Abstract: CHEN Hao，Physician，E-mail：mprmprvzdh@qq.com
Background　Bone marrow mesenchymal stem cells have a strong capacity of immunoregulation and anti-inflammation.Previous research identified that the conditioned medium derived from mesenchymal stem cells （MSC-CM） can downregulate various inflammatory responses and protect the small intestine.However，the effects and mechanisms of the MSC-CM in necrotizing enterocolitis（NEC） inflammation remain unknown.Objective　To investigate the role of MSC-CM with inflammatory pre-activation，in regulating NEC inflammatory response in an experimental newborn rat model of NEC.Methods　This study was carried out between July 2018 and April 2019.Eighty newborn SD rats（SPF grade） were randomly and equally divided into control group，NEC injury group，NEC injury+MSC-CMNOR group and NEC injury+MSC-CMTNF-α+IL-1β+NO group.DMEM-F12 medium，MSC-CMNOR and MSC-CMTNF-α+IL-1β+NO were given by intraperitoneal injection for the following 3 days to the NEC injury group，NEC injury+MSC-CMNOR group and NEC injury+MSC-CMTNF-α+IL-1β+NO group，respectively.The gross specimen of small intestine were observed，and the terminal ileum and blood samples were collected on the 4th day of intervention.H&E staining was used to observe the histological changes of the small intestine and determine the pathological scores.Inflammatory cytokines in small intestine tissue and serum samples were detected by ELISA.Results　Major changes in small intestine specimens：in the control group，the color of the tissue was ruddy and non-hyperemia with good elasticity，peristalsis，and no intestinal wall pneumatosis or necrosis.In both NEC injury and NEC injury+MSC-CMNOR groups，the color of the tissue was dull-red with distinct congestion，pneumatosis intestinalis，necrosis，low elasticity，and dilatation.In contrast，the NEC injury+MSC-CMTNF-α+IL-1β+NO group showed only mild congestion and fair elasticity，without pneumatosis intestinalis or necrosis.Intestinal histopathological score and incidence of NEC：the total intestinal histopathological scores for the control group，NEC injury group，NEC injury+MSC-CMNOR group and NEC injury+MSC-CMTNF-α+IL-1β+NO group were 16，51，43，and 16，respectively，showing a significant difference（H=41.70，P<0.01）.The control group showed lower mean intestinal histopathological score compared with NEC injury group and NEC injury+MSC-CMNOR group（P<0.01）.And NEC injury+MSC-CMTNF-α+IL-1β+NO group showed lower mean intestinal histopathological score compared with NEC injury group（P<0.01）.The incidence of NEC in the control group，NEC injury group，NEC injury+MSC-CMNOR group and NEC injury+MSC
-CMTNF-α+IL-1β+NO group was 0，85.0%（17/20），20.0%（4/20），and 80.0%（16/20），respectively，with a significant difference（χ2=44.00，P<0.01）.Mean levels of serum and intestinal pro-inflammatory and anti-inflammatory factors：the mean levels of serum TNF-α，IL-1β，IL-6，IL-8，IL-10 and IL-12 differed significantly across four groups（P<0.01）.The mean levels of intestinal TNF-α，IL-1β，IL-6，and IL-10 of four groups were significantly different（P<0.01）.Compared with the NEC injury group and NEC injury+MSC-CMNOR group，mean levels of serum TNF-α，IL-1β，IL-6，IL-8 and IL-12 decreased while mean level of serum IL-10 increased significantly in the NEC injury+MSC-CMTNF-α+IL-1β+NO group（P<0.01）；mean levels of intestinal TNF-α，IL-1β，IL-6  decreased while  mean level of intestinal IL-10 increased significantly in the NEC injury+MSC-CMTNF-α+IL-1β+NO group（P<0.01）.Conclusion　The conditioned medium from pre-activated MSCs has protective effects on the intestinal tract of newborn rat model of NEC，which can regulate the balance of inflammatory and anti-inflammatory factors and reduce intestinal damage.

