中国修复重建外科杂志

中国修复重建外科杂志

脱细胞气管基质材料的免疫原性及生物相容性研究

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目的探讨经高氯酸钠(NaClO4)处理后的脱细胞气管基质材料的免疫原性及其生物相容性。方法取 2 月龄新西兰兔胫骨骨髓,采用全骨髓贴壁筛选法分离培养 BMSCs。取 10 只 6 月龄成年新西兰兔气管,修剪至每段 1.5 cm,随机分为对照组(A1 组,n=5),仅剥离气管外表面疏松结缔组织;实验组(B1 组,n=5)采用改良 NaClO4 浸泡法脱细胞处理。MTT 法检测两组支架浸提液的细胞毒性;免疫组织化学染色观察支架主要组织相容性复合物(major histocompatibility complex,MHC)类抗原表达。取生长状态良好的第 4 代 BMSCs 接种至两组支架,制备细胞-支架复合物,培养 48 h 时行 Giemsa 染色,倒置显微镜观察材料周围的细胞活性;7、14 d 扫描电镜观察支架上的细胞状态。取 10 只 6 月龄成年新西兰兔,随机分成对照组(A2 组,n=5)和实验组(B2 组,n=5),分别于颈背部皮下皮囊埋植已制备的新鲜气管和脱细胞气管。术后行大体观察,并于术后 5、10、15、20、25、30 d 分析血清免疫球蛋白 IgM 和 IgG 含量的动态变化,术后 30 d 行 HE 染色观察。结果MTT 检测示,B1 组浸提液的细胞增殖情况与 A1 组或纯培养基阴性对照组比较,差异无统计学意义(P>0.05);免疫组织化学染色观察示,B1 组支架经脱细胞处理后可显著降低基质材料的抗原性。细胞-支架复合物培养 48 h Giemsa 染色示,两组材料周围的细胞贴壁生长良好。培养 7、14 d 扫描电镜观察示,细胞在 A1 组气管材料外壁上贴附良好,呈扁平的圆形、椭圆形,细胞排列紧密,成簇分布;细胞在 B1 组气管材料外壁上呈单片状生长,形态与 A1 组相似,生长趋势较好。同种异体动物体内实验显示,B2 组材料的排斥反应显著低于 A2 组;术后各时间点 A2 组的 IgM 和 IgG 含量均显著高于 B2 组(P<0.05);HE 染色示,B2 组未见炎性细胞深层渗透或破坏气管结构,未见钙化、排斥等不良反应。结论经 NaClO4 化学脱细胞处理后,兔气管支架材料具有良好的生物相容性,同时其免疫原性降低,适合作为构建组织工程气管的支架材料。

ObjectiveTo investigate the biocompatibility and immunogenicity of the tracheal matrix decellularized by sodium perchlorate (NaClO4).MethodsBone marrow mesenchymal stem cells (BMSCs) were divided from 2-month-old New Zealand white rabbits. The trachea of 6-month-old New Zealand white rabbits were trimmed to a length of 1.5 cm and randomly divided into control group (group A1, n=5, just stripped the loose connective tissue outside the trachea) and experimental group (group B1, n=5, decellularized by improved NaClO4 immersion method). The cytotoxicity of the scaffold leaching solution was detected by MTT assay, and the major histocompatibility complex (MHC) expression was detected by immunohistochemical method. The 4th generation of BMSCs were seeded onto the scaffold of 2 groups, and the cell activity around the material was observed by inverted microscope after Giemsa staining at 48 hours, while the cells states on the scaffold were observed at 7 and 14 days after culturing by scanning electron microscope. Another 10 6-month-old New Zealand white rabbits were randomly divided into control group (group A2, n=5) and experimental group (group B2, n=5), which implanted the native trachea and decellularized tracheal matrix into the subcutaneous sac of the back neck, respectively. The serum immunoglobulin IgM and IgG contents were analysed at 5, 10, 15, 20, 25, and 30 days after operation, and HE staining observation was performed at 30 days after operation.ResultsMTT assay showed that the proliferation activity of BMSCs cultured in the leach liquor of group B1 was well, showing no significant difference when compared with group A1 and negative control group with pure culture medium (P>0.05). The immunohistochemical staining showed that the decellularized process could significantly reducing the antigenicity of matrix materials. Giemsa staining showed that BMSCs grew well around the two tracheal matrixs (groups A1 and B1) in vitro. Scanning electron microscope observation showed that the cells were attached to the outer wall of the tracheal material in group A1, which present a flat, round, oval shaped, tightly arranged cells and cluster distribution; and in group B1, the cells formed a single lamellar sheet cover the outer wall of the tracheal material, whose morphology was similar to that in group A1, and the growth trend was better. In vivo experimental results showed that the rejection of group B2 was lower than that of group A2. The contens of IgM and IgG in group A2 were significantly higher than those in group B2 at each time point after operation (P<0.05). HE staining showed no signs of rejection, macrophagocyte, or lymphocyte infiltration occurred, and the collagen fibers maintained their integrity in group B2.ConclusionThe decellularized matrix treated by NaClO4 has a fine biocompatibility, while its immunogenicity decreased, and it is suitable for the scaffold material for constructing of tissue engineered trachea.

关键词: 组织工程气管; 脱细胞材料; 生物相容性; 免疫原性;

Key words: Tissue engineered trachea; decellularized material; biocompatibility; immunogenicity; rabbit

引用本文: 潘枢, 刘星辰, 史宏灿. 脱细胞气管基质材料的免疫原性及生物相容性研究. 中国修复重建外科杂志, 2018, 32(4): 441-447. doi: 10.7507/1002-1892.201710007 复制

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