目的 探讨新生儿坏死性小肠结肠炎(NEC)炎症损伤与肠道微生态-LPS-TLR4通路之间的关系。方法 本研究收集2019年3月1日—2021年1月31日在中山市人民医院新生儿监护室确诊为NEC新生儿11例为实验组,随机选取30 例同期在新生儿科病房住院喂养顺利,排除NEC及败血症诊断的新生儿为对照组。采集2组新生儿的粪便标本,进行Real-time PCR表达谱分析2组粪便肠道菌群;取2组外周静脉血检测外周血单核细胞Toll样受体4(TLR4)和血清PCT、CRP、IL-6、SAA等指标,对比2组肠道菌群、外周血单核细胞TLR4和炎症指标水平,通过统计学分析组间差异。结果 本研究结果提示实验组变形菌门占82%(9/11),厚壁菌门占9%(1/11),放线菌门占9%(1/11),对照组变形菌门占20%(6/30),厚壁菌门占73%(22/30),放线菌门占7%(2/30),2组患儿的粪便肠道菌群分布有差异(χ²=11.521,P<0.05);实验组患儿外周血单核细胞TLR4水平高于对照组,组间差异有统计学意义(P<0.001);实验组患儿血清PCT、CRP、IL-6和SAA等炎症指标高于对照组,组间差异有统计学意义(P<0.001)。结论 NEC患儿的肠道菌群以变形菌门为主,伴外周血单核细胞TLR4和外周血炎症指标升高。可见,肠道微生态-LPS-TLR4通路可能与新生儿坏死性小肠结肠炎炎症损伤相关,具体的机制仍需进一步深入研究。

Objective To investigate the relationship between intestinal flora-LPS-TLR4 pathway and the inflammatory injury of neonatal necrotizing enterocolitis (NEC). Methods Eleven neonates with NEC from March, 2019 to January, 2021 were enrolled as the experimental group, and 30 neonates without NEC and septicemia who were admitted in the department of neonatology in the same period were included as the control group. Faecal flora from the two groups were collected and analyzed by Real-time PCR. Toll-like receptor 4 (TLR4) and serum PCT, CRP, IL-6, SAA in peripheral blood were measured. The intestinal flora, the expression of TLR4 in peripheral blood leukocytes and inflammatory markers were compared between two groups. Results It showed that the ratio of Proteobacteria was 82% (9/11), Firmicutes was 9% (1/11), Actinobacteria was 9% (1/11) in the experimental group. In the control group, the ratio of Proteobacteria was 20% (6/30), Firmicutes was 73% (22/30), Actinobacteria was 7% (2/30). There was a significant difference in the distribution of faecal flora between the two groups (χ² = 11.521, P<0.05), and the level of TLR4 in peripheral blood of the experimental group was significantly higher than that of the control group (P＜0.001). The levels of serum PCT, CRP, IL-6 and SAA in the experimental group were significantly higher than those in the control group (P＜0.001). Conclusions The main intestinal flora of neonates with NEC is Proteobacteria, with elevated TLR4 expression and inflammatory markers in peripheral blood. Therefore, the intestinal flora-LPS-TLR4 pathway may be associated with inflammatory injury in neonatal necrotizing enterocolitis.The specific mechanism still needs further study.

目的 观察表皮生长因子(EGF)在新生儿坏死性小肠结肠炎(NEC)患儿肠组织中的动态表达情况,探讨EGF在NEC病程中起到的保护作用。方法 选取15例NEC患儿行一期回肠造瘘手术治疗的回肠组织为实验组(NEC组),将以上15例NEC患儿行二期回肠封瘘手术治疗的回肠组织为对照组(封瘘组),采用免疫组织化学技术检测,通过光密度测算软件(IPP)分析回肠组织中的EGF表达。结果 EGF主要表达于肠壁黏膜层,少量表达于黏膜下层、肌层。EGF在NEC组各层表达平均光密度值为:黏膜层(0.241±0.075),黏膜下层(0.213±0.061),肌层(0.1397±0.026),差异有统计学意义(P＜0.05);在封瘘组各层表达情况为:黏膜层(0.211±0.028),黏膜下层(0.119±0.022),肌层(0.097±0.007),差异有统计学意义(P＜0.05)。EGF在NEC组总体表达平均光密度值为(0.198±0.071),明显高于封瘘组(0.146±0.058),差异有统计学意义(P＜0.05)。结论 表皮生长因子(EGF)在新生儿坏死性小肠结肠炎(NEC)肠组织中的表达较封瘘组显著上调,推测EGF可能与NEC炎症相关,可能在NEC炎症过程中起到了一定的保护作用。

Objective We realized that EGF could play an important protective role against NEC. However, the practical condition of the distribution and expression of EGF in intestine of infants with NEC was indefinite. In order to figure out this problem,we carried out this experimentation. Methods The sample were divided into two group.The experimental group(necgroup) were composed of fifteen individual intestinal tissues after the ileostomy were performed on those infants suffered from NEC. The control group(sealing fistula group) were composed of fifteen individual intestinal tissues after the ileal closure fistula were performed on the same infants who were accepted the one-stage ileostomy in the period of NEC and were later accepted the two-stage operation on the condition that their bodies almost recovered from NEC after two to three months gone.Then, we utilized immunohistochemistry to test the distribution and quantities of EGF on those samples of the two group infants. Results The characteristic of EGF expression in intestine of the both group included strong positive expression in mucous layer and less expression in strata submucosum and muscular coat. The average optical density in nec group was mucous layer (0.241±0.075),strata submucosum(0.213±0.026),muscular coat (0.1397±0.022);In the control groupmucous layer (0.211±0.028),strata submucosum (0.119±0.022),muscular coat (0.097±0.007). The expression of EGF in intestinal tissues increased in the period of NEC0.198±0.071 by comparing with the control group (0.146±0.058). Conclusion There may be a correlation between the strong positive expression of EGF in intestinal tissues in the period of NEC and inflammation.By combining the result of this experiment and the research about EGF. We assumed that EGF is one factor of the protective mechanism by which injured intestinal mucous could be recovered and resist inflammation.