【作者】 刘娟；

【导师】 李廷玉；

【作者基本信息】 重庆医科大学 ， 临床医学（专业学位）， 2018， 博士

【摘要】 第一部分孤独症谱系障碍儿童肠道症状及肠道菌群与行为关系1背景:肠道症状及肠道菌群紊乱在孤独症谱系障碍(autism spectrum disorder,ASD)中常见,且肠道可以通过肠脑轴影响ASD的核心症状。目的:探索本地区ASD儿童胃肠道(gastrointestinal,GI)症状特点,GI症状与行为相关关系,以及无GI症状ASD儿童的相关肠道菌群;同时探索ASD最突出GI症状亚型儿童的行为损害特点及相关肠道菌群改变。方法:孤独症症状使用孤独症行为量表(autism behaviour checklist,ABC),儿童孤独症评估量表(childhood autism rating scale,CARS),社交反应量表(social responsiveness scale,SRS)进行评定,孤独症社交行为相关生物学指标使用CD38和视黄酸受体相关孤儿受体α(acid-related orphan receptor alpha,RORA)基因作为标志,引用国外(6-GI severity index,6-GSI)问卷作为GI症状评估工具。183例ASD儿童和66例CON儿童纳入横断面调查,完成上述指标,比较两组间GI症状差异。分析ASD儿童各GI症状与ABC、CARS、SRS、CD38和RORA基因表达水平的相关关系。分析ASD最突出GI症状儿童与无GI症状儿童行为差异。将收集到大便的ASD儿童按最突出GI症状、ASD无GI症状、对照组无GI症状进行分层,各分层中随机抽取24例,分别组成ASD+最突出GI症状、ASD-GI、CON-GI组,使用Illumina Mi Seq二代测序进行菌群分析,探索ASD+最突出GI症状组与ASD-GI,ASD-GI与CON-GI的菌群组成差异。结果:ASD较CON而言,GI总问题(52.46%VS.36.36%)及便秘(33.33%VS.15.15%)患病率显著增高(P均<0.05),校正混杂因素后ASD较CON GI总问题(1.08±1.44 VS.0.68±0.98)及便秘得分(0.40±0.62 VS.0.17±0.41)显著增高(校正P均<0.05)。ASD儿童GI症状与ABC、CARS、SRS量表得分之间无显著相关(P均>0.05)。ASD儿童RORA基因表达量与GI症状总得分(相关系数=-0.21,P=0.0053)、便秘得分(相关系数=-0.18,P=0.0184)显著负相关,CD38基因表达量与GI总得分及各GI症状得分无显著相关(P均>0.05)。ASD有便秘比ASD无GI症状儿童ABC、CARS、SRS量表得分无显著差异(P均>0.05),RORA基因表达显著降低(P=0.0075),CD38有降低趋势但无统计学差异(P>0.05)。随机抽样菌群亚组分析,LEf SE分析显示ASD-GI较CON-GI显著优势菌群为Prevotella、Bifidobacterium、Lactobacillales、Streptococcus、Erysipelotrichaceae等。经过Wilcoxon秩和检验及多重假设检验校正分析,显著优势菌群为Coriobacteriia、Coriobacteriales、Erysipelotrichales、Pseudomonadales。LEf SE分析显示ASD+便秘较ASD-GI儿童显著优势菌群为Prevotellaceae、Prevotella、Paraprevotella、Megasphaera、Christensenellaceae;相对丰度显著下降的菌群为Eggerthella。经Wilcoxon秩和检验以及多重假设检验校正分析,ASD+便秘与ASD-GI间显著差异菌群没有找到。结论:ASD较CON GI症状显著,GI症状与社交行为相关生物指标相关,Lactobacillales、Streptococcus、Bifidobacterium等在ASD-GI儿童中显著升高。便秘在ASD儿童GI症状中居首位,且ASD便秘亚型儿童伴随更多社交行为相关生物指标损害,尚未发现ASD便秘亚型相关的肠道菌群,需进一步研究。第二部分维生素A与孤独症谱系障碍儿童肠道症状、肠道菌群及行为的关系背景:第一部分研究发现ASD GI症状常见,且伴随肠道菌群改变,这可能加重孤独症社交行为相关生物学指标的损害。维生素A(vitamin A,VA)在神经系统发育,肠道机械屏障、免疫屏障、生物屏障方面均有一定调节作用。目的:此部分研究旨在探索VA在ASD肠道症状、肠道菌群及行为方面的作用。方法:VA使用高效液相色谱法(high performance liquid chromatography,HPLC)测定,孤独症症状使用ABC、CARS、SRS量表进行评定,孤独症社交行为相关生物学指标使用CD38和RORA基因作为标志,6-GSI问卷作为GI症状评估工具。首先,横断面调查的参与者同第一部分ASD儿童,其接受血浆VA水平、肠道症状、ABC、CARS、SRS、CD38和RORA基因表达水平的评估及测定。对VA与GI症状、ABC、CARS、SRS、CD38和RORA基因表达水平的相关关系进行分析。然后,建立64例ASD儿童的随访队列,完成VA大剂量20万IU干预,6月后随访。比较干预前后血浆VA水平、GI症状,ABC、CARS、SRS、CD38和RORA基因表达水平的差异。对64例中完成大便收集的20例儿童进行肠道Illumina Mi Seq二代测序菌群分析,了解VA干预前后肠道菌群差异。结果:ASD患者中VA与GI总得分、大便性状改变得分显著负相关(γs=-0.15,γs=-0.18,P=0.0449,P=0.0268)。VA与孤独症症状量表ABC、CARS、SRS得分无显著关系(全部P>0.05)。VA与孤独症社交行为相关生物学指标CD38和RORA基因表达量显著正相关(γs=0.30,γs=0.21,P=0.0001,P=0.0097)。VA干预6月后,64例队列儿童原有GI总问题,便秘、腹泻、大便气味改变等症状好转(全部P<0.05);血浆VA,CD38和RORA基因表达水平显著升高(全部P<0.05);ABC、CARS、SRS得分无显著差异(全部P>0.05)。20例肠道菌群分析儿童,LEf SE和Wilcoxon秩和检验均发现,VA干预6月后,Bacteroidetes/Bacteroidia/Bacteroidales(门纲目水平)相对丰度显著增加,Actinobacteria/Bifidobacteriales/Bifidobacteriaceae/Bifidobacterium(纲目科属水平)相对丰度显著降低。结论:VA对ASD儿童部分GI症状及孤独症社交行为相关生物学指标CD38及RORA有改善作用,Bacteroidetes/Bacteroidales可能是VA参与调解ASD的特异菌群,VA对ASD核心症状的作用需进一步大样本随机对照研究去验证。更多还原

