聚羟基脂肪酸酯的生物修饰及其生物相容性研究  

BIO-MODIFICATION OF POLYHYDROXYALKANOATES AND ITS BIOCOMPATIBILITY WITH CHONDROCYTES

作　　者：高天喜[1] 常会敏[2] 范敏杰[3] 卢晓云[4] 王正辉[1] 张向红[1] 井晓红[1] 史艳霞[1] 李智慧[1] 

机构地区：[1]西安交通大学第二附属医院耳鼻咽喉一头颈外科,西安710004 [2]西安医学院附属医院耳鼻喉科 [3]韩城市人民医院耳鼻喉科 [4]西安交通大学生命科学与技术学院

出　　处：《中国修复重建外科杂志》2014年第8期1023-1029,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81000416);中央高校基本科研业务费专项资金资助项目;西安交通大学第二附属医院人才培养基金资助项目[RC(XM)201102];重点基金资助项目[YJ(ZD)201301]~~

摘　　要：目的探讨经聚羟基脂肪酸酯表面颗粒结合蛋白(polyhydroxyalkanoates granule binding protein,PhaP)-精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)融合蛋白修饰后的聚羟基丁酸戊酸酯[poly(3-hydroxybutyrateco-3-hydroxyvalerate),PHBV]和聚羟基丁酸己酸酯[poly(3-hydroxybutyrate-co-3-hydroxyhexanoate),PHBHHx]的亲水性及其与软骨细胞的生物相容性。方法利用溶剂挥发法制备PHBV和PHBHHx生物膜材料,扫描电镜观察材料结构;通过蛋白工程技术表达纯化PhaP-RGD融合蛋白,按照3.5 mg/mL浓度对两种生物膜材料进行蛋白修饰,通过测量接触角检测蛋白修饰前后材料表面亲疏水性的改变。采用三步消化法体外分离培养人鼻中隔软骨细胞并传代。取第2代细胞分别接种于PHBV(A1组)、PHBV/PhaP-RGD(A2组)、PHBHHx(B1组)、PHBHHx/PhaP-RGD(B2组)、细胞培养板(C组)。培养3 d后行DAPI染色观察细胞增殖情况;3、7 d通过MTT法检测细胞增殖能力;7 d扫描电镜下观察细胞在生物膜材料表面的贴附及形态结构,并通过甲苯胺蓝染色初步检测细胞外基质分泌情况。结果扫描电镜观察示PHBV和PHBHHx生物膜材料表面为多孔结构;经融合蛋白修饰后PHBV、PHBHHx生物膜材料表面接触角均显著减小,差异有统计学意义(P<0.05)。细胞接种于生物膜材料表面后均能生长,培养3 d后B2组细胞增殖能力最强(P<0.05);7 d时各组软骨细胞增殖能力均较3 d增强(P<0.05),组间比较B1、B2组高于A1、A2、C组,B2组高于B1组、A1组高于A2组(P<0.05)。培养7 d,甲苯胺蓝染色示A1、A2、B1、B2组材料表面均可见蓝色异染,其中A1、A2组染色程度相似,B2组染色较B1组深;扫描电镜观察示各组细胞贴附良好,细胞之间形成连接,并伸入材料孔隙内。结论经PhaPRGD融合蛋白修饰的PHBHHx生物膜材料与软骨细胞有良好的生物相容性。Objective To study the hydrophilicity and the cell biocompatibility of the poly（3-hydroxybutyrate-co-3-hydroxyvalerate） （PHBV） and poly（3-hydroxybutyrate-co-3-hydroxyhexanoate） （PHBHHx） coated with a fusion protein polyhydroxyalkanoates granule binding protein （PhaP） fused with Arg-Gly-Asp （RGD） peptide （PhaP-RGD）. Methods PHBV and PHBHHx films were fabricated by solvent evaporation. Scanning electronic microscope （SEM） was used to study the morphology of the films. PhaP-RGD fusion proteins were expressed and purified by the technology of protein engineering; PHBV and PHBHHx films were immersed in the PhaP-RGD with an amount of 3.5 mg/mL protein/per sample respectively. The hydrophilicity of the surface were detected by the contact angle measurements. Septal cartilage cells obtained from human septal cartilage were cultured in vitro. The 2nd passage chondrocytes were incubated on PHBV unmodified with PhaP-RGD in group A1, PHBV modified with PhaP-RGD in group A2, PHBHHx unmodified with PhaP-RGD in group B1, PHBHHx modified with PhaP-RGD in group B2, and on the cell culture plates in group C. After cultured for 3 days, the proliferation of cells was detected by the DAPI staining; the proliferation viability of cells was detected by the MTT assay after cultured for 3 and 7 days; after cultured for 7 days, the adhesion and morphology of the cells on the surface of the biomaterial films were observed by SEM and the matrix of the cells was detected through the toluidine blue staining. Results SEM observation showed that PHBV and PHBHHx films had porous structures. The contact angle of the surface of the PHBV and PHBHHx films modified with PhaP-RGD fusion proteins were significantly reduced when compared with the films unmodified with PhaP-RGD fusion proteins （P 〈 0.05）. Chondrocytes of human nasal septal cartilage incubated on the films could grow in all groups. After 3 days of cultivation in vitro, the cell proliferation and viability of group B2 were the strongest among all group

关 键 词：软骨组织工程 聚羟基脂肪酸酯 聚羟基丁酸戊酸酯 聚羟基丁酸己酸酯PhaP-精氨酸-甘氨酸-天冬氨酸融合蛋白 生物相容性 

分 类 号：R318.08[医药卫生—生物医学工程]