The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts
The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts
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DOI：
10.1016/j.biomaterials.2011.09.015
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发表时间：
2011-12-01
期刊：
影响因子：
14
通讯作者：
Wilhelmi, Mathias
中科院分区：
文献类型：
--
作者：
Boeer, Ulrike;Lohrenz, Andrea;Wilhelmi, Mathias
脱细胞马颈动脉(DEAC)可能是血管替代治疗中替代同种异体材料的一种合理选择。用DNA定量来评价基质的无细胞性是标准的，但这可能不能充分反映基质的免疫原性程度。因此，我们的目标是分析低DNA含量的DEAC以检测残留的细胞蛋白。以洗涤剂为基础的脱细胞方案，包括内切酶处理，导致DEAC为0.6+/-0.15 ng DNA/mg干重，占天然组织DNA含量的0.33+/-0.14%。相反，当基质匀浆并用高浓度洗涤剂提取时，Western印迹分析显示胞浆和细胞质蛋白，如GAPDH和平滑肌肌动蛋白，分别减少到4.1+/-1.9%和13.8+/-0.55%。此外，假定的免疫原性MHC I复合体和α-Gal表位仅减少到14.8+/-1.2%和15.1+/-+2.05%。基质提取物的质谱仪鉴定了306种蛋白质，分别属于细胞质、细胞器、细胞核和细胞膜。此外，水基质提取物在小鼠体内注射时会引起显著的抗体形成，因此显示出高免疫原性潜力。我们的数据表明，一种已建立的脱细胞方案可以减少但不是消除体内可能有助于其免疫原性的细胞成分，这种脱细胞方案以低DNA含量评估脱细胞基质。(C)2011爱思唯尔有限公司。保留所有权利。
Decellularized equine carotid arteries (dEAC) may represent a reasonable alternative to alloplastic materials in vascular replacement therapy. Acellularity of the matrix is standardly evaluated by DNA quantification what however may not record sufficiently the degree of matrix immunogenicity. Thus, our aim was to analyze dEAC with a low DNA content for residual cellular proteins. A detergent-based decellularization protocol including endonuclease treatment resulted in dEAC with 0.6 +/- 0.15 ng DNA/mg dry weight representing 0.33 +/- 0.14% of native tissue DNA content. In contrast, when matrices were homogenized and extracted by high detergent concentrations westernblot analyses revealed cytosolic and cytosceleton proteins like GAPDH and smooth muscle actin which were depleted to 4.1 +/- 1.9% and 13.8 +/- 0.55%, resp. Also putative immunogenic MHC I complexes and the alpha-Gal epitop were reduced to only 14.8 +/- 1.2% and 15.1 +/- + 2.05%. Mass spectrometry of matrix extracts identified 306 proteins belonging to cytosol, organelles, nucleus and cell membrane. Moreover, aqueous matrix extracts evoked a pronounced antibody formation when administered in mice and thus display high immunogenic potential. Our data indicate that an established decellularization protocol which results in acellular matrices evaluated by low DNA content reduces but not eliminates cellular components which may contribute to its immunogenic potential in vivo. (C) 2011 Elsevier Ltd. All rights reserved.