纤维连接蛋白模拟肽P19的实验研究

Experimental study of a fibronectin like peptide P19

目的 研究无机骨修复材料的生物化,设计合成了纤维连接蛋白模拟肽P19,以便通过此途径改善羟基磷灰石(HA)类骨修复材料的生物活性。方法 运用固相多肽合成技术合成了P19,用Sephadex G 15对其进行初步纯化和空气氧化法修饰,并行质谱(TOF MS)鉴定,对其生物活性及与HA结合的特性进行了研究。结果 P19和HA具有较高的亲和力,能够影响凝胶系统体外矿化作用;对于HA的解离常数(Kd)为14 3±0 8μmol/L,100μg HA最大吸附多肽量为2 57±0 51nmol。吸附的多肽在生理条件下不能被洗脱。生理浓度的钙离子能够明显增加HA吸附的多肽量,磷酸根能够抑制多肽和HA的结合。P19能够明显增加培养板表面成骨细胞附着量。竞争性抑制实验结果表明,用P19处理的成骨细胞无法吸附到纤维连接蛋白包被的培养板表面,并表现为剂量效应,其作用强于GRGDSP六肽。结论 所合成的多肽分子具有纤维蛋白样活性,且能稳定的结合于HA,可作为HA类骨损伤修复材料以及种植体HA涂层的生物活性添加成分,有一定的临床应用前景。

Objective A fibronectin like peptide was designed and synthesized based on the structure and function relationship study of extra cellular matrix protein in order to search after a kind of new method to improve the bioactivity of hydroxyapatite based biomaterials. Methods A peptides named P19 with two functional groups was synthesized by employing solid phase peptide synthesis. The synthesized peptides were preliminarily purified with Sephadex G 15 column chromatography and further modified with air oxidation method. The purity of P19 were determined with RP HPLC. The molecular weight of peptide was identified with TOF MS. Its binding characteristics and bioactivity was investigated. Results P19 has high affinity with hydroxyapatite and cannot be eluted under physiological condition. Its dissociation constant is 14 3±0 8μmol/L and the maximum of attached peptides by 100μg HA is 2 57±0 51nmol. It can affect the in vitro calcification of gel system. The plate pretreated with P19 can enhance the number of attached cells and the proliferation of attached cells is also promoted. The fibronectin like activity was also proved by the competitive inhibition effect of P19 on the cell attachment. It also can promote the proliferation of UMR 106 cell line. Conclusion The synthesized peptide possess two function as design, it may have extensive application prospect as an ingredients of HA based materials or coating layer of implant