幼年成骨细胞体外培养传代衰老与70岁以上老年人成骨细胞的生物学特性比较

Comparison of biology characteristics between infant human osteoblasts of subculture senescence and ≥70 year-old human osteoblasts in Vitro

目的通过对幼年人成骨细胞（osteoblasts，OB）体外培养传代衰老与70岁以上老年人成骨细胞的生物学特性比较，观察培养代数与年龄的相关性，拟建立幼年人成骨细胞体外培养传代模拟衰老模型。方法选取≤5岁（2例，男1，女1）和≥70岁（14例，男5，女9）两个年龄组的股骨颈松质骨进行成骨细胞体外培养，幼年组细胞自第二代开始每5～10d传代一次，至第17代终止。培养期间观察第2，5，10，15代细胞的形态、增殖能力与碱性磷酸酶（alkaline phosphatase，ALP）活性，以及应用RT-PCR方法检测BGP，IGF-1，OPG，ODF基因mRNA表达，并与体外培养的70岁以上老年人成骨细胞进行比较。结果形态观察提示成骨细胞体外连续传代至第10-15代时，其胞体增大、折光性降低，呈塌陷状，细胞器稀少、糖原增多、溶酶体增多，与70岁以上老年人成骨细胞相似。MTT相对比值随传代次数增加而降低，第10-15代细胞明显低于第2-5代细胞，而与70岁以上老年人成骨细胞相似；ALP活性于10-15代时增高，与70岁以上老年人成骨细胞相似。幼年成骨细胞骨钙素（bone gla-protein／osteocalcin，BGP）、胰岛素样生长因子-1（insulin-like growth factor-1，IGF-1）、破骨细胞分化抑制因子（osteoprotegerin，OPG）、破骨细胞分化因子（osteoclast differentiation factor，ODF）、OPG／ODFmRNA表达随传代次数增加呈降低趋势，第10与15代细胞其表达量低于第2代细胞，也低于70岁以上老年人成骨细胞。结论幼年成骨细胞体外连续传代培养10-15代时，其形态结构、增殖能力与某些基因mRNA表达与体外培养第二代70岁以上老年人成骨细胞有相似性，有希望作为体外培养模拟衰老模型。

Purpose To compare the biology characteristics between serially passaged cultures of osteoblasts（OB） of 45 year-old donors and osteoblasts of ≥70 year-old donors, in order to establish the model of osteoblastic senescence in vitro. Methods Human osteoblasts from different age donors （2 aged 45 years, male 1, female 1 ; 14 aged≥70 years, male 5, female 9） were isolated and cultured in vitro. The osteoblasts of infant donors were serially subcultured every 5 - 10 days until to 17 th passage. The cell morphology, proliferation, ALP activity and mRNA expressions of BGP, IGF-1, OPG, ODF of the 2 nd, 5 th, 10 th and 15 th subcultures were compared with that of osteoblasts of ≥70 year-old donors. Results Morphology observation showed that the osteoblasts were larger, collapselike, with feeble refraction, few organelles, accumulated glycogen and more lysosomes in plasma when subcultured serially to 10 th- 15 th passage, being similar to osteoblasts of ≥70 year-old donors. MTT relative ratio was decreased with increasing subcultures. MTT relative ratio was much lower in 10 th- 15 th passage cells of infant donors than in 2 nd - 5 th passage cells, being similar to osteo-blasts of ≥70 year-old donors. ALP activity was higher in 10 th- 15 th osteoblasts, which was similar to osteoblasts of ≥70 year-old donors, mRNA expressions of BGP, IGF-1, OPG, ODF, OPG/ODF were decreased with increasing subcultures, and was lower in 10 th- 15 th passage cells of infant donors than in 2 nd passage cells, being also lower than in osteoblasts of ≥70 year-old donors too. Conclusions The 10 th- 15 th passage osteoblasts from infant donors are similar to the 2nd passage osteoblasts from ≥70 year-old donors in morphology characteristics, proliferation and some genes mRNA expression, which may be used as the aging model in vitro.