健脾方药提取物对小鼠小肠类器官的干预

目的探讨健脾方药四君子汤多糖提取物、黄芪甲苷对小鼠小肠类器官的干预作用。方法取4~6周龄小鼠小肠约20 cm,经过清洗、剪切、消化,分离出小肠隐窝细胞团后,接种于含有多种细胞因子的基质胶中,在基质胶3D结构支撑下,培养形成具有小肠上皮样形态的立体多叶结构,即小肠类器官。光镜下观察小肠类器官的形态特征;采用免疫荧光染色后在激光共聚焦显微镜下观察E-钙黏蛋白(E-cadherin)的表达;传代2天后的小鼠小肠类器官分为3组:对照组、四君子多糖组(终浓度为100 mg/L)以及黄芪甲苷组(终浓度为10μmol/L),继续培养48 h后观察四君子汤多糖提取物、黄芪甲苷对小肠类器官出芽生长及增殖细胞核核抗原Ki-67表达。结果分离出的小鼠隐窝细胞团1天类似囊状,中心具有单个内腔;2~3天开始出芽;4~5天出芽增多,管腔结构进一步清晰,形成小肠类器官,初步建立了小肠类器官培养模式;四君子多糖组(100 mg/L)小肠类器官出芽数量较对照组明显增多(2.31±1.60vs 4.15±1.91,P<0.05);黄芪甲苷组较对照组中小肠类器官Ki-67表达明显增高。结论小肠类器官模型的构建为探讨肠黏膜修复的病理生理机制及药物干预提供了更完善的体外研究模型。四君子汤多糖提取物、黄芪甲苷的肠黏膜保护作用可能与其促进隐窝干细胞更新能力有关。

Intervention of Jianpi Recipe Extract on Small Intestinal Organoids in Mice

Objective To investigate the intervention effect of polysaccharide from Sijunzi Decoction and Astroglosides(ASTs)on small intestinal organoids in mice.Methods The small intestine of mice aged 4-6 weeks was taken out about 20 cm.After washing,shearing and digesting,the small intestinal crypt cells were isolated and inoculated into matrigel containing various cytokines.Under the support of matrix 3D structure,a three-dimensional multi-leaf structure having a small intestinal epithelial-like morphology,that is,a small intestinal organoid was formed.The morphological characteristics of small intestine organoids were observed under a microscope.The expression of E-cadherin was observed under confocal microscopy after immunofluorescence staining.After 2 days of passage,the small intestinal organoids of the mice were divided into three groups:control group,Sijunzi polysaccharide group(final concentration 100 mg/L)and ASTs group(final concentration 10μmol/L),and observed the effect of polysaccharide extract of Sijunzi decoction and ASTs on budding growth and expression of Ki-67 in small intestinal organoids.Results The isolated mouse crypt cells were similar to sacs in 1 day,with a single lumen in the center;sprouting began in 2 to 3 days;4 to 5 days increased budding,and the lumen structure was further clear,forming small intestinal organoids.The intestinal organoids culture model was initially established;the Sijunzi decoction polysaccharide extract group(100 mg/L)was co-cultured for 48 h,and the number of small intestine organoids sprouts was significantly increased compared with the control group(2.31±1.60vs.4.15±1.91,P<0.05);The expression of Ki-67 in the small intestine organoids was significantly higher in ASTs group than control.Conclusions The construction of the small intestinal organoids model provides a more completein vitro study model for exploring the pathophysio logical mechanisms of intestinal mucosal repair and drug intervention. The intestinal mucosal protective effect of Sijunzi Decoctionpolysaccharide extract and ASTs may be related to its ability to promote crypt stem cell renewal.