新生鼠坏死性小肠结肠炎肠道菌群变化及意义

Change of intestinal flora in neonatal rat model of necrotizing enterocolitis

目的探讨新生鼠坏死性小肠结肠炎(NEC)时肠道菌群变化,旨在阐明肠道菌群在NEC发病中的作用,为探讨新生儿NEC的发病机制及寻求有效的防治措施提供理论依据。方法SD新生大鼠出生48 h开始给予鼠乳代用品人工喂养,100%氮气缺氧90 s,4℃冷刺激10 min,每天2次,连续3 d,建立新生SD大鼠NEC模型。40只新生SD大鼠随机分成NEC模型组(A组)和正常对照组(B组)。每组动物各20只。在最后一次缺氧、冷刺激后24 h空腹断头处死大鼠,留取回盲部近端肠管组织进行肠组织损伤评分,组织学评分≥2确定为NEC;实验前后留取两组新生鼠粪便,按照张秀荣方法进行肠道菌群检测。实验前后比较采用配对t检验,组间比较采用方差分析,α=0.05为显著性检验标准。结果模型组新生鼠相继出现腹泻、腹胀、萎靡、活动减少,生长减慢,对照组新生大鼠进食及排便均正常,无腹胀及胃潴留,活动度良好,皮下脂肪丰满。模型组新生大鼠NEC的发生率为100%(20/20),对照组无1例发生NEC。实验组和对照组肠损伤病理评分(x±s)分别为:3.25±0.85、0.45±0.51,t=12.622,P<0.01。模型组和对照组新生鼠实验前肠道细菌总数,杆菌、球菌总数,G+杆菌、G+球菌,G-杆菌、G-球菌数差异均无显著性,肠道菌群中杆菌和球菌的比例都在正常范围中。实验结束时,正常对照组新生大鼠肠道菌群总数明显增多,其中以G+杆菌增加为主;G-杆菌属及G+球菌属菌数占肠道菌群的比率在实验前后差异无显著性;模型组新生大鼠肠道群总数亦明显增多,而且明显高于正常对照组。实验结束时肠道菌群数量,其中主要是G+球菌显著增加,而G+杆菌却较对照组明显减少,与实验前相比,明显下降,差异有显著性;模型组实验结束时肠道菌群中杆菌和球菌比值减少、倒置。结论NEC发病前正常肠道菌群已发生质和量的变化,肠道菌群紊乱在NEC发病机制中起关键作用;及时纠正肠道菌群紊乱可能会降低新生大鼠发生NEC危险性。

Objective To investigate the change of intestinal flora and probe the effect of it on neonatal neerofizing enteroeolitis. Methods Forty neonatal rats were randomly divided into model group and normal control group （ n = 20 rats each group）. NEC model was established as follows：separate from mother rats and feeding with rat milk substitute, hypoxia （ 100% N2） for 90 and 4℃ cold exposure for 10 min, twice a day during 3 consecutive days. On the 4th day all the subjeets were sacrificed and intestinal tissues were obtained to examine. The intestine around ileoeeeal junction was for histolog- ical analysis and score. The histopathologieal changes were scored as 0 （normal villi）, 1 （epithelial cell sloughing） ,2 （midvillous necrosis）,3 （ complete villous necrosis） , or 4 （ transmural necrosis） in double-blind way. The mean score more than 2 were considered NEC. The intestinal flora in stools of neonatal rats were measured by Zhang Xiu Rang method. Match t test and ANOVA were used to analyze difference between two groups, α = 0.05 was considered significant. Results NEC model group appeared diarrhea, abdominal distension, dispirited, activating lessen and growth reducing. Control group showed norreal diet and stool, no abdominal distention and stomach storing up, good activity, subcutaneous fat well. Incidence of NEC in experimental group was 100% （20/20） and no ease in control group. The scores of histological evaluation（x±s） in experimental group and control group were 3.25± 0.85, 0.45 ±0.51, respectively （ t = 12. 622, P 〈 0.01 ）. The amount of total intestinal bacterial, total bacillus and coccus, G^＋ bacillus, G^- coccus, G^＋ bacillus and G^- coccus were no significantly different before the experiment between model group and control group, respectively. The proportion of bacillus and coccus in the intestinal flora was within the normal range. Experiment finishing, the intestinal flora total number of neonatal rats in control group increased. The ratio of G^- bacillus accounting for intestinal flora was no marked different with that of G ^＋ coccus accounting for intestinal flora before and after experiment. The intestinal flora total number of model group neonatal rats not only increased significantly,but also more higher than that of control group when experiment finishing,in which G^＋ coccus increased significantly and G^＋ bacillus decreased significantly compared with that of control group, it declined significantly compared with that of before experiment. There were significantly different among them. The ratio of bacillus to coccus in the intestinal flora decreased or inverted after experiment in model group. Conclusions The quantity and quality of the intestinal flora had been changed before NEC came on. The intestinal flora disorder probably play a key role at the development of NEC. Promptly correcting the disorder of intestinal flora may decrease the danger of NEC.