永生化软骨细胞体内软骨形成的实验研究

Experimental study of the cartilage formation of immortalized chondrocytes in vivo

目的 探讨以永生化软骨细胞作为种子细胞构建和形成软骨的可能性。方法 把人端粒酶逆转录酶基因(hTERT)导入兔髁状突软骨细胞 ,G418筛选 ,挑选阳性克隆扩增培养 ;实验组将扩增培养的转染细胞与细胞支架材料 β 磷酸三钙 ( β TCP)复合 ,对照组将正常软骨细胞与 β TCP复合或单纯 β TCP在体外培育后种植于裸鼠皮下 ,3和 6个月取材进行组织学和免疫化学检测 ,并与对照 1组和对照 2组进行比较。结果 实验组和对照 1组可见复合体包裹软骨样组织 ,对照 2组有少量纤维样组织形成。免疫组化染色显示 ,实验组番红“O”、甲苯胺蓝和Ⅱ型胶原染色呈强阳性 ,有明显软骨组织形成 ,与对照组有显著性差异 (P <0 0 1) ,对照 1组染色呈弱阳性 ,对照 2组则为阴性。结论 永生化软骨细胞可以作为软骨组织工程的种子细胞 。

Objective To explore the feasibility of the construction and formation of cartilage with immortalized chondrocytes by tissue engineering technology in vitro . Methods Human telomerase reverse transcriptase(hTERT) gene was introduced into rabbit mandibular condylar chondrocytes by eukaryotic vector. After screening with G418, the positive clones were amplified for culture. Normal or immortalized chondrocyte loaded cytoskeleton β tricalcium phosphate (β TCP) complexes were incubated in vitro for 1～2 d and then implanted into subcutaneous tissue of nude mouse. The complexes of immortalized chondrocyte β TCP and chondrocyte β TCP or β TCP alone were established as the experimental group and control groups respectively. The specimens were harvested within 3 and 6 months after surgical procedure for histological and immunohistochemical observation. Results In experimental groups and control group 1, the complexes packed with cartilage like tissue were found, but there was only a little fiber like tissue formed in control group 2. Immunohistochemistry revealed strong positive staining with safranine O, toluidine blue and collagen type II and obvious formation of cartilage. There was significant difference between the experimental group and the control groups( P <0.01). Weak positive staining was found in control group 1 but negative in control group 2. Conclusion Immortalized chondrocytes can be used as the seed cells for cartilage tissue engineering due to the better capacity of cartilage formation.