聚羟基脂肪酸酯的生物修饰及其生物相容性研究

发布时间：2018-03-24 21:22

本文选题：软骨组织工程　切入点：聚羟基脂肪酸酯　出处：《中国修复重建外科杂志》2014年08期

【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(P0.05)。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(P0.05);7 d时各组软骨细胞增殖能力均较3 d增强(P0.05),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(P0.05)。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。
[Abstract]:Objective to investigate the hydrophilicity of polyhydroxybutyrateco-3-hydroxyvaleratePHBV and polyhydroxybutyrate-co-hydroxyhexanoate (PHBHHX) modified by polyhydroxyalkanoates granule binding protein (PhaPN-Arg-Gly-AspRGDx) fusion protein modified by polyhydroxybutyrateco-3-hydroxyvaleratePHBHX (polyhydroxybutyrate-co-hydroxyhexanoate PHBHx) and polyhydroxybutyrate-co-hydroxyhexanoate polyhydroxybutyrate-co-hydroxyhexanoate (HHX). Methods PHBV and PHBHHx biofilm materials were prepared by solvent volatilization. Scanning electron microscope (SEM) was used to observe the structure of the material, and the PhaP-RGD fusion protein was expressed and purified by protein engineering technique. The two biofilm materials were modified with protein according to the concentration of 3.5 mg/mL. Human nasal septal chondrocytes were isolated and cultured by three-step digestion before and after modification by measuring contact angle. The second passage cells were inoculated in PHBV(A1 group with PHBV / PhaP-RGDX A2 group respectively. After 3 days of culture, the proliferation of cells was observed by DAPI staining. The ability of cell proliferation was measured by MTT method. The adhesion and morphological structure of cells on the surface of biofilm were observed under scanning electron microscope for 7 days. The secretion of extracellular matrix was detected by toluidine blue staining. Results the results showed that the surface of PHBV and PHBHHx biofilm was porous structure, and the contact angle of PHBV / PHBHHx biofilm material was significantly decreased after modified by fusion protein. The difference was statistically significant (P 0.05). Cells could grow after inoculation on the surface of biofilm. The proliferation ability of chondrocytes in group B2 was stronger than that in group B 2 at 7 days after culture for 3 days, and the proliferation of chondrocytes in group B 1 was higher than that in group A 1, and higher in group B 2 than that in group B 1. 7 days after culture, toluidine blue staining showed that the proliferation of chondrocytes in group B 2 was higher than that in group B 2. The surface of the group can be seen to be blue heterochromatic. The staining degree of A _ 1 A _ 2 group was similar to that of B _ 1 group, and the staining of B _ 2 group was deeper than that of B _ 1 group. Conclusion PHBHHx biofilm modified by PhaPRGD fusion protein has good biocompatibility with chondrocytes.
【作者单位】：西安交通大学第二附属医院耳鼻咽喉-头颈外科;西安医学院附属医院耳鼻喉科;韩城市人民医院耳鼻喉科;西安交通大学生命科学与技术学院;
【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]~~
【分类号】：R318.08

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