不同方法制备小肠黏膜下层脱细胞基质材料的对比研究  

Comparative study on different methods for preparing decellularized submucosal matrix materials of small intestine

作　　者：姬彦辉 燕淼恒 苏会芳 陈松峰 毛克亚[2] 郭晓东[3] 刘宏建[1] Ji Yanhui;Yan Miaoheng;Su Huifang;Cheng Songfeng;Mao Keya;Guo Xiaodong;Liu Hongjian(Department of Orthopedics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450002,China;Department of Orthopedics,the First Medical Center of the People’s Liberation Army General Hospital,Beijing 100039,China;Department of Orthopedics,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022,China)

机构地区：[1]郑州大学第一附属医院骨科医学部,郑州450002 [2]解放军总医院第一医学中心骨科医学部,北京100039 [3]华中科技大学同济医学院附属协和医院骨科,武汉430022

出　　处：《中华实验外科杂志》2024年第8期1789-1793,共5页Chinese Journal of Experimental Surgery

基　　金：河南省自然科学基金青年项目(212300410256)。

摘　　要：目的采用目前文献报道的3种制备小肠黏膜下层(SIS)的方法制备SIS, 并比较3种制备方法对SIS的脱细胞程度、结构、活性成分和生物相容性的影响。方法采用Badylak法、Abraham法和Luo法制备SIS。使用苏木精-伊红(HE)和4’, 6-二脒基-2-苯基吲哚(DAPI)染色及DNA含量检测其脱细胞是否完全、残余DNA量;蛋白组学检测其组成成分;扫描电镜检测其结构变化;并将3种SIS与骨髓间充质干细胞(BMSCs)共培养检测其细胞相容性。采用方差分析比较组间差异。结果 3种方法制备的SIS的HE和DAPI染色均未无肉眼可见的细胞核;Badylak法、Abraham法和Luo法制备的SIS中DNA含量检测分别为43.25、21.01 ng/mg和15.69 ng/mg ECM干重。蛋白组学分析结果:SIS-1、SIS-2和SIS-3中检测出的蛋白种类分别为644、717和135种。胶原蛋白的种类最多, 共有21个亚型, Ⅰ型胶原含量最大, 还含有纤维粘连蛋白(fibronectin)、蛋白多糖、血管内皮生长因子(VEGF)、成纤维细胞生长因子(FGF)、转化生长因子-β(TGF-β)和骨形态发生蛋白-2(BMP-2)。扫描电子显微镜显示3种SIS微观结构均有一定程度破坏, 表现为胶原纤维连续性中断与胶原纤维排列紊乱;与BMSCs共培养, 细胞能保持良好的形态和增殖活性。结论 3种脱细胞方法制备的SIS均达到标准, 具有良好的生物相容性, 并保留大部分活性成分。微观结构和生物活性成分都会受到不同程度的损伤。ObjectiveThree methods for preparing small intestinal submucosa(SIS)were applied to prepare SIS according to the current reported literatures.The effects of the three preparation methods on decellularization degree,structure,active ingredients and biocompatibility of SIS were compared.MethodsSIS was prepared using Badylak’s method,Abraham’s method and Luo’s method respectively.The degree of decellularization was detected with hematoxylin and eosin(HE)and 4′,6-diamidino-2-phenylindole(DAPI)and residual DNA content detected with standard method;Proteomic analysis was applied to detect components of SIS,and scanning electron microscopy to detect structural changes;And co-culture the three types of SIS with bone marrow mesenchymal stem cells(BMSCs)to test cell compatibility.Quantitative data was presented using an arithmetic mean±standard deviation table,and statistical significance was tested using analysis of variance.ResultsThe HE and DAPI staining of SIS prepared by three methods all showed no visible nuclei to naked eye;The DNA content detection in SIS prepared by Badylak’s,Abraham’s and Luo’s method were 43.25,21.01 and 15.69 ng/mg ECM dry weight,respectively.Proteomic analysis results:644,717,and 135 protein types were detected in SIS-1,SIS-2,and SIS-3,respectively.There are 21 subtypes of collagen with the most types.The content of Collagen I was the highest among all collagen types;and there were also fibronectin,proteoglycans,vascular endothelial growth factor(VEGF),fibroblast growth factor(FGF),transforming growth factor-β(TGF-β)and bone morphogenetic protein-2(BMP-2)within.SEM showed that the microstructures of all three types of SIS were damaged to a certain extent,with interrupted continuity of collagen fibers and disordered arrangement of collagen fibers;and BMSCs co-cultureed with all three types of SIS could maintained good morphology and proliferation activity.ConclusionThe SIS prepared by three decellularization methods all meet the standards,retain most of the active ingredients a

关 键 词：小肠黏膜下层 脱细胞基质 

分 类 号：R318.08[医药卫生—生物医学工程]