【作者】 胡锦华；

【导师】 王宇明；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 博士

【摘要】 当前研究显示肝脏和肠道微生态不仅在解剖结构上,而且在功能上都存在着密切的关系。肠道内寄居着种类繁多,数量巨大的细菌,是机体内最大的储菌库和内毒素池,是内源性感染和内毒素血症的主要来源。在正常情况下,肠道正常菌群与黏膜表面的粘蛋白、sIgA、完整的肠黏膜结构及肠壁免疫细胞共同构成了肠道的黏膜屏障系统,阻止了肠道细菌,内毒素向肠外移位。在某些病理情况下如重型肝炎,肝硬化,严重烧伤,小肠移植等均可以出现肠道微生态失衡,肠壁屏障功能损伤,细菌移位以及肠源性内毒素血症。近来实验研究和临床资料多集中于肝衰竭、肝硬化对肠道微生态的影响,但主要基于直肠粪便样本的分析,结果显示双歧杆菌、乳杆菌等有益菌下降而肠杆菌等有害菌升高的趋势。有关肝损伤的肠道微生态变化研究资料不多,各肠段正常菌群数量和分布规律未见报道。临床上对肠道正常菌群的定量分析主要采用传统分析方法,但费时、易受操作影响,近几年来有研究采用基于16S rRNA基因有关的实时定量PCR(Real-Time PCR)技术定量分析肠道菌群演替规律,资料不多且缺乏比较。为进一步弄清肝损伤时肠道微生态变化及大肠各节段正常菌群分布规律,我们采用种属特异性探针结合Real-Time PCR技术,定量分析正常和肝损伤动物模型的不同大肠节段微生态状况,设计进行了如下实验。第一部分:Real-Time PCR检测不同肠段肠道正常菌群及与传统培养结果的比较材料与方法1.研究对象SD大鼠6只,雌雄各半,体重150±10g。2.研究标本新鲜粪便3.研究方法3.1传统培养方法定量分析肠道正常菌群。3.2含有肠道目标细菌特异性16S rRNA基因片段的质粒DNA构建、克隆建库及测序。3.3荧光定量PCR定性、定量分析大肠不同节段内容物目的细菌群。3.4 SPSS for Windows用于统计分析。结果传统培养方法分析正常大鼠不同节段肠道正常菌群显示,各分析菌群在空肠、回肠迅速升高,盲肠达到峰值,结肠和直肠各菌群数量较盲肠变化不明显(p>0.05)。小肠内容物总DNA采用试剂盒未能提取。Real-Time PCR分析显示,各分析目的菌群在盲肠、结肠和直肠检测结果变化不明显。两种检测方法检测结果相比较,各菌群数值在大肠各节段变化规律是一致的。第二部分:Real-Time PCR定量分析急性肝损伤模型肠道微生态状况及相关干预的影响实验分组:将SD大鼠分成五组(每组10只动物)⑴正常对照组:正常大鼠,2ml灭菌水。⑵对照组:急性肝损伤大鼠模型,2ml灭菌水。⑶甘利欣组:急性肝损伤大鼠模型,甘利欣15mg/kg·d灌胃。⑷肝生元组:急性肝损伤大鼠模型,GSY-1 45mg/kg·d灌胃。⑸螺旋藻组:急性肝损伤大鼠模型,螺旋藻500mg/kg·d灌胃。各组于造模前5日开始灌胃,造模后48h处死动物采集标本,每组随机挑选3只雌鼠和3只雄鼠,共6只动物用于分析盲肠、结肠和直肠3个肠段正常菌群构成,并与传统方法结果比较。所有动物均检测血浆内毒素水平,肿瘤坏死因子水平,肝功能以及肝脏的病理改变。结果1.急性肝损伤对照组大鼠出现明显的肠道菌群紊乱,Real-Time PCR与传统方法定量分析盲肠、结肠和直肠内容物菌群状况,3个节段表现为肠杆菌科细菌和肠球菌的增加(与正常对照组比较P<0.01),双歧杆菌下降(与正常对照组比较P<0.01)。血清ALT,AST,血浆内毒素水平和肿瘤坏死因子α水平升高(与正常对照组比较P<0.01)。2.造模前给予甘利欣,肝生元和螺旋藻均可降低造模后2日的内毒素和肿瘤坏死因子水平(与对照组比较,P<0.01)。肝生元和螺旋藻组双歧杆菌数量增加(与对照组比较,P<0.01),肠球菌和杆菌降低(与对照组比较,P<0.01)。与对照组比较,甘利欣组调节菌群效果不明显(P>0.01)。在干预因素存在下,定量分析急性肝损伤动物模型不同肠段菌群,Real-Time PCR检测所获结果与应用传统方法获得的结果存在一致性。第三部分:Real-Time PCR定量分析慢性肝损伤模型肠道微生态状况及相关干预的影响实验分组:将SD大鼠分成五组(每组10只动物)⑴正常对照组:正常大鼠,2ml灭菌水。⑵对照组:慢性肝损伤大鼠模型,2ml灭菌水。⑶甘利欣组:慢性肝损伤大鼠模型,甘利欣15mg/kg·d灌胃。⑷肝生元组:慢性肝损伤大鼠模型,GSY-1 45mg/kg·d灌胃。⑸螺旋藻组:慢性肝损伤大鼠模型,螺旋藻500mg/kg·d灌胃。各组于造模前3月前开始灌胃,皮下注射CCl4每周1次造模,剂量为0.2ml/kg;每次注射前CCl4用花生油稀释,浓度逐渐递增,即每2周递增5%,依次为5%、10%、15%、20%、25%、30%;连续3个月,共注射12次。末次造模后48h处死动物采集标本,每组随机挑选3只雌鼠和3只雄鼠,共6只动物用于分析盲肠、结肠和直肠3个肠段正常菌群构成。所有动物均检测血浆内毒素水平,肿瘤坏死因子水平,肝脏纤维化,肝功能以及肝脏的病理改变。结果1.实验性慢性肝损伤对照组大鼠出现明显的肠道菌群紊乱,Real-Time PCR与传统方法定量分析盲肠、结肠和直肠内容物菌群状况,3个节段均表现为肠杆菌科细菌和肠球菌的增加(与正常对照组比较P<0.01),双歧杆菌下降(与正常对照组比较P<0.01)。血清ALT,AST,血浆内毒素水平和肿瘤坏死因子α水平升高(与正常对照组比较P<0.01),PLD、TGF-β1和HA三个肝纤维化指标亦非常显著增高(P<0.01)2.造模前给予甘利欣,肝生元和螺旋藻均可降低造模后2日的内毒素和肿瘤坏死因子水平(与对照组比较,P<0.01)。肝生元和螺旋藻组双歧杆菌数量增加(与对照组比较,P<0.01),肠球菌和杆菌降低(与对照组比较,P<0.01)。与对照组比较,甘利欣组调节菌群效果不明显(P>0.01)。在干预因素存在下,定量分析慢性肝损伤动物微生态,Real-Time PCR检测结果与传统方法结果存在一致性。结论应用Real-Time PCR技术分析证实,急性和慢性肝损伤后均存在较明显的肠道微生态的紊乱;而肝生元和螺旋藻能在一定程度上改善实验性肝损伤后肠道菌群失衡,增加肠道的定植抗力,降低血内毒素水平,起到一定护肝作用。本研究结果提示,Real-Time PCR技术分析不同肠大肠节段正常菌群所获得的结果,在正常动物,实验性肝损伤动物模型以及干预因素存在条件下,与传统培养方法有很好的一致性,且具有便捷、省时等优点,可成为肠道菌群紊乱及相关干预效果的评价体系之一,并有望成为一种客观分析肠道正常菌群变化规律的常规方法。更多还原

