永生化骨骺软骨细胞的生物学性状研究

目的比较永生化骨骺软骨细胞与原代骨骺软骨细胞的生物学性状,为细胞替代疗法治疗小儿身材矮小提供理论依据。方法利用SV40LTag诱导骨骺软骨细胞永生化,经G418稳定筛选后,观察细胞形态,应用MTT、血清依赖性实验、软琼脂克隆形成试验、裸鼠致瘤试验分析其生物学性状。结果原代软骨细胞体外传代至第6代左右即出现衰老现象,而转化软骨细胞可连续传代,未出现衰老迹象。转化软骨细胞更趋向于长梭形。两者的生长曲线比较相似,但转化软骨细胞的饱和密度明显高于正常软骨细胞。两者都对血清有依赖性,都为二倍体核型,均不能在软琼脂上形成克隆。经8周观察两者都不能使裸鼠致瘤。结论永生化骨骺软骨细胞为良性转化,为转基因细胞移植治疗身材矮小等疾病的研究提供了安全的细胞来源。

Comparative study of biological characters between the primary and the immortalized epiphysis cartilage cell strain

Objectives To compare biological characters between immortalized epiphysis cartilage cells and primary epiphysis cartilage cells,and to provide stable cell resource for cell-substitution and gene therapies of growth retardation with immortalized epiphysis cartilage cells. Methods Plasmid pEGFP-IRES2-SV40LTag containing the simian virus 40 large T antigen gene was transfected into the primarily cultured epiphysis cartilage cell of newborn rat using lipofection transfection method. Cellular morphology,MTT,serum-dependent growth character,clone forming test,nude mice tumor forming test were employed to investigate cytobiological characters of transfection. Results Immortalized epiphysis cartilage cells could be subcultured successively,while primary epiphysis cartilage cell strain was subcultured for only 6 passages replicative senescence began. The immortalized cells were elliptic or triangular cells with two or three short axons. The growth curve was similar but the saturation density of transfected cells was higher than primary cells. Both type of cells had serum-dependent character and were diploid karyotype. Neither of them formed clone in soft agar nor tumor developed in nude mice after eight weeks. Conclusions Immortalized epiphysis cartilage was optimum transfection. It could be safe source for cell-substitution and gene therapies.