一种优化的猪骨骼肌来源无细胞基质支架材料制备方法及鉴定  

EVALUATION OF AN OPTIMIZING PROTOCOL FOR FABRICATING A SCAFFOLD DERIVED FROM PORCINE SKELETAL MUSCLE EXTRACELLULAR MATRIX

作　　者：张迪[1,2] 谭秋雯 汤沈力 罗静聪[2] 张忆[2] 吕青[1] 

机构地区：[1]四川大学华西医院乳腺外科专病中心,成都610041 [2]四川大学华西医院生物治疗国家重点实验室.干细胞与组织工程研究室,成都610041

出　　处：《中国修复重建外科杂志》2016年第10期1282-1289,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：成都市科技局-华西医院转化医学创新基金项目(ZH13033)~~

摘　　要：目的 探讨一种优化的猪骨骼肌脱细胞制备方法,以期制备理想的猪骨骼肌来源无细胞基质（decellularized porcine muscle tissue,DPMT）支架材料。方法 取市售新鲜健康成年家猪腰部骨骼肌组织,采用匀浆-球磨-去垢剂多步骤脱细胞方案制备DPMT。行组织学观察脱细胞、脱脂效果,天狼猩红染色分析胶原成分,扫描电镜观察材料内部微结构;测定材料中DNA含量及总蛋白质含量。取人脂肪来源干细胞（adipose derived stem cells,ADSCs）、小鼠成纤维细胞（NIH3T3）和人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）,置于不同浓度DPMT浸提液中,培养后第1、3、5天采用细胞计数试剂盒8法测算细胞相对增殖率,评价材料细胞毒性;通过DPMT表面接种HUVECs体外共培养3 d后,行活死细胞染色评价其体外细胞相容性。采用SPF级雄性SD大鼠建立DPMT大鼠背部皮下植入模型,于植入3、7、14、28 d取材,大体观察材料降解情况,并测量其横径;行HE染色和CD31免疫组织化学染色观察其生物相容性和血管新生情况。结果 匀浆-球磨-去垢剂多步骤脱细胞方法操作简便,制备DPMT仅需1 d时间;检测显示DPMT脱细胞、脱脂完全,主要成分为Ⅰ型胶原及Ⅳ型胶原;DPMT中DNA含量为（15.902±1.392）ng/mg干重,总蛋白质含量占其干重的68.94%,显著低于新鲜猪骨骼肌组织[分别为（140.724±10.422）ng/mg干重及93.14%]（P〈0.05）;DPMT内部呈均质疏松网状结构。体外细胞相容性实验显示DPMT细胞毒性评级均为0~1级,HUVECs在DPMT表面能稳定生长。DPMT大鼠皮下植入后14 d内可保持大体结构的稳定性,至28 d时完全降解;炎性反应在3 d时最明显,7 d时明显减轻,两时间点炎性细胞数量比较差异有统计学意义（P〈0.05）;植入后7 d即可见DPMT内部新生血管环结构形成,14 d时新生血管环数量显著增加,比较差异有统计学意义（P〈0.05）。结论 匀浆-球磨-去垢剂�Objective To explore an optimized protocol of decellularization to fabricate an ideal scaffold derived from porcine skeletal muscle acellular matrix. Methods Serial-step protocol ofhomogenating-milling-detergent method was used to fabricate decellularized porcine muscle tissue （DPMT） derived from native porcine skeletal muscle tissue from adult pig waist. Histological method was used to assess the effects of decellularization and degreasing. Sirius red staining was used to analyze collagen components. Scanning electron microscopy, BCA assay, and PicoGreen assay were used to evaluate the ultrastructure, total protein content, and DNA content in DPMT. The adipose derived stem cells （ADSCs）, NIH3T3 cells, and human umbilical vein endothelial ceils （HUVECs） were cultured in extraction liquor of DPMT in different concentrations for 1, 3, and 5 days, then the relative growth rate was calculated with cell counting kit 8 to assess the toxicity in vitro. Live/dead cell staining was used to evaluate the cytocompatibility by seeding HUVECs on the surface of DPMT and co-cultured in vitro for 3 days. For in vivo test, DPMT was subcutaneously implanted at dorsal site of male specific-pathogen free Sprague Dawley rats and harvested after 3, 7, 14, and 28 days. Gross obersvation was done and transverse diameter of remained DPMT in vivo was determined. HE staining and immunohistochemical staining of CD31 were used to assess inflammatory response and new capillary rings formation. Results Decellularization of the porcine skeletal muscle tissue by homogenating-milling-detergent serial steps protocol was effective, time-saving, and simple, which could be finished within only 1 day. The decellularizarion and degreasing effect of DPMT was complete. The main component of DPMT was collagen type I and type IV. The DNA content in DPMT was （15.902±1.392） ng/mg dry weight, the total protein content was 68.94% of DPMT dry weight, which was significantly less than those of fresh skeletal muscle tissue [（140.727±10.422） n

关 键 词：组织工程 支架材料 猪骨骼肌 无细胞基质 生物相容性 血管新生 

分 类 号：R318.08[医药卫生—生物医学工程] R737.9[医药卫生—基础医学]