摘要目的:探讨外源性小肠类器官(intestinal organoid，IO)肠道内灌注对坏死性小肠结肠炎(necrotizing enterocolitis，NEC)新生小鼠肠道损伤的影响及相关机制，为探索NEC新型防治方法及临床转化提供依据。方法:取9日龄健康C57BL/6新生小鼠末端回肠组织体外培养IO，培养7 d后鉴定IO并将其重悬于磷酸盐缓冲溶液用于干预实验。选用5日龄C57BL/6新生小鼠，按照随机数字法随机分成4组：正常对照组(control组，仅母鼠母乳喂养，无其他干预措施)、实验对照组(control+IO组，母鼠母乳喂养，于生后第6天进行类器官干预)、NEC组(于生后第5至9天之间，诱导新生鼠NEC发生)、实验干预组(NEC+IO组，诱导NEC同时于生后第6天进行类器官干预)，每组5只小鼠。生后第9天收集各组新生小鼠末端回肠组织，分析比较各组小鼠肠道损伤程度，肠道炎症反应，肠道干细胞活性、肠上皮细胞增殖能力变化并进一步比较各组Wnt/β-catenin通路活性。计量资料多组间比较采用ANOVA检验，单因素方差分析后进一步两两比较使用LSD检验；计数资料多组比较采用Kruskal-Wallis非参数检验；生存分析采用Log-rank(Mantel-cox)检验。结果:与control组相比，NEC组小鼠累积存活率显著降低(NEC组比control组：100.00%比42.42%， P<0.001)，肠道组织病理评分[NEC组比control组：3(2.5~3)比0(0~0.5)， P<0.01]与IL-6 mRNA表达水平[NEC组比control组：(4.52±0.82)比(1.01±0.23)， P<0.001]均明显升高，而Lgr5 mRNA表达水平[NEC组比control组：(0.02±0.01)比(1.03±0.40)， P<0.001]、Ki-67 mRNA表达水平[NEC组比control组：(0.18±0.06)比(1.00±0.15)， P<0.001]显著下降，单个隐窝内Ki-67 +细胞个数[NEC组比control组：(3.17±2.04)比(9.17±1.47)， P<0.001]明显减少，β-catenin mRNA表达水平[NEC组比control组：(0.01±0.01)比(1.23±0.42)， P<0.05]及β-catenin蛋白活化水平(即非磷酸化β-catenin)[NEC组比control组：(0.14±0.04)比(0.92±0.41)， P<0.001]明显降低。而相较于NEC组，NEC+IO组小鼠累积存活率显著提高(NEC组比NEC+IO组：42.42%比69.84%， P<0.001)，NEC病理评分[NEC组比NEC+IO组：3(2.5~3)比0(0~1.5)， P<0.05]与IL-6 mRNA表达水平[NEC组比NEC+IO组：(4.52±0.82)比(1.04±0.18)， P<0.001]均显著降低，Ki-67 mRNA表达水平[NEC组比NEC+IO组：(0.18±0.06)比(0.37±0.10)， P<0.05]升高，单个隐窝内Ki-67 +细胞个数明显增加[NEC组比NEC+IO组：(3.17±2.04)比(12.67±1.51)， P<0.001]。此外，肠道β-catenin mRNA表达水平[NEC组比NEC+IO组：(0.01±0.01)比(2.44±1.19)， P<0.001]及β-catenin蛋白活化水平[NEC组比NEC+IO组：(0.14±0.04)比(0.54±0.25)， P<0.05]均升高。 结论:外源性IO可以缓解NEC肠道损伤，抑制肠道炎症反应，提高肠道内细胞增殖水平，改善肠道干细胞活性，Wnt/ β-catenin信号通路可能参与介导了上述IO干预作用。

abstractsObjective:To explore the effect and mechanism of exogenous small intestinal supplementation in a neonatal murine model of necrotizing enterocolitis(NEC)and to provide rationales for exploring novel methods for preventing and treating NEC.Methods:Terminal ileum of 9-day-old normal mice was selected for extracting intestinal crypt.Intestinal organoid was cultured in vitro for 7 days, identified and resuspended in PBS for intervention of organoid enema(2.5 organoids/μL of 50 μL organoid suspension instilled into the intestine of experimental mice through anal opening). Five-day-old C57BL/6 neonatal mice were randomized into four groups: 1)control group: neonates were fed by maternal mice without any other interventions; 2)experimental control group(control+ IO): neonates were fed by maternal mice and received organoid enema on postnatal day six; 3)NEC group: neonates were induced to NEC development from postnatal day five to nine; 4)experimental NEC group(NEC+ IO): besides stimuli of NEC modeling, intestinal organoid suspension was given to neonates on postnatal day six.Tissue from terminal ileum of each group was collected on postnatal day nine and utilized for comparing the extent of intestinal damage, intestinal inflammatory response, intestinal stem cell activity, intestinal epithelial cell proliferation capacity and Wnt/β-catenin pathway activity of each group.ANOVA test was employed for comparing measurement data between multiple groups and LSD test for further comparison with one-way ANOVA analysis.Kruskal-Wallis nonparametric test was performed for multi-group comparison of count data.Log-rank(Mantel-cox)test was performed for survival analysis.Results:As compared with control group, NEC group showed obviously lower survival rate(NEC vs control: 100% vs 42.42%, P<0.001), marked intestinal epithelial injury with higher NEC score[NEC vs control: 3(2.5~3)vs 0(0~0.5), P<0.01]and significantly higher expression level of IL-6 mRNA[NEC vs control: (4.52±0.82)vs(1.01±0.23), P<0.001]while Lgr5 mRNA[NEC vs control: (0.02±0.01)vs(1.03±0.40), P<0.001]and Ki-67 mRNA[NEC vs control: (0.18±0.06)vs(1.00±0.15), P<0.001]were down-regulated and number of Ki-67 + cell decreased markedly[NEC vs control: (3.17±2.04)vs(9.17±1.47), P<0.001]. The expression levels of β-catenin mRNA[NEC vs control: (0.01±0.01)vs(1.23±0.42), P<0.05]and active non-phosphorylated β-catenin protein decreased obviously[NEC vs control: (0.14±0.04)vs(0.92±0.41), P<0.001]. In contrast, as compared with NEC group, experimental NEC group showed that survival rate(NEC vs.NEC+ IO: 42.42% vs 69.84%, P<0.001)improved obviously while NEC score[EC vs NEC+ IO: 3(2.5~ 3)vs 0(0~1.5), P<0.05]and IL-6 mRNA[NEC vs NEC+ IO: (4.52±0.82)vs(1.04±0.18), P<0.001]dropped markedly.The expression level of Ki-67 mRNA[NEC vs NEC+ IO: (0.18±0.06)vs(0.37±0.10), P<0.05]and number of Ki-67 + cell[NEC vs NEC+ IO: (3.17±2.04)vs(12.67±1.51), P<0.001]were elevated.In addition, intestinal Wnt/β-catenin pathway activity was improved, including increased β-catenin mRNA[NEC vs NEC+ IO: (0.01±0.01)vs(2.44±1.19), P<0.001]and active β-catenin protein[NEC vs NEC+ IO: (0.14±0.04)vs(0.54±0.25), P<0.05]. Conclusions:Enema treatment of intestinal organoid can alleviate intestinal injury of neonatal mice with NEC through reducing the level of intestinal inflammation, promoting intestinal epithelial cell proliferation and intestinal stem cell activity.And Wnt/β-catenin signaling pathway may be involved in mediating the above effects of organoid intervention.