铅对乳鼠成骨细胞活力和功能标志物及形态学的影响

Effects of Lead on Vitality, Differentiation, Special Markers and Morphological Changes of Osteoblasts in Newborn Rats

目的研究铅对乳鼠颅骨成骨细胞活力、分化、特异的功能标志物及形态学的影响,以探讨铅对骨骼发育的毒作用机制。方法分离并培养Wistar胎鼠颅骨原代成骨细胞,加入不同浓度Pb(Ac)2(0、0.1、0.5、1、5、10、50、100μmol/L),分别培养24、48、72h,用噻唑蓝(MTT)法检测成骨细胞活力;对-硝基苯磷酸盐(pNPP)法检测碱性磷酸酶(ALP)活力;考马斯亮蓝法测定细胞蛋白含量;放射免疫法测定培养液及细胞骨钙素水平;倒置相差显微镜观察铅对细胞光镜形态的影响。结果100μmol/L醋酸铅染毒组成骨细胞细胞活力下降,染毒72h时更明显。0.5～100μmol/L醋酸铅染毒组ALP活力均低于对照组(P<0.05)。50、100μmol/L醋酸铅染毒组蛋白质水平低于对照组(P<0.05)。各染毒组细胞内骨钙素水平无明显差异;培养液中分泌型骨钙素水平随染铅浓度的增加呈显著下降趋势(P<0.05)。光镜下可见醋酸铅对成骨细胞有毒性作用。结论醋酸铅对成骨细胞有毒性作用,可影响其光镜下结构;铅在达到较高剂量时可引起成骨细胞一般指标(细胞活力和蛋白质含量)的改变;而较低剂量即能引起特异性功能标志物(ALP和骨钙素)损伤,可能是铅影响成骨细胞乃至骨骼系统发育的机制之一。

Objective To investigate the effects of lead on the vitality, differentiation, special functional markers and morphological changes of the osteoblasts in newborn rats, which helps to clarify the toxicity mechanism of lead on the skeleton development in children. Methods The osteoblasts were isolated from the calvariae in newborn Wistar rats and cultured in DMEM medium. Lead acetate at different concentration (0, 0.1, 0.5, 1, 5, 10, 50, 100 μmol/L) was added for 24 h, 48 h, 72 h. The vitality of the osteoblasts was measured by MTT methods; The activity of alkaline phosphatase(ALP) was measured by pNPP method. The protein content of the osteoblasts was determined by Coomassie brilliant blue method. The levels of osteocalcin in both secreted (in medium) and cellular of osteoblasts were determined by radiate immunochemistry methods. The optical structure was determined by LM. Results The vitality decreased significantly when the dose was at more than 50 μmol/L, especially after 72 h. The activity of ALP decreased significantly when more than 0.5 μmol/L, whereas the levels of secreted osteocalcin decreased significantly when at 0.1 μmol/L. It appeared lead had some toxicity on the shape and number on the osteoblasts. Conclusion Lead acetate has adverse effect, at higher doses, on the vitality, protein content, ALP, osteocalcin and optical structure, maybe it is one of the mechanisms of lead influencing the development of the osteoblasts even the skeleton.