低免疫原性猪真皮支架制备及其细胞相容性评价  

Preparation of low immunogenic porcine dermal scaffold and evaluation of its cytocompatibility

作　　者：邵阳[1] 武岩 贾军[1] 马印东[1] 辛乃军[1] 常朋飞[1] 宋国栋[1] 

机构地区：[1]山东大学附属济南市中心医院烧伤科,250013 [2]手术室

出　　处：《中华烧伤杂志》2012年第5期329-335,共7页Chinese Journal of Burns

基　　金：山东省医药卫生科技发展计划（2011HZ008）;济南市留学人员创业计划（20080405）

摘　　要：目的观察基质金属蛋白酶7（MMP-7）、京尼平及真空冷冻干燥处理对猪脱细胞网状层真皮基质组织结构及细胞相窬性的影响。方法取清洁级健康大约克夏猪边腹部皮肤，采用机械法制作54块网状层真皮，面秋10．0mm×5．0mm，厚0．5～0．6mm，按随机数字表法分为正常对照绀（A。组，小仃处娌）、脱细胞组（B组，行脱细胞处理）、脱细胞＋MMP-7组（C组，行脱细胞和MMP-7处理）、脱细胞＋MMP-7＋京尼平组（D组，脱细胞处理后用MMP-7和京尼平处理）和脱细胞＋MMP-7＋康皑平＋真窄冷冻干燥组（E组，除行D组处理外，加真空冷冻了：燥处理），A，组6块，其余每组均为12块、，乃取6块卡相同面积人ADM没为人ADM组（A，组，不行处理），即细胞相容性实验中的对照组、采用HE染色、扫描电镜检测A1、B～E组真皮支架中细胞数量及组织结构变化，采用免疫组织化学染色法俭测A1、B、C组样本中波形蛋白、层粘连蛋白（LN）、Ⅳ型胶原蛋白残留情况，采用细胞毒性试验榆测B～E组浸提液的细胞毒性。接种人Fb于B～E组和A，组真皮支架表面培养，观察培养3、7、14d时Fb生长情况，采用ELISA法检测培养3、7d上清液中IL-6、IL-8含量。对数据进行双因素办差分忻及LSD-t检验。结果A1组可见含毛囊的浅黄色颗粒状结构，B组标本毛囊内仍有少艟毛发、上皮根鞘、细胞核、细胞碎片样结构及波形蛋白、LN、1V型胶原蛋白残留，C～E组经MMP-7处理后上述物质被完个去除。大体观察显示，D、E组真皮支架韧性较B、C组增加。C～E组胶原纤维结构亢褴，排列与A．组卞日似儿胶原纤维之间间隙增大，其中C、D组间隙卡H近但均大于B组，E组最大．B～E组真皮支架浸捉液细胞毒性为0或1级。F11于A2和B～E组真皮支架上均能增殖，并且随培养时问延长逐渐由单层变成复层生长进而向标本内部生长。Objective To observe the changes in the structure and cytocornpatibility of porcine a-cellular dermal matrix, which was prepared with dermal reticular layer, treated with matrix metalloproteinase 7 （MMP-7） , genipin, and vacuum freeze-drying. Methods Fifty-four pieces of porcine dermal reticular layer, prepared with lateral abdominal skin were obtained from healthy large Yorkshire pig with mechanical method under sanitary condition, each 10.0 mm ± 5.0 mm in size and 0.5-0.6 mm in thickness. They were divided into normal control group （ A1 , without treatment, n = 6） , decellularization group （ B, decellulized, n = 12）, decellularization ＋ MMP-7 group （C, treated with MMP-7 after decellularization, n = 12）, decel- lularization ＋ MMP-7 ＋ genipin group （ D, treated with MMP-7 and genipin after decellularization, n = 12） , and decellularization ＋ MMP-7 ＋ genipin ＋ vacuum freeze-drying group （ E, treated with MMP-7, genipin, and vacuum freeze-drying after decellularization, n = 12） according to the random number table. Mean- while, 6 pieces of human acellular dermal matrix, with the same size and thickness as listed above, were taken as control group （A2 , without treatment） in the cytocompatibility tests. HE staining and scanning e- lectron microscope were used to detect the cell number and the change in tissue structure in dermal scaffold in groups AI and B-E. Immunohistochemical staining was used to determine residual vimentin, laminin and collagen 1V in groups A1 , B, and C. Cytotoxicity tests were employed to test the cytotoxicity of the leaching solutions of groups B-E. Human fibroblasts were seeded on the surface of dermal scaffold in groups A2 and B-E. The proliferation of fibroblasts were determined on post culture day （PCD） 3, 7, and 14, and the con- tent of IL-6 and IL-8 in the supernatant were determined on PCD 3 and 7 with enzyme-linked immunosorbent assay. Data were processed with two-way analysis of variance and LSD- t test. Results Granular structure with ha

关 键 词：摹质会属蛋白脯7 细胞毒性试验 免疫 冷冻干燥法 脱细胞真皮基质 细 胞相容性 京尼平 

分 类 号：R318.08[医药卫生—生物医学工程]