低渗刺激对成骨样细胞MG63功能的影响

Effects of Hypotonic Solution on Function of Osteoblast-like Cell Line MG63

目的本试验拟通过研究低渗透压的静牵张作用,对成骨样细胞MG63的增殖能力、碱性磷酸酶活性以及[Ca2+]i的影响,探讨该应力形式下成骨样细胞MG63的力学响应特征。方法采用人成骨肉瘤来源的成骨样细胞MG63作为细胞源进行细胞培养传代。用不同渗透压的低渗透液,对成骨样细胞MG63分别进行2 h、4 h、8h、12 h和24 h持续牵张作用后,用免疫组化法检测ALP表达的情况,用ALP试剂盒检测ALP活性的变化,用钙试剂盒检测[Ca2+]i含量波动情况。结果成骨样细胞MG63的ALP免疫细胞化学染色为阳性。随着作用时间的延长,成骨样细胞MG63在277 mOsm和240 mOsm低渗透液的持续性膨胀作用下,其细胞的[Ca2+]i、ALP活性缓慢增高,但240 mOsm组ALP活性始终低于对照组(p<0.01);而细胞在163 mOsm低渗液作用下,8 h时出现[Ca2+]i急剧升高(11.383±0.111),ALP活性(0.326±0.002)明显高于其对照组(p<0.01)。结论结果提示不同水平的低渗膨胀对MG63的增殖分化、ALP活性以及Ca2+-ATPase都有一定的影响作用,且Ca2+内流与ALP活性之间存在一定的相关性。低渗膨胀法作为一种应力形式有一定的实验意义。

Objective To investigate the effects of static- stretch from hypotonic sulution on proliferation ,alkaline phosphatase activity and [Ca^2＋ ]iin osteoblast- like cells. Method Human osteosarcoma originated MG63 osteoblast cell line was used in this experiment. Mechanical loading was applied by swelling in hypotonic solution. In vitro cultured MG63 were incubated under continuous swelling of 277, 240 mOsm for 2h, 4h, 8h, 12h and 24h. The cell morphology was observed by inverted microscope. ALP expression was confirmed by immunocytochemical methods. ALPase and [Ca^2＋ ]i were determined by modified enzyme dynamic method and OCPC respectively, and all of results were normalized to total cellular protein values. All data were analyzed with SPSS 12.0 software package. Results The cell AI.P expression was positive in both of experiment and control groups. ALP activity and [Ca^2＋]i increased slowly under continuous staticstretch of 277, 240 mOsm, while [Ca^2＋ ]i showed a significant increasing at 8h under 163 mOsm（11. 383±0.111）, ALPase activity（0. 326±0. 002）was higher than that of control group. Conclutions The study has demonstrated that different levels of hypotonic swelling could influence ALPas and Ca^2＋-ATPase of osteoblasvlike cell llne MG63 , as well as the level of [Ca^2＋-]i was correlated with the ALPase activity in cell. The hypotonic swelling ,which was a stress method for osteoblast, exhibited a definite advantage.