小鼠骨髓基质细胞Kusa-A1体外成骨活性评价

Evaluation of the osteogenic potentiality of Kusa-A1 cells in vitro

目的：检测骨髓基质细胞系Kusa-Al体外成骨活性，分析其用于成骨细胞功能和分化机制研究的价值。方法：培养Kusa-Al细胞，在常规培养和成骨诱导培养条件下，分别检测细胞的体外成骨活性，包括碱性磷酸酶(ALP)活性、细胞钙沉积能力、体外矿化结节(CN)形成能力、骨桥蛋白(OPN)和骨钙素(0c)表达水平、NF-κB受体激活因子(RANKL)水平。结果：常规培养下，Kusa-Al细胞表现出较高碱性的ALP活性(约为0．75IU／mg)、一定的钙沉积能力，并可检测到OPN和OC表达，但无CN形成，RANKL蛋白低于可检测水平。诱导培养条件下，细胞ALP活性先升高后降低、钙沉积量持续升高、OPN和OC表达水平后期显著提高，汇片后3～7d检测到大量CN形成，汇片后24h可检测到RANKL蛋白。结论：Kusa-Al可能是一种成骨前体细胞，经诱导可以向成骨细胞分化，在体外表现出良好的成骨细胞活性。Kusa-Al细胞可作为成骨细胞功能及其分化机制研究的模型细胞。

AIM:To examine the osteogenic potentiality of mouse bone marrow stromal cell line,the Kusa-A1,and analyse its value in the research of osteoblast differentiation.METHODS:Kusa-A1 cells were cultured and its osteogenic activities were measured,including the alkaline phosphatase (ALP) activity,calcium deposition ability, calcified nodules (CN) formation, osteopontin (OPN) and osteocalcin (OC) expression level examined with real time RT-PCR.The receptor activator of NF-κB ligand (RANKL) level was detected by western blot.RESULTS:Under standard culture condition, Kusa-A1 cells showed high ALP activity and calcium deposition,also exhibited slight OPN and OC expression,but no CN formation and RANKL expression.Addition of L-ascorbic acid-2-phosphate and β-glycerophosphate strikingly up-regulated ALP activity,calcium deposition,OPN and OC expression,and a mass of CN were observed.RANKL protein was also detected 24 h after confluence.CONCLUSION:Kusa-A1 is osteoblastic precursor cell.It is useful as a model cell line in the study of osteoblast function and mechanisms of osteoblastic differentiation.