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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。结论体外培养不同年龄组人成骨细胞的形态与增殖能力随年龄增加而发生衰老改变
Objective To observe the morphology, proliferation and function of osteoblasts in different age groups cultured in vitro. Methods The ilium, proximal femur or femoral neck cancellous bone in four age groups ≤5 years old, 30 ~ 40 years old, 50 ~ 60 years old and ≥ 70 years old were selected for culture. The biological characteristics of osteoblasts were observed by in vivo phase contrast microscope and alkaline phosphatase staining. The generation of osteoblasts grew to the confluence, digestion count continued to be cultured, the second generation of osteoblasts were cultured for 1 day, 5 days detection of cell proliferation rate (MTT method), 5 days detection of alkaline phosphatase (ALP) activity, 14 days to send Electron microscope room for transmission electron microscopy. Results The results of in vivo observation showed that the number of osteoblasts increased, the number of vacuoles increased, the number of osteoblasts increased, and the protuberances became longer and fusiform when confluent. The alkaline phosphatase staining of osteoblasts was positive in all age groups. Observations suggest that osteoblasts become elongated with age, organelles decreased, the Golgi apparatus underdeveloped, glycogen increased and deposited, lysosome increased vacuoles, increased nuclear heterochromatin. Compared with MTT, the ratio of cell number to MTT increased with increasing age (P <0.01). The ALP activity increased with age, but the difference was not statistically significant. Conclusion The osteoblast morphology and proliferative ability of different age groups in vitro are aging change with age