脱细胞基质生物材料DNA去除标准和免疫原性研究

The biosafety and biocompatibility of acellular tissue matrix (ACTM) have gained increasing attention. However, well-recognized decellularization standard has not been established. Low residual DNA (below 50ng/mg) was set as criteria of complete cell removal by domestic ACTM manufacturer. In our previous research, skeletal muscle whole organ acellular matrix with residual DNA over 50ng/mg has shown improved in vivo tissue reconstruction and myogenesis, which is contradicted with the previous mentioned standards. Further, the in vitro behaviors of ACTM manufactured by ultra-high pressure processing and perfusion-decelluarization technique revealed that it is endotoxin, not DNA residue that related to the high cytotoxicity and T cells activation. Thus we aimed to evaluate the effect of residual DNA, lipid and endotoxin on in vitro immune response and in vivo host response, tissue reconstruction and regeneration of ACTM, confirming the impact of residual DNA on the in vivo immune response of ACTM following implantation. Novel criteria of DNA residue for successful decellularization should be raised. The second object of the research is to figure out a new method to dissocaite endotoxin from ACTM and accomplish complete detection of endotoxin from FDA proved products. The current research would point out novel directions and standards of ACTM evaluation and manufacture. The applicant and co-researchers were experienced in fundamental research and clinical application of ACTM, goals of the research can be accomplished.

至今尚无制备脱细胞基质（ACTM）指示彻底脱细胞的公认标准，而国内常以DNA残留量少于50ng/mg为主要指标。课题组既往发现高DNA残留的骨骼肌全器官ACTM体内炎症反应轻微、材料重塑和肌肉再生效果佳，与上述标准矛盾；进一步研究利用自主知识产权超高压联合灌注新型脱细胞技术制备的不同DNA/脂质/内毒素残留量的ACTM，发现非残留DNA和脂质、而可能是内毒素与ACTM体外免疫原性相关。本课题拟深入比较DNA和内毒素与ACTM体外免疫原性、体内宿主免疫应答、材料重塑和组织再生的相关性，确认影响ACTM免疫原性的关键因素、建立新的DNA残留标准，确认现有ACTM中内毒素分离和检测结果存在假阴性并探索新的方法、彻底清除内毒素可降低ACTM所致的血清肿/发热等并发症率。预期结果将为规范脱细胞技术制备ACTM提供新的方向和标准。申请者在ACTM制备和基础研究、临床应用领域有一定积累，研究目标均能实现

至今尚无制备脱细胞基质（ACTM）指示彻底脱细胞的公认标准，而国内常以DNA残留量少于50ng/mg为主要指标。课题组在既往研究中发现高DNA的ACTM体内炎症反应轻微，提示DNA可能不是评判脱细胞彻底性的唯一指标。在此基础上，课题组①完成不同DNA/脂质/内毒素残留量的ACTM体内外免疫原性、组织修复效果研究，确认残留内毒素与ACTM的免疫原性密切相关；②完成DNA残留标准的建立，确认DNA含量低于500ng体内外免疫反应低、组织修补效果好；③确认适合于ACTM的内毒素检测方法：选用磷酸酯盐溶液进行浸提同时检测介质选择脱氧胆酸、十二烷基硫酸钠、Pyrosperse（Lonza）合并Ca离子或镁离子，可以有效增加浸提效果并避免假阴性的结果。④研究成果已发表研究论文3篇，另有2篇SCI已投稿。申请国家发明专利3项。主办、参加多次国际、国内学术会议交流。项目组负责人晋升正高级技术职称，获批国防科技卓越青年科学基金项目、国防科技基础加强计划、交通战备科研项目、应用推进计划，入选上海市东方学者，培养博士生1名、硕士生2名。