Effects on gene expression of bone and cell-adhesive proteins by biomaterials

生物材料对骨和细胞粘附蛋白基因表达的影响

• 批准号: 11671920
• 负责人: YOKOYAMA Atsuro
• 金额: $ 2.24万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671920/
• 关键词: biomaterials; osteoblast; gene transfection; osteopontin; adhesive activity; 骨芽細胞様細胞; チタン; ハイドロキシアパタイト; α-3リン酸カルシウム

The purpose of this study was to investigate the mechanism of adhesion of osteoblast to biomaterials. Firstly, the effect of the difference of the biomaterials on the adhesion of osteoblasts to them was investingated. Cell adhesion rate of a human osteosarcoma derived cell line (Saos2) to hydroxyapatite was same to that to titanium incubated without serum, whereas cell adhesion rate of Saos2 to hydroxyapatite was higher than that to titanium with serum. From these results, we speculated that the proteins within serum effected cell adhesion to biomaterials. We carried out the tranfection of osteopontin (OP) into Saos2. OP is a noncollagenous protein in bone matrix. It is reported that OP enhanced adhesive activity of osteoblast-like cells in vitro. We investigated the effect on adhesive activity of osteoblast-like cell to titanium by transfection of OP. Human OP cDNA given FLAG tag to C-terminus was subcloned to retrovirus vector (pBabe-puro). Selection was carried out with DMEM contained ouromyciiL Saos2 cell line that expressed OP-FLAG was cloned (Saos2-OP). Expression of OP was observed in only Saos2-OP by the RT-PCR and production of OP included FLAG tag was confirmed in only Saos2-OP. There was a significant difference between the numbers of Saos2-OP and parental cells attached to the titanium disc and polystyrene dish after 12 and 24 hours of incubation in serum-free DMEM, although there was no significance among the numbers of attached cells after 6 hours. Observation of SEM revealed the difference between the shapes of Saos2-OP and Saos2. The shape of Saos2-OP was smaller and thicker, compared with Saos2, and there were small global structure on the surface of Saos2-OP, but not on the surface of Saos2. These results suggested that human osteopontin is a potent enhancer of osteoblast-like cell adhesion to titanium, and that transfection of osteopontin influenced the shape of Saos2.

本研究的目的是探讨成骨细胞与生物材料的粘附机制。首先，研究了生物材料的差异对其成骨细胞粘附的影响。人骨肉瘤细胞系(Saos2)对羟基磷灰石的细胞粘附率与无血清培养的钛的细胞粘附率相同，而Saos2对羟基磷灰石的细胞粘附率高于对有血清的钛的细胞粘附率。根据这些结果，我们推测血清中的蛋白质影响细胞对生物材料的粘附。我们将骨桥蛋白（OP）转染到 Saos2 中。 OP是骨基质中的非胶原蛋白。据报道，OP在体外增强了成骨细胞样细胞的粘附活性。我们研究了OP转染对成骨样细胞对钛粘附活性的影响。将 C 末端带有 FLAG 标签的人 OP cDNA 亚克隆至逆转录病毒载体 (pBabe-puro)。用含有ouromyciiL的DMEM进行选择，克隆了表达OP-FLAG的Saos2细胞系(Saos2-OP)。通过RT-PCR仅在Saos2-OP中观察到OP的表达，并且仅在Saos2-OP中确认了包含FLAG标签的OP的产生。在无血清 DMEM 中孵育 12 小时和 24 小时后，附着在钛盘和聚苯乙烯培养皿上的 Saos2-OP 和亲本细胞数量之间存在显着差异，但 6 小时后附着细胞数量之间没有显着差异。 SEM观察揭示了Saos2-OP和Saos2形状的差异。与Saos2相比，Saos2-OP的形状更小、更厚，并且Saos2-OP表面存在小的全局结构，但Saos2表面没有。这些结果表明人骨桥蛋白是成骨细胞样细胞与钛粘附的有效增强剂，并且骨桥蛋白的转染影响Saos2的形状。