【作者】 刘社兰；

【导师】 阮冰；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 原位肝移植术是治疗重型肝炎、肝硬化肝功能失代偿等终末期肝病最有效的治疗手段。随着现代医学技术的发展,肝移植手术的成功率不断提高,术后生存时间不断延长。但是,肝移植术后各种并发症仍然是影响手术成功的一个重要因素,其中术后感染是继移植排异后的另一常见的并发症,发生率约为47%～80%,病死率达13%～36%。肝移植术后感染也是导致移植物慢性失功的重要原因之一,并将增加移植病人住院时间及医疗费用。肝移植术后感染的常见病原体包括细菌(48%)、真菌(22%)、病毒(12%),以术后30天内发生的内源性感染最为常见,约31%的肝移植患者术后至少发生1次由多重耐药细菌引起的感染,约69%的病原菌呈多重耐药。由于肝脏与肠道在解剖结构及功能上有着密切的联系,肠道作为人体最大的贮菌所和内毒素库,在特定条件下可以释放多种毒性物质,导致隐匿的内源性感染。肠道微生态失衡、肠黏膜屏障损伤和肠渗透性增加、免疫功能失调是肝移植感染发生的重要原因。本研究重点是肝硬化大鼠在肝移植前后肠道细菌的分子生态结构以及血清代谢谱的变化规律,旨在探明肝移植后肠道微生态变化与感染的关系,寻找与肝移植相关的特异性代谢靶分子,为进一步探索通过重建、优化肠道微生态,减少肝移植后感染的发生,促进移植受者长期健康存活提供新思路和新途径。本研究首先建立肝硬化大鼠的肝移植模型,继而应用16S rDNA分子微生态研究方法和核磁共振法(~1H NMR),对肠道细菌组成和血清代谢谱变化进行了动态观察,在国内外未见类似的报道。研究方法1.四氯化碳(CCL4)诱导SD大鼠肝硬化模型清洁级Sprague-Dawley(SD)大鼠50只,雌雄各半,体重为180g～200g。分为两组:对照组10只,造模组40只。模型制备方法:对照组大鼠无菌腹腔皮下注射花生油溶液;造模组大鼠无菌腹腔皮下注射40%CCL4花生油溶液。于给药后4周、8周、12周、16周,分别处死2只造模大鼠,取肝,观察肝脏病理进展情况,持续16周复制大鼠肝脏病变模型。造模第16周处死造模组大鼠10只和对照组大鼠5只。剩余的造模组大鼠22只和对照组大鼠5只肝移植备用。2.肝硬化SD大鼠肝移植模型的建立选择清洁级、体重为250g～300g的SD雄性大鼠19只作为供体;从CCL4造模组剩余的22只SD大鼠中,选择体重相近的肝硬化大鼠14只,以及造模对照组剩余的5只SD大鼠作为受体。分为三组:肝硬化肝移植组(正常大鼠一肝硬化大鼠,9只),肝移植对照组(正常大鼠-造模对照组大鼠,5只),假手术组(肝硬化大鼠,5只,只打开腹腔便缝合)。大鼠原位肝移植模型采用经典的Kamada二套袖法。所有的肝移植受体鼠移植前后均不使用抗生素,免疫抑制剂等。3.标本的收集(1)血与粪便:在大鼠CCL4给药前、CCL4给药后4周、肝硬化时/移植前、肝移植术后7天、肝移植术后30天五个时间点,收集所有大鼠的粪便。除CCL4给药后4周外、其他的4个时间点同时采集全血。(2)下腔静脉血、肝、脾、肾组织:在肝硬化/肝移植前(造模组10只、对照组5只)以及肝移植术后30天(所有19只移植受体鼠)两个时间点处死大鼠,收集下腔静脉血、肝、脾、肾组织。4.标本检测方法(1)鲎试验法:检测大鼠血浆内毒素水平。(2)核磁共振法(~1H-NMR):检测大鼠血清代谢谱。(3)细菌需氧与厌氧培养法:取大鼠肝、脾、肾组织,立即研磨,做细菌需氧与厌氧培养及生化鉴定,分析细菌易位发生率及易位细菌种类。(4)细菌荧光定量PCR法:从大鼠粪便抽提总DNA,分别采用肠道六大菌属(肠球菌、肠杆菌、拟杆菌、梭菌、双歧杆菌和乳酸菌)的特异性引物,进行荧光定量PCR检测。(5)聚合酶链反应-变性梯度凝胶电泳指纹图谱(PCR-DGGE)分析:用16SrDNA V3区的通用引物和拟杆菌属、梭菌属和双歧杆菌属特异性引物作PCR-DGGE指纹图谱,对肠道细菌总体变化趋势和以上三种菌属的分子生态结构进行主成分分析并作克隆测序。5.数据统计分析方法计数资料采用t检验,率的统计用χ2检验,采用MATLAB软件进行主成分分析(PCA)。研究结果1.肝硬化SD大鼠肝移植术后细菌易位情况肝移植术后30天,大鼠肝、脾、肾细菌易位率达66%,远高于肝硬化/移植前细菌易位率(10%),两者有统计学差异(P<0.05)。细菌易位部位以肝脏最为常见,脾脏次之,肾脏少见。肝硬化肝移植组的易位细菌种类以粪肠球菌、铜绿假单孢菌、大肠埃希菌为主,而肝硬化/肝移植前的易位细菌种类则以铜绿假单孢菌为主。2.肝硬化SD大鼠肝移植术后肠道细菌的定量变化采用实时荧光定量PCR方法,选择5个时间点(CCL4给药前、给药后4周、肝硬化/肝移植前、肝移植术后7天、肝移植术后30天),动态观察大鼠粪便中六大优势菌的定量变化。结果显示,与肝移植对照组相比,肝硬化肝移植组在肝硬化发展过程中,肠道拟杆菌属、梭菌属、肠杆菌属细菌数量不断下降,肝移植术后细菌数量逐步回升,至肝移植术后30天基本恢复至造模前水平。肠道双歧杆菌属在肝硬化造模过程中菌量升高,但在肝移植术后7天开始显著下降,术后30天部分恢复。肠道乳酸菌属和肠球菌属在肝硬化造模过程中细菌含量改变不明显。3.肝硬化SD大鼠肝移植术后肠道细菌总体分子生态结构变化采用PCR-DGGE方法,借助通用引物,对肠道所有细菌的16S rDNAV3区(可变区)进行扩增,选择4个时间点(CCL4给药前、肝硬化/肝移植前、肝移植术后7天、肝移植术后30天),观察大鼠肠道菌群总体分子生态结构变化。结果发现:(1)组内比较结果显示,CCL4给药前,大鼠肠道细菌的总体分子生态组成差异小,基本聚成一类;肝硬化/肝移植前,肠道细菌的总体分子生态组成差异增大,分布弥散,不能聚成一类;至肝移植术后30天,肠道细菌的总体分子生态组成差异减小。(2)组间比较结果显示,肝移植术后30天组与CCL4给药前组相比差异最大,但与肝移植术后7天组相比差异不明显。4.肝硬化SD大鼠肝移植术后肠道拟杆菌属、梭菌属、双歧杆菌属的分子生态结构变化应用类群特异性PCR-DGGE方法,进一步观察肠道各优势菌属(拟杆菌属、梭菌属、双歧杆菌属)在4个时间点(CCL4给药前、肝硬化/肝移植前、肝移植术后7天、肝移植术后30天)的分子生态结构变化,结果发现:(1)组内比较结果显示,CCL4给药前,大鼠肠道拟杆菌属、梭菌属、双歧杆菌属的组内分子生态组成均相似,基本上均聚成一类;CCL4给药后,上述各菌属的组内分子生态组成差异均增大,分布弥散。(2)组间比较结果显示,拟杆菌属肝硬化/肝移植前组、肝移植术后术后7天、肝移植术后术后30天组与CCL4给药前组相比差异均明显,特别是术后30天组最为显著,而术后7天组与肝硬化/肝移植前组的距离较近;梭菌属肝硬化/肝移植前组、肝移植术后30天组与CCL4给药前组相比差异均明显,特别是术后30天组,而在术后7天组基本恢复到CCL4给药前组水平;双歧杆菌属肝硬化/肝移植前组与CCL4给药前组相比差异明显,分子生态结构不能聚类,分布在各个象限,但术后7天组与术后30天组相比,能聚集在一起,提示在肝移植术后,肠道双歧杆菌属的组成能较快恢复稳定。5.