的软骨形成情况；Real-time PCR检测软骨分化相关基因的表达；生物力学分析新生软骨的弹性模量。结果各代复合物体外培养4周时均不能形成软骨组织，但第3-5代残耳软骨细胞COL 2A1、第3-4代的SOX 9和第3代的DLK 1仍可维持较高的表达水平（P〈0.05）；体内植入8周后，第3-6代复合物均有不同程度的弹性软骨结构形成，并随代次增高而减少，第3-6代复合物的弹性模量明显高于第7、8代。结论残耳软骨细胞传至第4代仍能保持良好的体内软骨形成能力，但扩增传代对残耳软骨细胞软骨表型去分化的影响在第7代后已无法逆转。

Objective To investigate the cartilage forming ability of human remnant ear chondrocytes with continuous passage in vivo. Methods Human remnant ear chondrocytes were isolated and cultured, then seeded onto Polyglycolic acid/Polylactic acid scaffolds. The cell-scaffold complexes were cultured for 4 weeks in vitro and then were implanted into nude mice for 8 weeks. The in vivo fates of cell-scaffold complexes constructed by P3-8 remnant ear chondrocytes and Polyglycolic acid/Polylactic acid scaffolds were evaluated by histological assays, quantitative analysis of gene expression and biomechanical test. Results All the complexes constructed by P3-8 remnant ear chondrocytes were unable to form cartilaginous structure in 4 weeks in vitro. However, the genes expression of COL2A1 in P3-5, SOX9 in P3-4, and DLK1 in P3 were significantly higher （P〈0.05）. After 8 weeks in vivo, P3-6 complexes could partially form cartilage tissue, which showed less and less with passage. The elastic modulus were significantly higher in P3-6 complexes （P〈0.05） than in P7-P8 complexes. Conclusion The cartilage forming ability was well maintained in remnant ear chondrocytes within P4 in vivo and was totally lost after P7.