神经化肠黏膜组织工程模型的构建及体外评价  

Construction and evaluation of a neuralized intestinal mucosal tissue engineering model in vitro

作　　者：王明琦 冯诗雅 韩银河 于朋鑫 郭丽娜 贾子萱 王秀丽[1] Wang Mingqi;Feng Shiya;Han Yinhe;Yu Pengxin;Guo Lina;Jia Zixuan;Wang Xiuli(Dalian Medical University,Dalian 116044,Liaoning Province,China)

机构地区：[1]大连医科大学,辽宁省大连市116044

出　　处：《中国组织工程研究》2026年第4期892-900,共9页Chinese Journal of Tissue Engineering Research

基　　金：国家重点研发计划-政府间国际合作项目(2019YFE0117700),项目负责人:王秀丽。

摘　　要：背景:组织工程化肠道模型的体外构建在肠道再生与肠道疾病研究中均扮演重要角色。肠道神经系统和肠上皮屏障相互作用共同维持机体稳态,是组织工程化肠道仿生构建中的研究热点。目的:体外构建可模拟体内肠道神经系统的仿生模型。方法:以带有绒毛结构的丝素蛋白为支架,待人诱导神经干细胞与胶原固化后(培养7 d)加入肠上皮细胞(Caco-2细胞和HT29-MTX-E12细胞)培养3 d,构建肠道上皮细胞与神经细胞共培养体系(肠上皮细胞-人诱导神经干细胞共培养组),同时将单独培养的人诱导神经干细胞或肠上皮细胞接种于丝素蛋白支架设为对照。通过扫描电镜与苏木精-伊红染色观察细胞形态,Live/Dead细胞活性染色检测细胞活性,β-微管蛋白免疫荧光染色检测人诱导神经干细胞分化情况,微绒毛蛋白、蔗糖酶-异麦芽糖酶、紧密连接蛋白1、E-钙黏蛋白及黏蛋白2免疫荧光染色检测肠上皮组织学特性与屏障功能,阿利新蓝染色检测肠上皮细胞分泌黏液的功能,碱性磷酸酶染色检测肠上皮细胞的分化情况,同时进行蔗糖酶-异麦芽糖酶、紧密连接蛋白1及碱性磷酸酶RT-qPCR检测。结果与结论:实验成功构建了带有绒毛结构的神经化肠黏膜共培养模型,丝素蛋白支架上的神经干细胞和肠上皮细胞均具有良好的细胞活性。神经化后的肠上皮细胞蔗糖酶-异麦芽糖酶、碱性磷酸酶含量增多,同时紧密连接蛋白1表达水平上调。结果表明,神经化的仿生肠上皮模型有利于肠黏膜上皮细胞的功能成熟和屏障功能形成。BACKGROUND:In vitro construction of tissue-engineered intestinal models plays an important role in intestinal regeneration and intestinal disease research.The interaction of intestinal nervous system and intestinal epithelial barrier to maintain body homeostasis is a hot topic in the bionic construction of tissueengineered intestinal tract.OBJECTIVE:To construct a bionic model that can mimic the enteric nervous system in vivo.METHODS:Using fibroin protein with villus structure as scaffold,human induced neural stem cells solidified with collagen were added to intestinal epithelial cells(Caco-2 and HT29-MTX-E12)for 3-day culture to construct a co-culture system of intestinal epithelial cells and nerve cells(co-culture group).Human induced neural stem cells or intestinal epithelial cells cultured alone that were inoculated with fibroin scaffolds were set as controls.Cell morphology was observed by scanning electron microscopy and hematoxylin-eosin staining.Cell activity was detected by Live/Dead cell staining.Human induced neural stem cell differentiation was detected byβ-microtubulin immunofluorescence staining.Intestinal epithelial histological properties and barrier function were detected by microvillin,sucrase-isomaltase,tight junction protein 1,E-calmodulin,and mucin-2 immunofluorescence staining.The function of mucus secretion from intestinal epithelial cells was detected by Alcian blue staining.Alkaline phosphatase staining was performed to detect differentiation of intestinal epithelial cells,at the same time,sucrase-isomaltase,tight junction protein 1,and alkaline phosphatase mRNAs were detected by RT-qRCR.RESULTS AND CONCLUSION:The neuralized intestinal mucosal co-culture model with villi structure was successfully constructed,and neural stem cells and intestinal epithelial cells on the fibroin scaffold showed good cellular activities.After neuralization,the activity of alkaline phosphatase and sucrase-isomaltase in intestinal epithelial cells was enhanced,while the expression level of tight junction prot

关 键 词：肠黏膜 组织工程 肠道神经系统 肠上皮细胞 工程化组织构建 

分 类 号：R459.9[医药卫生—治疗学] R319[医药卫生—临床医学] R329