冠心病人源肠道菌群小鼠模型的建立及评价

Establishment and evaluation of a mouse model of human gut microbiota transplanted from patients of coronary heart disease

目的通过粪菌移植方法建立冠心病人源肠道菌群(human flora-associated,HFA)小鼠模型并对模型进行评价。方法28只无菌雌性C57BL/6J小鼠分为对照(CON)组和模型(CAD)组,分别接种健康志愿者和冠心病患者新鲜粪便悬液,移植后6周和10周每组安乐7只动物,取粪便、血液、主动脉进行16S rDNA、血脂、细胞因子和组织病理学检查。结果从第5周开始CAD组动物体重增长加快(P<0.05)。α-多样性分析,CAD组的Simpson指数在两个时间点的数值均升高(P<0.05,P<0.01),Shannon指数(P<0.05,P<0.01)、ACE(P<0.05)和Chao1指数均降低(P<0.05)。CON组reads主要分布在厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrobia)、梭杆菌门(Fusobacteria)、放线菌门(Actinobacteria)、蓝藻菌门(Cyanobacteria)、软壁菌门(Tenericutes)。造模6周时,CAD组厚壁菌门、软壁菌门丰度降低(P<0.05),拟杆菌门、疣微菌门丰度升高(P<0.01);造模10周时,CAD组厚壁菌门丰度降低(P<0.01),拟杆菌门、疣微菌门丰度升高(P<0.01)。β-多样性分析,CAD组、CON组的肠型分布在不同区域,同一组的不同阶段则分布在相同区域。CAD组与CON组在微生物物种构成方面存在显著性差异(P<0.05)。CAD组小鼠在建模6周和10周时血液中甘油三酯(TG)(P<0.05,P<0.01),胆固醇(TC)(P<0.05),乳酸脱氢酶(LDH)(P<0.01,P<0.0001)和磷酸激酶(CK)(P<0.01,P<0.05)值均升高。低密度脂蛋白(LDL-C)在建模6周时升高(P<0.05)。细胞因子检测,IL-6在建模6周时浓度升高(P<0.05),10周时减低(P<0.00001);IL-10在建模10周时浓度降低(P<0.05);IL-2、IL-4、IL-5、IL-1β在建模10周时浓度升高(P<0.0001,P<0.05,P<0.0001,P<0.01)。IL-12p70、TNF-α、INF-γ在两个时间点差异无统计学意义。CAD组及CON组小鼠的冠状动脉苏木素-伊红(H.E)染色在建模后两个时间点均未见泡沫细胞形成等动脉粥样硬化改变。结论本研究利用粪菌移植方法获得冠心病人源菌群小鼠,肠道菌群结构丰度、体重、血脂、细胞因子含量等指标出现了与临床类似的改变。

Objective To establish and evaluate a mouse model of human flora-associated(HFA)from patients of coronary heart disease via fecal microbiota transplantation.Methods Twenty-eight female germ-free(GF)C57 BL/6 J mice were divided into the healthy control(CON)and coronary heart disease(CAD)groups.Eight-week-old mice were orally inoculated with 0.4 mL of stool suspension from healthy participants or CAD patients to build the HFA mouse model.At 6 and 10 weeks post-inoculation,fresh fecal samples were collected and examined for the V3 region of the 16 S rDNA gene.Blood sera were collected and examined for blood lipid,cholesterol,myocardial enzymes and cytokine levels.Coronary arteries were collected,processed and stained with hematoxylin and eosin(H&E)for pathological examination.Results The average body weight of the CAD group was significantly higher than that of the CON group(P<0.05)from 5 weeks post-inoculation.α-diversity analysis showed that the Simpson(P<0.05,P<0.01),Chao1(P<0.05)and ACE indices(P<0.05)were significantly lower in the CAD group than that in the CON group.The Shannon index(P<0.05,P<0.01)was higher in the CAD group than in the CON group at 6 and 10 weeks post-inoculation.The intestinal florae were mainly comprised of the phyla Firmicutes,Proteobacteria,Bacteroidetes,Verrucomicrobia,Fusobacteria,Actinobacteria,Cyanobacteria and Tenericutes.At six weeks post-inoculation,the relative abundances of Firmicutes and Tenericutes were lower(P<0.05),and those of Bacteroidetes and Verrucomicrobia were higher(P<0.01)in the CAD group than in the CON group.At ten weeks post-inoculation,the relative abundance of Firmicutes(P<0.01)was lower,and the relative abundances of Bacteroidetes and Verrucomicrobia(P<0.01)were higher in the CAD group than in the CON group.β-diversity analysis showed that the CON and CAD groups were distributed in different quadrants,but the same groups at different stages were distributed in the same quadrants,with a significant difference between the groups(P<0.05).The serum levels of TG(P<0.05,P<0.01),TC(P<0.05),LDH(P<0.01,P<0.0001)and CK(P<0.01,P<0.05)were significantly higher in the CAD group than in the CON group at 6 and 10 weeks post-inoculation.LDL-C levels were significantly higher(P<0.05)in the CAD group than in the CON group at 10 weeks post-inoculation.IL-6 levels were higher at 6 weeks(P<0.05)and lower at 10 weeks(P<0.01)post-inoculation in the CAD group than in the CON group.The IL-2,IL-4,IL-5 and IL-1βlevels were significantly higher(P<0.0001,P<0.05,P<0.0001,P<0.01,respectively)in the CAD group than in the CON group at 10 weeks post-inoculation.IL-12 p70,TNF-αand INF-γlevels did not differ between the CAD and CON groups.Pathological examination using HE staining of the coronary arteries showed no obvious atherosclerotic changes(e.g.,foam cell infiltration).Conclusions A mouse model of HFA from CAD patients was established via fecal microbiota transplantation.The main advantages of using bacteria from CAD patients are that the GF mice were well colonized,and the animals have similar body weights and serum levels of blood lipid,cholesterol and cytokines.