人釉原蛋白亮氨酸富集片段的冷冻电子显微镜观察及其引导矿化性能研究  

Study of human leucine-rich amelogenin peptide and its regulation of mineralization by cryogenic transmission electron microscopy

作　　者：田鲲[1] 冯小云[1] 杜芹[1] 廖楚航[1] 任小华[1] 

机构地区：[1]电子科技大学附属医院.四川省人民医院口腔科,成都610072

出　　处：《华西口腔医学杂志》2017年第1期63-67,共5页West China Journal of Stomatology

基　　金：国家自然科学基金(81100786)~~

摘　　要：目的采用冷冻电子显微镜观察体外重组的人釉原蛋白富亮氨酸片段(LRAP),分析其细微结构和聚集状态,结合矿化实验评估LRAP体外引导羟磷灰石生长的定向成核能力。方法人工合成LRAP基因,与原核表达载体p Cold-SUMO行质粒构建,于宿主菌大肠杆菌BL21plys中诱导表达并纯化,在冷冻电子显微镜下观察LRAP在p H值从3.5到8.0的环境中聚合自组装的过程,并在人工唾液中通过透射电镜观察羟磷灰石晶体的生长规律。结果通过原核表达成功得到纯度90%以上的LRAP蛋白提取物,当p H值为8.0时,LRAP能聚集成多聚体和纳米小球等功能结构,在人工唾液中能诱导羟磷灰石晶体生长成熟。结论作为简化的釉原蛋白功能域,LRAP兼备了自组装为纳米小球和c轴诱导晶体矿化的特性,可作为运用釉基质蛋白多聚物行无细胞修复牙体硬组织缺损的备选材料之一。Objective Recombinant human leucine-rich amelogenin peptide （LRAP） was studied by cryogenic transmission electron microscopy （TEM）; evaluation focused on its self-assembly and crystal growth in vitro. Methods Human LRAP was recombined through prokaryotic expression vector pCold-SUMO and transformed into Escherichia coli BL2 lplys to acquire purified proteins. Cryogen TEM recorded assembly and self-assembling of LRAP from pH 3.5 to pH 8.0, and the hydroxyapatite crystal growth in the mixture of LRAP protein solution and artificial saliva was observed using TEM and selected area electron diffraction. Results More than 90% purity LRAP was expressed, purified and identified as described in methods. LRAP linked into oligomers, nanospheres, nanochains, and microribbons, whereas pH value increased from 3.5 to 8.0. Mature hydroxyapatite crystal growth was guided in artificial saliva filled with calcium phosphate. Conclusion LRAP is simplified amelogenin functional domain and conserved the basic characters of amelogenin such as self-assembling and inducing crystallization along c axis. In the area of acellular synthesis of hydroxyapatite using extracellular enamel matrix protein, LRAP is one of candidate repair materials for irregular hard tissue defection.

关 键 词：冷冻电子显微镜 釉原蛋白 釉原蛋白亮氨酸富集片段 羟磷灰石 

分 类 号：R783[医药卫生—口腔医学]