灌注法构建小肠黏膜全层去细胞外基质

背景：对于解剖层次复杂的细胞外基质,单纯浸泡法则很难达到理想的去细胞效果。目的：观察灌注法去细胞技术制备小肠黏膜全层去细胞外基质的效果。方法：采用灌注法去细胞技术,经成年雄性新西兰大白兔小肠黏膜上动脉灌注去离子剂后处理获得小肠黏膜全层去细胞外基质。采用MTT法检测小肠黏膜全层去细胞外基质浸提液(实验组)或含体积分数20%小牛血清的DMEM(对照组)与1 月龄新西兰大白兔骨髓间充质干细胞共培养后的细胞相对增殖率。结果与结论：①大体观察：小肠黏膜上动脉经灌注30 min 后变得苍白透明,经2 h 灌注后肠段变得苍白剔透,清楚可见脉管纹理。②组织学与扫描电镜观察：灌注法构建的小肠黏膜全层去细胞外基质的去细胞程度均匀,胶原纤维未受损,孔隙多,孔隙率为(86.72±2.98)%。③MTT 检测实验：培养2,4,7 d,实验组细胞A 值明显高于对照组细胞A值(P < 0.05),且实验组细胞相对增殖率均>1。表明采用灌注法构建小肠黏膜全层去细胞外基质可操作性强,安全性好,且小肠黏膜全层去细胞外基质对骨髓间充质干细胞无毒性,并有一定的促生长作用。

Reconstructing the perfused-decellularized full-thickness small intestine extracellular matrix

BACKGROUND:Immersing method is difficult to construct the ideal acellular matrix for the complex organ.OBJECTIVE:To investigate the effect of preparing the acellular full-thickness small intestine scaffold byperfusion method.METHODS:All the small intestines were excised in a sterile fashion in adult male New Zealand rabbits andthe acellular small intestine scaffolds were obtained by perfusing ionic detergents through the superiormesenteric arteries. Relative growth rates were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method after bone marrow mesenchymal stem cells from 1-month-old NewZealand rabbits were co-cultured with decellularized full-thickness small intestine extracellular matrix(experimental group) or Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum (control group).RESULTS AND CONCLUSION:The macroscopic view showed that the perfused-decellularized small intestineartery became translucent after 30 minutes of perfusion, and the small intestine segment became translucentafter 2 hours of perfusion. The vessels were seen distinctly. Histological view and scanning electron microscoperesults indicated that the perfused-decellularized full-thickness small intestine showed perfect acellular effect.Many pores and collagen fibers were retained and the porosity was (86.72±2.98)%. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that the relative growth rate of the experimental group overtopped1. The absorbance values in the experimental group were significantly higher than those in the control group at 2,4 and 7 days of coculture (P < 0.05). These findings suggest that the perfusion method is a better way toconstruct acellular full-thickness small intestine scaffold because it can achieve better acellular effect and retainscaffold vitality at the greatest extent in the acellular small intestine scaffold that can promote the growth of bonemarrow mesenchymal stem cells and not add toxicity to the cells.