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

doi:10.12307/2025.960

Abstract

Abstract: 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 tissue-engineered 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 by β-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 protein 1 was up-regulated. To conclude, the neuralized bionic intestinal epithelial model is beneficial to the maturation of intestinal mucosal epithelial cells and the formation of barrier function.

Key words: intestinal mucosa, tissue engineering, enteric nervous system, intestinal epithelial cells, engineered tissue construction

CLC Number:

Wang Mingqi, Feng Shiya, Han Yinhe, Yu Pengxin, Guo Lina, Jia Zixuan, Wang Xiuli. Construction and evaluation of a neuralized intestinal mucosal tissue engineering model in vitro[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(4): 892-900.

Figures/Tables 4

展呈多角形，培养至第3天细胞紧密排列形成铺路石状；单独培养的人诱导神经干细胞和与肠上皮细胞共培养的人诱导神经干细胞均有突触伸出，见图3B。进一步采用免疫荧光染色检测共培养基条件下，人诱导神经干细胞中β-微管蛋白表达，结果显示人诱导神经干细胞分化为β-微管蛋白阳性表达的神经元，并可见梭形或圆形的神经元胞体和细长的突触，见图3C。上述结果说明，在共培养基条件下，3种细胞生长、神经干细胞分化状态良好，最终确定共培养基为神经分化培养基与DMEM培养基等体积混合。 2.3 肠黏膜上皮-神经细胞共培养模型的形态学表征将人诱导神经干细胞接种于支架表面，倒置相差显微镜下可见人诱导神经干细胞分化，并且在胶原内伸出突触，培养至第5天时突触逐渐变长交织形成神经网络，并且随着培养时间的增加，人诱导神经干细胞突触增多且增长(图4A)。将人诱导神经干细胞培养至第7天，加入Caco-2和HT29-MTX-E12细胞共培养3 d，可见肠上皮细胞在人诱导神经干细胞上密集排列，形成连续完整的单层(图4A)。进一步对接种于丝素蛋白支架中的细胞进行形态学表征。扫描电镜观察结果显示，单独人诱导神经干细胞培养组和肠上皮细胞-人诱导神经干细胞共培养组中的人诱导神经干细胞形成突触，共培养组中肠上皮细胞呈鹅卵石状，沿支架增殖并延拓扑结构迁移(图4B-i)。苏木精-伊红染色显示，单独人诱导神经干细胞培养组人诱导神经干细胞生长于胶原层内，肠上皮细胞-人诱导神经干细胞共培养组中肠上皮细胞增殖形成单层覆盖于胶原层之上(图4B-ii)，模拟人体生理状态下黏膜下神经丛和肠上皮细胞的解剖学位置。进一步采用Live/Dead细胞活性染色检测两组中的细胞活性，结果显示3种细胞活性良好(绿色荧光标记)，少见凋亡细胞(红色荧光标记)，并且人诱导神经干细胞形成细长的突触(图4B-iii)。为了进一步评估人诱导神经干细胞的分化情况，对单独人诱导神经干细胞培养组和肠上皮细胞-人诱导神经干细胞共培养组进行β-微管蛋白免疫荧光染色，结果显示两组中神经元均相互连接形成神经网络，共培养组中神经元突触数目更多且更细长(图4B-iv)，提示与肠上皮细胞共培养可能促进神经元的分化。以上结果表明，实验成功构建了小肠黏膜下神经丛模型，肠上皮细胞和人诱导神经干细胞生长状态良好，并且人诱导神经干细胞可分化为β-微管蛋白阳性表达的神经元。 2.4 肠黏膜上皮-神经细胞共培养对上皮细胞的影响为明确与人源诱导神经干细胞共培养后，肠上皮细胞功能的改变，结果见图5。采用免疫荧光染色检测单独肠上皮细胞培养组与肠上皮细胞-人诱导神经干细胞共培养组肠上皮细胞中微绒毛蛋白、蔗糖酶-异麦芽糖酶、紧密连接蛋白1、E-钙黏蛋白以及杯状细胞的标志性黏蛋白2表达情况，见图5A。结果显示，两组均能表达微绒毛蛋白、蔗糖酶-异麦芽糖酶、紧密连接蛋白1、E-钙黏蛋白和黏蛋白，但相较于单独肠上皮细胞培养组，肠上皮细胞-人诱导神经干细胞共培养组中紧密连接蛋白1蛋白表达增加。紧密连接蛋白1检测能够反映肠上皮细胞的屏障功能，其表达越高说明肠上皮细胞屏障功能越完整。碱性磷酸酶是一种表达于肠纹状缘的酶，可水解单磷酸盐，用于评估肠上皮细胞分化过程[15]。蔗糖酶-异麦芽糖酶为肠上皮细胞中负责将蔗糖和异麦芽糖分解为葡萄糖和麦芽糖的关键酶，该酶表达升高有助于肠上皮细胞功能成熟。RT-qPCR检测结果显示，肠上皮细胞-人诱导神经干细胞共培养组中紧密连接蛋白1、蔗糖酶-异麦芽糖酶与碱性磷酸酶mRNA表达均高于单独肠上皮细胞培养组(P < 0.001，P < 0.01，P < 0.01)，见图5C。阿利新蓝染色结果显示，肠上皮细胞-人诱导神经干细胞共培养组肠上皮细胞具有分泌黏液的功能，见图5B-i。碱性磷酸酶染色结果显示，两组均具有碱性磷酸酶活性，其中肠上皮细胞-人诱导神经干细胞共培养组碱性磷酸酶表达强于单独肠上皮细胞培养组，见图5B-ii。以上结果说明，相较于单独肠上皮细胞培养组，肠上皮细胞-人诱导神经干细胞共培养模型可以有效促进肠上皮细胞紧密连接蛋白1的表达，形成更紧密的上皮屏障，并促进肠上皮细胞黏液分泌及肠黏膜功能成熟。"

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