人源脱细胞羊膜与脱细胞羊膜下层组织修复皮肤缺损  

Human acellular amniotic membrane and acellular subamniotic membrane in repairing skin defects

作　　者：王丹 尹晶 王玉英 杜勇 李京玲 俞舜 Wang Dan;Yin Jing;Wang Yuying;Du Yong;Li Jingling;Yu Shun(Affiliated Hospital of Jiangnan University,Wuxi 214000,Jiangsu Provinee,China)

机构地区：[1]江南大学附属医院,江苏省无锡市214000

出　　处：《中国组织工程研究》2020年第34期5570-5576,共7页Chinese Journal of Tissue Engineering Research

基　　金：无锡市科学技术局项目(BE2018626),项目参与者:俞舜。

摘　　要：背景:羊膜与羊膜下层具有同源性且均属于医疗废物,均具有较低的免疫原性与一定的韧性,羊膜作为组织工程支架材料已有较多报道,但羊膜下层作为组织工程支架的研究较少。目的:对比脱细胞羊膜与脱细胞羊膜下层分别复合骨髓间充质干细胞用于皮肤修复的差异。方法:采用十二烷基硫酸钠+核酸酶法制作脱细胞羊膜下层材料,采用TritonX-100+胰酶法制作脱细胞羊膜材料。采用两种脱细胞材料浸提液分别培养骨髓间充质干细胞,利用CCK-8法检测两种脱细胞材料的细胞毒性。将骨髓间充质干细胞接种于2种脱细胞材料表面,光学显微镜与扫描电镜下观察细胞的生长与黏附。在每只SD大鼠一侧脊柱背部制作深达真皮层的皮肤缺损,将36只造模大鼠随机分为3组处理,每组12只:空白组不植入任何材料,对照组植入脱细胞羊膜与骨髓间充质干细胞复合物,观察组植入脱细胞羊膜下层与骨髓间充质干细胞复合物,术后7,14,21d分别进行创面愈合率、创面病理及创面基因与蛋白检测。动物实验获得江南大学动物伦理委员会批准。结果与结论:①在培养的3-7d内,两种脱细胞材料浸提液均可促进骨髓间充质干细胞的增殖;②光学显微镜与扫描电镜显示,骨髓间充质干细胞在两种脱细胞材料表面黏附、生长良好,细胞形态与培养瓶中培养的相同;③观察组与对照组术后各时间点的创面愈合率均高于空白组(P<0.05),且观察组高于对照组(P<0.05);④术后14 d的病理观察显示,观察组与对照组的皮肤修复优于空白组,且观察组优于对照组;⑤观察组、对照组术后各时间点的Ⅲ型胶原、CK18、Ⅰ型胶原与血管内皮生长因子基因表达量均高于空白组(P<0.05),且观察组高于对照组(P<0.05);⑥观察组、对照组各时间点的CK18、血管内皮生长因子蛋白表达均高于空白组(P<0.05),观察组高于对照组(P<0.05);⑦相较于脱细BACKGROUND:Amniotic membrane and subamniotic membrane are homologous and belong to medical waste.They have low immunogenicity and certain toughness.Amniotic membrane as tissue-engineered scaffold material has been reported more,but the research of subamniotic membrane as tissue-engineered scaffold is less.OBJCETIVE:To compare the difference between acellular amniotic membrane and acellular subamniotic membrane in combination with bone marrow mesenchymal stem cells for skin repair.METHODS:The acellular subamniotic membrane materials were prepared by sodium dodecyl sulfate+nuclease method,and the acellular amniotic membrane materials were prepared by Triton X-100+pancreatin method.Bone marrow mesenchymal stem cells were cultured with two kinds of acellular materials.The cytotoxicity of the two kinds of acellular materials was detected by CCK-8 method.Bone marrow mesenchymal stem cells were seeded on the surfaces of two kinds of acellular materials.The growth and adhesion of cells were observed under light microscope and scanning electron microscope.Skin defects deep into the dermis were made on the back of one spine of each Sprague-Dawley rat,and 36 rats were randomly divided into three groups(n=12).No materials were implanted in the blank group.Acellular amniotic membrane and bone marrow mesenchymal stem cell complex were implanted in the control group.Acellular subamniotic membrane and bone marrow mesenchymal stem cell complex were implanted in the observation group.Wound healing rate,wound pathology,wound gene and protein were detected at 7,14 and 21 days after operation,respectively.This study was approved by the Animal Ethics Committee of Jiangnan University.RESULTS AND CONCLUSION:(1)In the 3-7 days of culture,two kinds of acellular materials could promote the proliferation of bone marrow mesenchymal stem cells.(2)The results of light microscope and scanning electron microscope showed that bone marrow mesenchymal stem cells grew well on the surfaces of two kinds of acellular materials,and the morphology of bon

关 键 词：材料 脱细胞 羊膜 羊膜下层 创面 皮肤缺损 干细胞 大鼠 基因 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]