肝硬化SD大鼠肝移植术后血清代谢谱的变化应用核磁共振法(~1H-NMR),结合多元统计分析,选择4个时间点(CCL4给药前、肝硬化/肝移植前、肝移植术后7天、肝移植术后30天),动态观察22只大鼠的血清代谢谱(18种物质)变化。结果显示,大鼠肝硬化/肝移植前,血清乳酸、高密度脂蛋白、不饱和脂肪酸、甜菜碱明显上升,而谷氨酸、谷氨酰胺、缬氨酸、O-乙酰糖蛋白、N-乙酰糖蛋白、葡萄糖、低密度脂蛋白、极低密度脂蛋白、脂肪酸、乙酸出现下降。随着供肝的植入,大鼠血清代谢物的异常逐渐消失,代谢物恢复正常所需的时间各不相同。用主成分分析(PCA)发现,CCL4给药前组、肝硬化/肝移植前组、肝移植术后7天组的血清代谢谱完全分离,分布在不同的区域,说明它们的代谢谱完全不同,但肝移植术后30天组与CCL4给药前组的血清代谢谱有部分重叠,提示此时的代谢谱已有部分恢复。结论本研究采用系统生物学方法,对SD大鼠肝硬化前后、肝移植前后不同时间点肠道细菌的分子生态组成及血清代谢谱进行动态观察,证实大鼠肝移植术后存在肠道菌群与血清代谢失衡,发现了肠道六大类优势菌属及血清代谢谱的变化规律与变迁模式,为下一步寻找功能菌以及用微生态制剂(包括益生菌等)恢复正常菌群结构、保护肠黏膜屏障、减少细菌易位和移植感染的发生提供了一个创新的思路。更多还原

【Abstract】 Orthotopic liver transplantation(OLT) is to date the best and most durable treatment for patients with end-stage liver disease such as acute or chronic hepatic failure and cirrhosis.For various improvements have been made in both surgical and therapeutic techniques,the successful rate of liver transplantation increased and the survive time became longer.All complications are still major causes of post-transplant mortality.The infection is the second cause next to the acute rejection.The incidence or mortality is 47%- 80%or 13%- 36%respectively.The post-transplant infection not only lead to rise the duration and fee of hospitalization,but also it adds the high risk of graft liver dysfunction.The most often pathogen infected liver transplantation include the bacteria(48%),fungi(22%) and virus(12%).The Endogenous infection often occurred in 30 days after liver transplantation.About 31%of liver transplant patients with at least one time caused by multiple drug-resistant infections.The 69%of bacterial infections in post liver transplantation are caused by multi-drug resistant bacteria. Because of the anatomical structures and functions in close contact between liver and intestinal tract,intestine as the body’s largest reservoir of bacteria and endotoxin,it can release a variety of toxic substances under certain conditions,which will bring the occult cause of endogenous infection.Gut microflora imbalances,intestinal barrier damage and increased intestinal permeability,immune dysfunction are the major reasons for infection of liver transplantation.This study focused on the gut microflora and serum metabolic changes before and after liver transplantation.Aimed at finding the changes in gut microflora of liver transplantation infection,as well as searching specific metabolic target related liver transplantation.It can provide of new ideas and new ways for future exploration through the reconstruction,to optimize gut microflora,to reduce the incidence of infection after liver transplantation,and to promote long-term health of transplant recipients survive.In this study,there are three distinctive characteristics.Firstly,we chose the liver transplantation model of rat with liver cirrhosis induced by carbon tetrachloride. Secondly,we discovered the changes of the bacterial composition and serum metabolism of dynamic range by using 16S rDNA molecules microecological technology and H~1 NMR.It was different from the traditional bacteria culture.There were no similar reports up to now.Research Methods1.The rat liver cirrhosis