自分泌骨桥蛋白在Nfl＋／-小鼠破骨细胞功能增强中的作用

Role of autoerine osteopontin in promoting multiple functions of murine Nil＋/- osteoclast

目的研究体外培养的Nfl＋／-小鼠破骨细胞合成、分泌骨桥蛋白（osteopontin，OPN）的能力，应用OPN中和抗体抑制破骨细胞分泌的OPN，测定破骨细胞功能，并与野生型破骨细胞比较，分析OPN在Nfl＋／-小鼠破骨细胞功能增强中的作用。方法配对选取神经纤维瘤病基因野生型（Nf＋/＋）和杂合型（Nf＋／-）基因型小鼠，采集骨髓单个核细胞，体外培养破骨细胞，获取体外培养破骨细胞不同时期的上清液，做ELISA分析检测OPN浓度，并与Nfl＋／＋破骨细胞比较；在加入OPN中和抗体或等量PBS的条件下，测定体外培养的Nfl＋／-小鼠破骨细胞的形成数量，应用Transwell小室方法测定破骨细胞迁移功能，应用自然贴壁法测定破骨细胞黏附功能，应用骨片吸收法测定破骨细胞骨吸收功能，并进行数据分析。结果在体外培养的Nfl＋／-小鼠破骨细胞上清液中，OPN含量比野生型小鼠破骨细胞上清液中含量明显增高，培养6d的破骨细胞上清液中OPN含量的差异更为明显。体外培养Nfl＋／-破骨细胞形成数量比Nfl＋／＋破骨细胞明显增多，破骨细胞的黏附功能、迁移功能以及骨吸收功能较Nfl＋／＋破骨细胞明显增强，差异有统计学意义；在培养时加入有活性的OPN中和抗体，可以抑制Nfl＋／-破骨细胞的形成，破骨细胞的黏附能力、迁移能力以及骨吸收功能均降低，恢复到野生型水平。结论自分泌OPN在Nfl＋／-小鼠破骨细胞功能增强过程中发挥重要作用，OPN可能是治疗1型神经纤维瘤病骨质疏松的靶向治疗候选靶分子之一，其具体分子机制还需进一步研究。

Objective To detect the osteopontin （OPN） autocrine function of the osteoclasts in neurofibromatosis type 1 heterozygote （Nfl＋/-） and wild type （Nfl＋/＋） mice. Test the osteoclasts function of neurofibromatosis type 1 heterozygote （Nfl＋/-） and wild type （Nfl＋/＋） mice with exogenous neutralizing OPN antibody, analysis the role of autocrine OPN in the hyperfunction of osteoclast in neurofibromatosis type 1. Methods Culture the low density bone marrow cells from Nfl heterozygote （Nfl＋/-） and wild type （Nfl＋/＋） mice （ 4-6 weeks old ） with macrophage colony-stimulating factor （M-CSF） and receptor activator of NF-KB lig-and（RANKL）, Measure the OPN concentration in osteoclast culture superenant with ELISA. Culture the low density bone marrow ceils from Nfl ＋/- and Nfl ＋/＋ mice with or without exogenous neutralizing antibody for OPN. The function of osteoclasts and os-teoclast progenitors in formation, migration, adhesion, and bone absorption were tested. Results A significantly higher concen- tration of OPN was detected in the Nfl ＋/- osteoclast cuhure media as compared to that of wild type. In control, Osteoclast func-tions, including migration, adhesion, and bone resorption of Nfl＋/-were higher than that of wild type. Addition OPN neutralizing antibody to the Nfl＋/- OCL significantly reduced OCL formation. Neutralizing OPN antibody diminished both wild type and Nfl＋ /- OCL adhensiontion, Anti-OPN minimized OCL migration in both wild type and Nfl＋/- OCL cultures as measured by the tran- swell assays. Neutralizing OPN antibody diminished both wild type and Nfl＋/- OCL pit formation,P〉0.05 for comparing Nfl＋/- vs. wild type OCLs with anti-OPN antibody. Conclusion The hyperfunction of osteoclast in Nfl heterozygote is related with au- tocrine osteopontin, inhibition of OPN may be an effective treatment for bone destruction of neurofibromatosis type 1.