【Abstract】 Recent studies showed that intestinal microecology and liver tie up closely not in anatomical structure but also in functional status, they can influenced each other. It can exacerbate the injury of the liver and subsequently induce various complications. Recent experiments and clinic trials are mainly concerned with the effect of hepatic failure, liver cirrhosis on the intestinal flora and based on the specimen examination of rectum feces, and yield out the decrease of beneficial bacteria like Bifidobacterium and Lactobacillus whereas the increase of harmful bacteria like Enterobacteriaceae. Datas of the intestinal flora changes related to chronic hepatic injury are rare, and the colony number and distribution of intestinal flora haven’t been reported yet. Thus the present experiment is designed as follows:Materials and MethodsPart one: Quantitative Analysis of Intestinal Flora Status in Different Large Intestinal Segments of SD Rats By Real-Time PCRAnimals and Sample:SD rats, male vs. female 1:1, weight 150±10g, were enrolled in this study. The fresh faeces were analyzed as samples.Methods:1. Quantitative analysis of intestine flora by traditional method.2. Standard plasmid DNAs containing different interest fragments according to the aim of investigation were constituted and transformed into DH5αcells(E.coli)3. The real-time PCR was established with SYBR GreenⅠ, and we quantified the targeted faecal bacteria of all subjects.4. All results were analyzed by SPSS(Version 10.0).Results The results analyzed by traditional method showed that intestine flora in the Normal Group reached the peak in almost equivalent amount in the following three segments: the caecum, the colon and the rectum, which consistents with the results by other researches. The results analyzed by real-time PCR showed intestine flora were equivalent amount in the following three segments: the caecum, the colon and the rectum. The results were relative to that from traditional culture method.Part two: Quantitative Analysis of Intestinal Flora in Rats with Acute Hepatic Injury By Real-Time PCR and Effect of“GANSHENGYUAN”or Spirulina on itExperiment design: SD mice were divided into five group(Normal group, Model group,“GANLIXIN”(GLX) group,“GANSHENGYUAN”(GSY-1) group and Spirulina group), each group contained 10 rats. Normal group received 2m1 sterile water by gavage from 5th day before injection of 25% CCl4(5ml/kg). While GLX group received GLX (15mg/kg·d ), GSY-1 group received GSY-1(45mg/kg·d), Spirulina group received Spirulina (500mg/kg·d ). Gavage lasts until the sacrifice of the animals.Specimen collection was collected on the 2th day after injection of 25% CCl4(5ml/kg) (n=10). The rat was sacrificed and liver enzyme, plasma endotoxin, tumour necrosis factorα(TNF-α) and liver histological changes were measured. Six mice of each group were random selected for investigating bacterial counts in different intestinal segments (caecum, colon, and rectum).ResultModel group showed a decrease of Bifidobacterium counts and a increase of Enterobacteriaceae and Enterococcus in intestine fragments: caecum, colon, and rectum. This results analyzed by traditional method were similar to those