不同方法制备大鼠肝脏脱细胞支架及其免疫原性研究  

The Preparation and Immunoreactivity Analysis of Rat Liver Bioscaffolds Decellularized by Three Different Protocols

作　　者：姜楠[1] 季茹[1] 王建锋[1] 韩炜[1] 李伟民[1] 曹阳[1] 王德盛[1] 窦科峰[1] 

机构地区：[1]第四军医大学附属西京医院肝胆胰脾外科,陕西西安710032

出　　处：《现代生物医学进展》2013年第5期844-849,共6页Progress in Modern Biomedicine

基　　金：国家高技术研究发展计划(863项目)(2012AA021005)

摘　　要：目的:肝脏脱细胞支架(decellularized liver bioscaffold,DLB)在组织工程研究中具有良好前景,但对DLB的免疫原性尚未见探讨。本研究利用不同方案制备大鼠DLB,检测支架成分和体内重塑反应,为制备出低免疫原性的DLB提供改进依据。方法:选取文献报道的三种方案(分别以SDS,Triton X-100和NP-40为主要洗脱成分),制备F344大鼠DLB,进行HE和Masson染色,检测DLB中DNA、氨基葡聚糖(GAG)以及羟脯氨酸(HYP)含量;再将DLB埋植于C57BL/6小鼠的背部皮下,于术后第3、7和14天取出后进行形态学观察和组织学评分。结果:三种方案均成功制备出符合脱细胞标准的大鼠DLB;形态学结果提示Triton X-100和NP-40方案较SDS方案能更好地保护肝脏超微结构;成分检测结果发现NP-40方案去除DNA的能力显著强于SDS和Triton X-100方案(P<0.05),而对GAG含量的洗脱作用最弱(P<0.05);异种体内埋置实验提示NP-40方案制备的DLB引起的免疫反应强度明显弱于SDS和Triton X-100方案,体内重塑结局评分显著高于SDS和Triton X-100方案。结论:与SDS和Triton X-100方案相比,NP-40方案能更有效地去除肝脏DNA,较好地保留GAG,诱导移植受体对DLB的良性重塑反应,为进一步的体内研究提供更优化的支架。Objective： Decellularized liver bioscaffold（DLB） holds great potential in tissue engineering.However,the published methods for DLB vary widely,and there has been no comparative study with regard to the host immune response in vivo.The objective of the present study was to characterize the rat DLB treated with different decellularization methods to determine their structural and biochemical properties for the optimal of DLB derivation.Methods： Livers of adult F344 rats were decellularized using three published methods based on sodium dodecyl sulfate（SDS）,Triton X-100 and nonyl phenoxylpolyethoxylethanol（NP-40） combined with enzymes.The obtained DLB were submitted to HE and Masson staining.The contents of residual DNA,GAG and HYP were determined.Following subcutaneous implantation into C57BL/6 mice,the DLB were collected and histological analysis on day 3,7 and 14.Results： All three methods succeed in the production of DLB that satisfied the current decellularization criteria.The histological results suggested that Triton X-100-and NP-40-based methods protected the liver ultrastructure better than SDS-based method.The results of composition analysis showed that NP-40-based method exhibited the greatest potential of DNA removal and GAG retention compared to SDS-and Triton X-100-based methods（P0.05）.The results of xenogeneic implantation of DLB suggested the DLB generated by NP-40-based method elicited lower level of host immune response and resulted in stronger remodeling results compared to other two methods.Conclusion： Compared to SDS-and Triton X-100-based methods,the rat DLB treated with NP-40-based method has less residual DNA content and more GAG content,and induces more positive host remodeling response following xenogeneic implantation.These data may provide an optimized DLB for further in vivo study.

关 键 词：脱细胞肝支架 肝组织工程 细胞外基质 免疫原性 

分 类 号：Q95-3[生物学—动物学] R318.14[医药卫生—生物医学工程]