摘要
目的建立体外小肠类器官培养体系,探讨脂多糖(LPS)对小肠类器官生长和炎性因子分泌的影响。方法体外无菌分离并收集C57BL/6小鼠小肠隐窝细胞团,使用类器官基质胶进行包埋,并在完全培养基的支撑下,培养形成具有小肠上皮样结构的立体多叶结构的小肠类器官。小肠类器官培养5~7 d或类器官中央区域变黑时传代,传代后3 d将小肠类器官随机分为不同质量浓度LPS组(0、150、175、200、225、250、275、300 mg/L)。在LPS诱导24 h和48 h后观察小肠类器官生长和形态特征变化;用CCK-8法检测不同时间和质量浓度LPS对小肠类器官诱导炎症后增殖活力的影响;采用酶联免疫吸附试验检测4种不同质量浓度LPS(0、175、200、225 mg/L)在不同时间对类器官培养上清液中粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素(IL)-1α、IL-6、IL-10水平的影响。结果初步构建了小鼠小肠类器官培养体系。不同时间和质量浓度LPS作用小肠类器官诱导炎症后,通过形态学观察到小肠类器官会有不同程度的膨胀和内腔凋亡现象的发生,受损的肠上皮中隐窝或肠干细胞的增殖分化和出芽数量也受到不同程度的抑制。小肠类器官在175~225 mg/L的LPS诱导24 h和48 h后,其增殖活力呈现不同程度的降低(P<0.05),但细胞活力仍大于50%。200 mg/L和225 mg/L的LPS诱导24 h和48 h后,IL-1α、IL-6和GM-CSF水平部分升高(P<0.05);200 mg/L的LPS诱导24 h和48 h后,IL-10水平降低(P<0.05)。结论本研究初步构建了不同质量浓度和时间的LPS诱导小肠类器官体外肠道炎症损伤模型,为今后肠道疾病的机制研究和有效药物的筛选提供了更为可靠的研究平台。
Objective To establish in vitro the small intestinal organoid culture system and to investigate the effect of lipopolysaccharide(LPS)on the growth of small intestinal organoids and the secretion of inflammatory factors.Methods In vitro,the small intestinal crypt cell mass of C57BL/6 mice was aseptically isolated,collected and embedded in organoid matrix.Under the support of complete medium,the small intestinal organoids with three-dimensional multi-leaf structure with small intestinal epithelioid structure were formed.The small intestinal organoids were subcultured after 5-7 d culture.On the third day after passage,the small intestinal organoids were randomly divided into different mass concentrations of LPS groups(0,150,175,200,225,250,275 and 300 mg/L).After 24 h and 48 h of LPS induction,morphological changes of small intestinal organoid growth and differentiation were observed.CCK-8 method was used to detect the effect of different time points and mass concentrations of LPS on the proliferative activity of small intestinal organoids after induction of inflammation.The effects of four different mass concentrations of LPS(0,175,200 and 225 mg/L)on expression levels of granulocyte-macrophage colony stimulating factor(GM-CSF),interleukin(IL)-1α,IL-6 and IL-10 in organoid culture supernatant at different times were detected by enzyme-linked immunosorbent assay(ELISA).Results The mouse small intestinal organoid culture system was preliminarily constructed.After different time and mass concentration of LPS induced inflammation of small intestinal organoids,it was observed by morphology that small intestinal organoids would have different degrees of expansion and apoptosis in lumen.The proliferation,differentiation and budding of damaged intestinal epithelial crypts or intestinal stem cells were also inhibited to varying degrees,indicating that the growth of small intestinal organoids would be limited to varying degrees after induced inflammation.The proliferation activity of small intestinal organoids decreased to
作者
陈浩
李蕊
易菲
周丽
陈嘉琪
朱璠
管城艳
吴娜
CHEN Hao;LI Rui;YI Fei;ZHOU Li;CHEN Jiaqi;ZHU Fan;GUAN Chengyan;WU Na(Department of Pharmacy,Jiangxi University of Chinese Medicine,Nanchang 330004,China;Department of Pharmacy,Affiliated Hospital of Jiangxi University of Chinese Medicine;College of Computing,Jiangxi University of Chinese Medicine;Clinical Medical College,Jiangxi University of Chinese Medicine;The First Department of Digestive,Affiliated Hospital of Jiangxi University of Chinese Medicine)
出处
《天津医药》
CAS
2024年第1期16-21,共6页
Tianjin Medical Journal
基金
国家自然科学基金资助项目(82360966,82160903)
江西省中医药科技计划重点项目(2023Z009)
江西省中医药中青年骨干人才(第四批)培养计划(赣中医药科教字[2022]7号)。
关键词
小肠
类器官
脂多糖类
生物因子
隐窝
intestine,small
organoids
lipopolysaccharides
biological factors
crypt