【Abstract】 PART I THE RELATIONSHIP BETWEEN GASTROINTESTINAL SYMPTOMS,GUT MICROBIOTA AND BEHAVIORS IN CHILDREN WITH AUTISM SPECTRUM DISORDERBackground: Gastrointestinal(GI)symptoms and dysbiosis of gut microbiota are commonly reported in autism spectrum disorder(ASD),which may contribute to behavioral impairment via gut-brain axis.Objective: To find the GI symptoms in ASD and analyze the relationship between GI symptoms and behaviors.To find the related gut microbiota in ASD without GI symptoms.To find the most common GI symptom in ASD,analyze the damage of behaviors in this subtype and related gut microbiota.Methods: The autism behaviour checklist(ABC),childhood autism rating scale(CARS)and social responsiveness scale(SRS)were used to assess autism symptoms.CD38 and acid-related orphan receptor alpha(RORA)mRNA levels were used to assess autism behavior related biochemical indicators’ changes,6-GI severity index(6-GSI)was used to assess GI symptoms.183 children with ASD and 66 typical developmental children participated in this study,completed all above.The difference of GI symptoms between ASD and CON,the relationship between GI symptoms and ABC,CARS,SRS,CD38,RORA mRNA levels in ASD were analyzed.The difference of ABC,CARS,SRS,CD38,RORA mRNA levels were evaluated between the subtype of ASD with most common GI symptom and ASD without GI symptom.Illumina MiSeq for 16 S rRNA genes was used to compare the differences in gut microbiota between ASD-GI and ASD+most common GI symptom,ASD-GI and CON-GI in the subsets,with each group of 24,randomly selected from the ASD children who finished the stool collection.Results: The prevalence of total GI symptoms(52.46% VS.36.36%)and constipation(33.33% VS.15.15%)were significantly more common in ASD than CON(all P < 0.05).After adjusting the confounding factors,the GI socres(1.08 ± 1.44 VS.0.68 ± 0.98)and constipation scores(0.40 ±0.62 VS.0.17 ± 0.41)were significantly higher in ASD than CON(all adjust P < 0.05).In children with ASD,GI scores were not significantly correlated with ABC,CARS,SRS scores(all P > 0.05);RORA mRNA level were significantly negatively correlated with total GI score(γs=-0.21,P=0.0053)and constipation(γs=-0.18,P=0.0184);CD38 mRNA level werenot significantly correlated with GI scores(all P >0.05).There were no significant differences in ABC,CARS,SRS scores between ASD with constipation and ASD without GI symptoms(all P >0.05),neither in CD38 mRNA level(P >0.05).RORA mRNA level was significantly lower in ASD with constipation than ASD without GI symptoms(P=0.0075).In the gut microbiota analysis subgroups,ASD-GI were enriched in Prevotella,Bifidobacterium,Lactobacillales,Streptococcus,Erysipelotrichaceae,etc than CON-GI in LEfSE analysis,while were enriched in Coriobacteriia,Coriobacteriales,Erysipelotrichales,Pseudomonadales than CON-GI in Wilcoxon analysis.ASD+constipation were enriched in Prevotellaceae,Prevotella,Paraprevotella,Megasphaera,Christensenellaceae and lowered in Eggerthella than ASD-GI in LEfSE analysis.But the significant differences between ASD+constipation and ASD-GI can not be repeated in Wilcoxon analysis.Conclusions: GI symptoms were more common in ASD than CON;GI symptoms positively correlated with social behavior related marker