镉对体外培养成骨细胞增殖、凋亡、分化及矿化的影响

Effects of cadmium on proliferation,apoptosis,differentiation and mineralization in osteoblast in vitro

目的探讨镉对体外培养成骨细胞(osteoblast,OB)增殖、凋亡、分化及矿化的影响。方法用混合酶消化法分离大鼠颅盖骨成骨细胞,进行原代培养;成骨细胞与不同浓度的Cd2+作用后,MTT法测定细胞增殖;AO/EB双染法进行凋亡形态学观察;PNPP法检测碱性磷酸酶活性;矿化结节计数和面积测量分析镉对成骨细胞矿化能力的影响。结果各剂量组氯化镉均可抑制成骨细胞增殖,其中2.0μmol/L以上剂量组与对照组相比差异有统计学显著意义(P<0.01);16.0μmol/L、32.0μmol/L剂量组OB可见较多凋亡细胞;各时点、各剂量组氯化镉均能抑制ALP活性,尤其是48h以上影响作用更明显(P<0.01);0.5μmol/L、1.0μmol/L浓度的氯化镉可明显抑制成骨细胞矿化能力。结论镉离子可以抑制成骨细胞增殖、分化及矿化能力,促进成骨细胞凋亡,对成骨细胞的骨形成能力有明显抑制作用。

Purpose To investigate the effects of cadmium on proliferation,apoptosis,differentiation and mineralization in osteoblast in vitro. Methods The osteoblastic cells were harvested by sequential enzyme digestion from calvaria of Sprague Dawley rats and then cultured. They were treated with Cd^2＋ ,then the proliferation was analyzed by MTT methods cell apoptosis was observed by fluorescence microscopy-acridine orange/ethidium bromide double staining;PNPP method was used to assay the ALP activity of osteoblastic cell; to analyze the mineralization ability of osteoblastic cells,the number and area of mineralization nodes were observed. Results Cd^2＋ （2.0-32 μmol/L） significantly inhibited the proliferation of osteoblastic cells （P〈0.01）;cell apoptosis was observed in the osteoblastic cells exposed to 16.0 μmol/L and 32.0 μmol/L C^d2＋ ;Cd^2＋ inhibited the ALP activity at 24 h,48 h and 72 h,particularly at 48 h and 72 h （P〈0.01）;Cd^2＋ significantly inhibited osteoblast mineralization at the concentration of 0.5 μmol/L and 1.0μmol/L. Conclusions Cd^2＋ significantly inhibits boneformation of osteoblastic cells by inhibiting the proliferation, differentiation and mineralization of osteoblastic cells and promoting osteoblastic apoptosis.