from real-time PCR. The levels of the plasma endotoxin and TNF-αincreased.Pretreatment with GLX, GSY-1 and Spirulina can both lowed the levels of the plasma endotoxin and TNF-α48h after injection of 25% CCl4(5ml/kg). In GSY-1 group and Spirulina group showed a increase in Bifidobacterium counts and a decrease in Enterobacteriaceae and Enterococcus; GLX, GSY-1 and Spirulina can lowed the levels of the plasma endotoxin and TNF-α24h after acute liver injury, which were associated with decreased liver function such as ALT, AST and DBIL.Part three: Quantitative Analysis of Intestinal Flora in Rats with Chronic Hepatic Injury By Real-Time PCR and Effect of GSY-1 or Spirulina on itExperiment design: SD mice were divided into five group(Normal group, Model group, GLX group, GSY-1 group and Spirulina group), each group contain 10 mice. Four groups except Normal group were subcutaneously injected with CCl4, diluted with peanut oil in advance, at a dosage 0.2ml/kg. The concentration of CCl4 ascended 5%: 5%, 10%, 15%, 20%, 25%, 30%. CCl4 injection lasted 3 months, totally 12 times. Normal group received 2m1 sterile water by gavage from 5th day before injection of CCl4. While GLX group received GLX (15mg/kg·d ), GSY-1 group received GSY-1(45mg/kg·d), Spirulina group received Spirulina (500mg/kg·d ). Gavage lasts until the sacrificed of the animals.Specimen collection was collected on the 2th day after injection of 25% CCl4(5ml/kg) (n=10). The rat were sacrificed and liver enzyme, plasma endotoxin, tumour necrosis factorα(TNF-α) and liver histology were investigated. 6 mice were random selected for investigating bacterial counts in different intestine segments (caecum, colon, and rectum).Model group showed a decrease of Bifidobacterium counts and a increase of Enterobacteriaceae and Enterococcus in intestine fragments: caecum, colon, and rectum. This results analyzed by traditional method were similar to that from real-time PCR. The levels of the plasma endotoxin and TNF-αwere increased, t the levels of PLD,TGF-β1 and HA were also increased.Pretreatment with GLX, GSY-1 and Spirulina can both lowed the levels of the plasma endotoxin, TNF-α, PLD, TGF-β1 and HA. In GSY-1 group and Spirulina group showed a increase in Bifidobacterium counts and a decrease in Enterobacteriaceae and Enterococcus; GLX, GSY-1 and Spirulina can lowed the levels of the plasma endotoxin and TNF-α24h after chronic liver injury.ResultsAccording to quantitative analysis by real-time PCR, the results of intestinal flora were consistent with the results by traditional culture.The above results also showed that disturbance of intestinal flora was observed in rats with acute or chronic liver injury. The elevation of plasma endotoxin and TNF-αwas also found. Supplemented with prebiotics, probiotics or symbiotics, such as GSY-1 and Spirulina, can decrease the levels of plasma endotoxin liver injury through improving intestinal microflora. Real-Time PCR is an effective method to analyze the intestinal flora.更多还原