damage and Lactobacillales,Streptococcus,Bifidobacterium significantly increased in ASD-GI.Constipation was the most common GI symptom in ASD and ASD with constipation may have more damage in social behavior related marker.Constipation related gut micriobiota were not foud,it’s open to futher study.PART Ⅱ RELATIONSHIP BETWEEN VITAMIN AAND GASTROINTESTINAL SYMPTOMS,GUT MICROBIOTA,BEHAVIORS IN CHILDREN WITH AUTISM SPECTRUM DISORDERBackground: Our preliminary work showed that GI symptoms were common in ASD,along with gut microbiota changes,which may contribute to autism behavior related biochemical marker changes.Vitamin A(VA)plays a role in regulation of both central nervous system(CNS)development and intestinal mechanical barrier,immune barrier,biological barrier.Objective: This study was performed to investigate the role of VA in the changes of GI symptoms,gut microbiota and behaviors in children with ASD.Methods: High performance liquid chromatography(HPLC)was used to assess plasma retinol levels.ABC,CARS and SRS were used to assess autism symptoms.CD38 and RORA m RNA levels were used to assess autism behavior related biochemical indicators’ changes.6-GSI was used to assess GI symptoms.Fisrt,all ASD participants from part I accepted tests about plasma retinol,GI symptoms,ABC,CARS,SRS,CD38 and RORA m RNA levels.Relationship between VA levels and GI scores,VA levels and autism symptoms,VA levels and biochemical indicators were analyzed.Second,sixty four children with ASD completed a 6-month follow-up study with VA intervention.Evaluations of plasma retinol,GI symptoms,ABC,CARS,SRS,CD38 and RORA m RNA levels were performed before and after 6 months of intervention in the 64 children.Illumina Mi Seq for16 S r RNA genes was used to compare the differences in gut microbiota before and after 6 months of treatment in the subset 20 of the 64 children.Results: In the 64 autistic children,VA were significantly negatively correlated with total GI scores,stool consistency scores(γs=-0.15,γs=-0.18,P=0.0449,P=0.0268);VA were not significantly correlated with ABC,CARS,SRS scores(all P > 0.05);VA were significantly correlated with CD38 and RORA m RNA levels(γs=0.30,γs=0.21,P=0.0001,P=0.0097).After 6 months of intervention,the total GI problems,constipation,diarrhea,stool smell scores significantly decreased(all P < 0.05);plasma retinol,CD38 and RORA m RNA levels significantly increased(all P <0.05);the scores of ABC,CARS and SRS scales showed no significant differences(all P > 0.05).Meanwhile,in the subgroup of 20,after 6 months of intervention,the proportion of Bacteroidetes/Bacteroidia/Bacteroidales(phylum to order level)significantly increased and the proportion of Actinobacteria/Bifidobacteriales/Bifidobacteriaceae/Bifidobacterium(class to genus level)significantly decreased no matter using LEf SE or Wilcoxon tests(all P< 0.05).Conclusions: Bacteroidetes/Bacteroidales maybe the key taxa related to VA.Moreover,VA played a role in the changes in GI symptoms and autism social behavior related biomarkers.It remains unclear whether the VA concentration is associated with autism symptoms.更多还原