【摘要】 目的　探讨不同缓冲液和不同裂解方法对体外培养成骨细胞内碱性磷酸酶 (alkalinephosphatase,ALPase)活性的影响 ,建立较为理想的检测方法和条件 ,为研究骨代谢异常及骨质疏松症等骨病提供简便可靠的检测手段。方法　采取经体外培养 72h的成骨细胞 ,制备上清液、细胞裂解液和细胞反复冻融液样本 ,分别用二乙醇胺 (DEA)、碳酸盐 (CO=3)、2 氨基 2 甲基 1 内醇 (AMP)等 3种不同缓冲液种类的试剂 ,测定上述液体中的ALPase活性。结果　使用DEA缓冲液的 3种样本的测量值都为最高 ,且不精密度 (CV)较其他两种缓冲液小 ,各组比较分析 ,差异均有显著性 (P <0 .0 1 )。使用 3种缓冲液试剂 ,均以裂解液中ALP活性最高 ,而冻融液次之 ,上清液最低 ,分析结果 ,差异均有显著性 (P <0 .0 1 )。结论　用DEA缓冲液测量细胞裂解液体外培养成骨ALPase活性的效果较理想更多还原

【Abstract】 Objective Studying the effects of different buffers and lytic methods in detecting the activities of alkaline phosphatase(ALPase) in cultured osteoblasts in vitro , and finding an ideal assay to research metabolic disorders of bone and osteoporosis simply and reliably.Methods Osteoblasts cultured in vitro for 72 h were collected and made to different samples of supernate, cell lysis buffer and cell freezing melting sample. Using three different buffers,diethanolalmine(DEA), carbonate and 2 amino 2 methyl 1 propanol (AMP) respectively, to detect the activities of ALPase in solution mentioned above.Results The highest detected values in all three samples were with DEA, then were with AMP the lowest values were with carbonate. Analytic results showed significant differences among all of these groups ( P <0.0l). Detected with DEA, carbonate and AMP respectively, the activities of ALPase in cell lysis buffer were the highest, then were in cell freezing melting buffer, the lowest activities were in supernate. Significant differences were also seen among all these groups ( P <0.01). Conclusion For cultured osteoblasts, detecting the activities of ALPase in lysis buffer with DEA is recommended.更多还原