本文選題：軟骨組織工程　切入點：聚羥基脂肪酸酯　出處：《中國修復(fù)重建外科雜志》2014年08期

【摘要】：目的探討經(jīng)聚羥基脂肪酸酯表面顆粒結(jié)合蛋白(polyhydroxyalkanoates granule binding protein,PhaP)-精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)融合蛋白修飾后的聚羥基丁酸戊酸酯[poly(3-hydroxybutyrateco-3-hydroxyvalerate),PHBV]和聚羥基丁酸己酸酯[poly(3-hydroxybutyrate-co-3-hydroxyhexanoate),PHBHHx]的親水性及其與軟骨細(xì)胞的生物相容性。方法利用溶劑揮發(fā)法制備PHBV和PHBHHx生物膜材料,掃描電鏡觀察材料結(jié)構(gòu);通過蛋白工程技術(shù)表達(dá)純化PhaP-RGD融合蛋白,按照3.5 mg/mL濃度對兩種生物膜材料進(jìn)行蛋白修飾,通過測量接觸角檢測蛋白修飾前后材料表面親疏水性的改變。采用三步消化法體外分離培養(yǎng)人鼻中隔軟骨細(xì)胞并傳代。取第2代細(xì)胞分別接種于PHBV(A1組)、PHBV/PhaP-RGD(A2組)、PHBHHx(B1組)、PHBHHx/PhaP-RGD(B2組)、細(xì)胞培養(yǎng)板(C組)。培養(yǎng)3 d后行DAPI染色觀察細(xì)胞增殖情況;3、7 d通過MTT法檢測細(xì)胞增殖能力;7 d掃描電鏡下觀察細(xì)胞在生物膜材料表面的貼附及形態(tài)結(jié)構(gòu),并通過甲苯胺藍(lán)染色初步檢測細(xì)胞外基質(zhì)分泌情況。結(jié)果掃描電鏡觀察示PHBV和PHBHHx生物膜材料表面為多孔結(jié)構(gòu);經(jīng)融合蛋白修飾后PHBV、PHBHHx生物膜材料表面接觸角均顯著減小,差異有統(tǒng)計學(xué)意義(P0.05)。細(xì)胞接種于生物膜材料表面后均能生長,培養(yǎng)3 d后B2組細(xì)胞增殖能力最強(qiáng)(P0.05);7 d時各組軟骨細(xì)胞增殖能力均較3 d增強(qiáng)(P0.05),組間比較B1、B2組高于A1、A2、C組,B2組高于B1組、A1組高于A2組(P0.05)。培養(yǎng)7 d,甲苯胺藍(lán)染色示A1、A2、B1、B2組材料表面均可見藍(lán)色異染,其中A1、A2組染色程度相似,B2組染色較B1組深;掃描電鏡觀察示各組細(xì)胞貼附良好,細(xì)胞之間形成連接,并伸入材料孔隙內(nèi)。結(jié)論經(jīng)PhaPRGD融合蛋白修飾的PHBHHx生物膜材料與軟骨細(xì)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.
【作者單位】： 西安交通大學(xué)第二附屬醫(yī)院耳鼻咽喉-頭頸外科;西安醫(yī)學(xué)院附屬醫(yī)院耳鼻喉科;韓城市人民醫(yī)院耳鼻喉科;西安交通大學(xué)生命科學(xué)與技術(shù)學(xué)院;
【基金】：國家自然科學(xué)基金資助項目(81000416) 中央高校基本科研業(yè)務(wù)費專項資金資助項目 西安交通大學(xué)第二附屬醫(yī)院人才培養(yǎng)基金資助項目[RC(XM)201102] 重點基金資助項目[YJ(ZD)201301]~~
【分類號】：R318.08

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