大鼠肠道平滑肌胶原条带构建及周期性拉伸培养的体外评价  

The construction of rat intestinal smooth muscle collagen band and evaluation of periodic stretching culture in vitro

作　　者：于朋鑫 韩禹秋 郭丽娜 王秀丽[1] Yu Pengxin;Han Yuqiu;Guo Lina;Wang Xiuli(Dalian Medical University,Dalian 116000,Liaoning Province,China)

机构地区：[1]大连医科大学,辽宁省大连市116000

出　　处：《中国组织工程研究》2024年第35期5630-5635,共6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:肠道平滑肌层是肠道管壁结构的重要组成部分,在组织工程化肠道的仿生构建中备受关注。目的:探索周期性机械拉伸对胶原条带内肠道平滑肌细胞生长活性以及功能基因表达的影响。方法:以自制鼠尾胶原为支架,以大鼠原代肠道平滑肌细胞为种子细胞,同时结合自主设计的胶原条带拉伸培养装置,构建肠道平滑肌细胞胶原条带培养体系,采用Eth D-1/Calcein-AM细胞活性染色、洋红染色、细胞骨架-Ki67免疫荧光染色观察细胞的生长活性与增殖情况,定量RT-PCR检测desmin、α-sma、vimentin功能基因的表达。结果与结论:(1)成功构建肠道平滑肌细胞胶原条带培养体系,条带内肠道平滑肌细胞具有良好的细胞活性;(2)与静态培养相比,周期性拉伸动态培养条件下Ki67阳性表达的细胞数量增多且显著高表达标志性基因desmin、α-sma、vimentin(P<0.001);(3)结果表明:机械力学刺激有利于肠道平滑肌细胞在体外培养过程中维持生长状态,并促进其功能性分化。BACKGROUND:The in vitro construction of intestinal smooth muscle layer,as an important component of the intestinal wall,has attracted much attention in the bionic construction of tissue-engineered intestinal canal.OBJECTIVE:To explore the effects of cyclic mechanical stretching on the growth activity of intestinal smooth muscle cells and the expression of functional genes within collagen strips.METHODS:The collagen band culture system of intestinal smooth muscle cells was constructed using a self-designed collagen strip stretching culture device with self-made rat tail collagen as a scaffold and primary rat intestinal smooth muscle cells as seed cells.EthD-1/Calcein-AM cell activity staining,magenta staining,cytoskeleton-Ki67 immunofluorescence staining were used to observe the growth activity and proliferation of the cells,and quantitative RT-PCR was used to detect the expression of desmin,α-sma,and vimentin functional genes.RESULTS AND CONCLUSION:The collagen band culture system of intestinal smooth muscle cells was successfully constructed,and intestinal smooth muscle cells in the band had good cell activity.The number of Ki67 positive cells increased and desmin,α-sma and vimentin were significantly overexpressed under cyclic stretching and dynamic culture conditions(P<0.001).To conclude,mechanical stimulation is beneficial to maintain the growth phenotype of smooth muscle cells and promote their functional differentiation during three-dimensional culture in vitro.

关 键 词：组织工程 肠道平滑肌 胶原条带 动态拉伸 胶原支架 三维培养 

分 类 号：R459.9[医药卫生—治疗学] R329.3[医药卫生—临床医学] Q811.9[生物学—生物工程]