体外培养人成骨细胞随供体的增龄而改变

Influence of increasing donor age on human osteoblastic cells cultured in vitro

目的 观察体外培养的不同年龄组人成骨细胞形态、增殖与功能改变。方法 选取≤5岁 ,30～ 4 0岁 ,5 0～ 6 0岁和≥ 70岁四个年龄组的髂骨、股骨近端或股骨颈松质骨进行培养。培养期间应用相差倒置显微镜活体观察与碱性磷酸酶染色观察成骨细胞的生物学特性。一代成骨细胞生长至汇合时消化计数继续培养 ,二代成骨细胞分别于培养 1天 ,5天检测细胞增殖率 (MTT法 ) ,5天时检测碱性磷酸酶 (ALP)活性 ,14天时送电镜室进行透射电镜观察。结果 活体观察提示成骨细胞随增龄其胞体增大、变薄、空泡增多、突起拉长 ,汇合时趋向于长梭形 ,各年龄组成骨细胞碱性磷酸酶染色均呈阳性反应 ;电镜观察提示成骨细胞随增龄变得瘦长、细胞器减少、高尔基体不发达、糖原增多并呈堆积现象、溶酶体增多、出现空泡 ,细胞核内异染色质增多。成骨细胞生长至汇合时的细胞数与MTT相对比值随增龄而降低 (P <0 .0 1) ;ALP活性随增龄呈增高趋势 ,但差异无统计学意义。

Objective To investigate the differences in morphology, proliferation and function of cultured human osteoblasts in different age donors in vitro. Methods Human osteoblasts from different age donors (≤5 years, 30～40 years, 50～60 years and ≥70 years) were isolated and cultured by the tissue fragment migrating growth method. Their biological characteristics were observed by phase contrast microscope, alkaline phosphatase (ALP) staining and electromicroscope. The osteoblasts in the first passage were cultured to confluence, then numbered by cell counting method. The proliferation ratio of osteoblasts in the second passage was examined with thiazolyl blue reagents when cultured for 1 day and 5 days. ALP activity was examined in the osteoblasts of the second passage cultured for 5 days. Results Phase contrast inverted microscope showed that the aging osteoblasts were larger, thinner, containing more vacuole and with elongated cell apophyses. The osteoblasts of all age donors showed positive ALP staining. Electromicroscope showed that the osteoblasts with donor aging become thin and elongated, with fewer cell organelles and undeveloped Golgi complex, with accumulated glycogen and more lysosomes in the plasma, and with more heterochromatin in the nuclear. Thecellnumberatconfluenceandcell proliferation ratio were decreased with donor aging (P<0.01, respectively). ALP relative ratio did not show any statistically significant increase with donor aging. Conclusion Human osteoblasts cultured in vitro manifaste senescence in morphology and proliferation with donor aging.