脐带间充质干细胞对新生小鼠坏死性小肠结肠炎的保护作用

Protective effects of umbilical cord mesenchymal stem cells on necrotizing enterocolitis in neonatal mice

目的 探讨脐带间充质干细胞(umbilical cord mesenchymal stem cells, UC-MSCs)对坏死性小肠结肠炎(necrotizing enterocolitis, NEC)小鼠模型肠组织的保护作用及可能的分子机制。方法 选取新生C57BL/6小鼠,采用高渗配方喂食、缺氧和冷刺激的方法,构建NEC小鼠模型,随机分为对照(母乳喂养)组、坏死性小肠结肠炎(NEC+PBS)组和间充质干细胞治疗(NEC+MSC)组,每组10只。UC-MSCs干预后,进行肠道宏观评估及HE染色观察肠组织病理形态学变化,免疫荧光染色检测上皮细胞增殖标志物KI-67和中性粒细胞浸润标记物髓过氧化物酶(myeloperoxidase, MPO)的表达;实时荧光定量PCR检测肠干细胞标记物富含亮氨酸的G蛋白偶联受体5(leucine-rich repeat containing G protein-coupled receptor 5,Lgr5)、炎症指标白介素6(interleukin 6,IL-6)和肿瘤坏死因子(tumor necrosis factor, TNF-α)的mRNA表达。结果 UC-MSCs经腹腔注射给药后,NEC发生率由50%降低为20%(P<0.05);肠道损伤、小肠宏观学评分、组织病理学评分均低于NEC+PBS组,差异均有统计学意义(P=0.048 0和0.024 3,P均<0.05);肠道上皮细胞增殖标记物KI-67阳性细胞数量及肠道干细胞标记物Lgr5基因表达量较NEC+PBS组均增加,差异均有统计学意义(P=0.012 9、0.030 4,P均<0.05);肠道中性粒细胞浸润指标MPO、炎症指标IL-6和TNF-α较NEC+PBS组均降低,差异均有统计学意义(P=0.020 1、0.000 5和0.001 5,P均<0.05)。结论 UC-MSCs可通过增加肠道上皮细胞增殖、恢复肠道干细胞活性和减少炎症因子来减轻NEC小鼠肠道损伤,具有较好的保护作用。

Objective To investigate the molecular mechanism and protective effect of umbilical cord mesenchymal stem cells(umbilical cord mesenchymal stem cells, UC-MSCs) on necrotizing enterocolitis(necrotizing enterocolitis, NEC) in neonatal mice. Methods Neonatal C57BL/6 mice were selected to develop NEC mouse model by using hypertonic formula feeding, hypoxia and cold stimulation. 30 mice were randomly divided into three groups(10 mice in each group): control group(breast feeding),necrotizing enterocolitis group(NEC+PBS) and mesenchymal stem cell therapy group(NEC+MSC). After treatment with UC-MSCs, the pathomorphological changes in intestine were evaluated by HE staining. The expression of KI-67,a marker of epithelial cell proliferation, and myeloperoxidase(MPO),a marker of neutrophil infiltration, were detected by immunofluorescence staining;and the mRNA expression of leucine-rich repeat containing G protein-coupled receptor 5(Lgr5),interleukin 6(IL-6) and tumor necrosis factor(TNF-α) were detected by real-time fluorescence quantitative PCR. Results After UC-MSCs administration by intraperitoneal injection, the incidence of NEC decreased from 50% to 20%(P <0.05);the intestinal injury, macroscopic score and histopathological score were lower than the control group,(P=0.048 0 and 0.024 3,P<0.05). The number of KI-67 positive cells and Lgr5 gene expression increased significantly compared with the control group(P=0.012 9 and 0.030 4,P<0.05);MPO,IL-6 and TNF-α were significantly reduced compared with the control group(P=0.020 1,0.000 5 and 0.001 5,P<0.05). Conclusion UC-MSCs can reduce intestinal injury by promoting the proliferation of intestinal epithelial cells, modulating the activities of intestinal stem cells and reducing inflammation in NEC mice.