Key words: Enterocolitis, necrotizing；Mesenchymal stem cells；Culture media, conditioned；Inflammation；Models, animal

摘要： 背景　骨髓间充质干细胞具有强大的免疫调节和抗炎能力。既往研究发现不同状态间充质干细胞条件培养基能下调多种小肠损伤炎性反应，对肠道起到保护作用，但对于坏死性小肠结肠炎（NEC）炎性反应的作用和机制尚未明确。目的　建立新生大鼠NEC模型，探讨炎症预激活间充质干细胞条件培养基调控NEC炎性反应的作用。方法　2018年7月—2019年4月，建立新生大鼠NEC模型，随机选取造模后的80只SPF级新生Sprague-Dawley（SD）大鼠，根据处理方式分为对照组、单纯NEC损伤组、NEC损伤+正常间充质干细胞条件培养基（MSC-CMNOR）组、NEC损伤+经肿瘤坏死因子α（TNF-α）、白介素（IL）-1β和一氧化氮（NO）联合培养诱导的炎症预激活间充质干细胞条件培养基（MSC-CMTNF-α+IL-1β+NO）组，每组20只。采用腹腔注射给药的方式，对照组、单纯NEC损伤组、NEC损伤+MSC-CMNOR组、NEC损伤+MSC-CMTNF-α+IL-1β+NO组分别给予等量的0.9%氯化钠溶液、DMEM-F12培养基、MSC-CMNOR及MSC-CMTNF-α+IL-1β+NO。在注射药物后第4天开腹观察小肠大体标本，收集回肠末端和血液标本，行苏木素-伊红（HE）染色观察小肠病理学变化并进行病理学评分，采用酶联免疫吸附试验（ELISA）对小肠组织及血清标本行炎性因子检测。结果　小肠大体标本变化：对照组新生大鼠小肠色泽红润，无充血，弹性好，有蠕动，未见肠壁积气或坏死。单纯NEC损伤组及NEC损伤+MSC-CMNOR组新生大鼠肠道色泽呈暗红色，充血明显，可见肠壁积气、坏死，弹性差，肠管扩张。NEC损伤+MSC-CMTNF-α+IL-1β+NO组新生大鼠肠道轻度充血，未见肠壁积气或坏死，弹性尚可。小肠组织病理学评分及NEC发生率：对照组、单纯NEC损伤组、NEC损伤+MSC-CMNOR组、NEC损伤+MSC-CMTNF-α+IL-1β+NO组病理学评分总分为16、51、43、16分；4组病理学评分比较，差异有统计学意义（H=41.70，P<0.01）。其中单纯NEC损伤组、NEC损伤+MSC-CMNOR组病理学评分高于对照组（P<0.01），NEC损伤+MSC-CMTNF-α+IL-1β+NO组病理学评分较单纯NEC损伤组降低（P<0.01）。对照组无NEC发生，单纯NEC损伤组、NEC损伤+MSC-CMTNF-α+IL-1β+NO组、NEC损伤+MSC-CMNOR组分别有17、4、16只诊断为NEC（NEC发生率为85.0%、20.0%、80.0%）；4组NEC发生率比较，差异有统计学意义（χ2=44.00，P<0.01）。4组血清和小肠组织中促炎因子和抑炎因子水平：4组血清中TNF-α、IL-1β、IL-6、IL-8、IL-10、IL-12水平比较，差异有统计学意义（P<0.01）；4组小肠组织中TNF-α、IL-1β、IL-6、IL-10水平比较，差异有统计学意义（P<0.01）。其中，与单纯NEC损伤组、NEC损伤+MSC-CMNOR组相比，NEC损伤+MSC-CMTNF-α+IL-1β+NO组血清中TNF-α、IL-1β、IL-6、IL-8、IL-12水平降低，IL-10水平升高，MSC-CMTNF-α+IL-1β+NO组小肠组织中TNF-α、IL-1β、IL-6水平降低，IL-10水平升高（P<0.01）。结论　炎症预激活间充质干细胞条件培养基对NEC新生大鼠的肠道具有保护作用，能够调控促炎因子和抑炎因子的平衡，减轻肠道的损伤。

关键词: 小肠结肠炎, 坏死性；间质干细胞；培养基, 条件性；炎症；模型, 动物