model induced by Carbon tetrachloride (CCL4)Class clean Sprague-Dawley(SD) rats with 50,were equally divided between male and female,weight 180g-200g.Dividing into two groups:control group(10) and model group(40).Model preparation methods:the control group of rats with sterile peanut oil solution subcutaneously;model in rats by subcutaneous injection in sterile 40%solution of carbon tetrachloride in peanut oil.After administration of 4 weeks,8 weeks,12 weeks,16 weeks,the two were put to death in model rats,observing the progress of liver pathology,continuous 16-week rat model of liver disease.10 Models and 5 control group were executed at the time of the first 16 weeks.Last,the remaining 22 liver cirrhosis in rats and 5 control rat liver transplantation standby.2.Liver transplantation model after cirrhotic ratChoose a clean class,weight 250g - 300g Sprague-Dawley(SD) male rats for 19 as the donor.Receptor:CCL4-induced SD rat model of liver cirrhosis 14 and SD rats in control group 5.Divided into three groups:the same gene liver transplantation group(9 rats),the control group(5 rats),sham operation group(liver cirrhosis in rats,it is only open abdominal suture,5 rats).The classic two Kamada Sleeve law was applied in the orthotopic liver transplantation model in rats. 3.Specimen collection(1) Blood and feces:all the blood and feces were collected CCL4 before delivery, liver cirrhosis,the seven days and 30 days after liver transplantation.(2) The liver cirrhosis model 10 and 5of the control group were killed when the cirrhosis model became successful;and the 19 mouse receptors were put to death 30 days post-transplant treatment.Remove the blood cavity,liver,spleen and kidney.4.Detection methods(1)~1H-NMR for detection of serum metabolites.(2) Liver,Spleen and Kidney immediately to do aerobic and anaerobic grinding culture,biochemical identification of bacterial types,analysis of the incidence of translocation.(3) Extraction from the faeces of total DNA,using specific primers for the case of Enterococci,Enterobacter, Bacteroides,Clostridium,Bifidobacterium,Lactobacillus quantitative fluorescent PCR; on the levels of intestinal bacteria to change six clear of bacteria make further 16S DNA PCR-DGGE,analysing of different groups of bacteria molecular structure of ecological patterns and differences in the strains,using cloning and sequencing analysis.5.Data analysis methodsCategorical variables and measurement data were compared using the chi-square and T test respectively.MATLAB software was used to do principal component analysis(PCA).Results1.There is significant difference in the rate of bacterial translocation to liver, spleen,kidney between liver cirrhosis group(10%) and liver transplantation group(66%) (P<0.05).The liver was the most common site of bacterial translocation,followed by the spleen and the kidney.The often translocation types of bacteria were Enterococcus faecalis,Pseudomonas aeruginosa and Escherichia Coli in the liver transplantation group,while Pseudomonas aeruginosa dominated in liver cirrhosis group.2.We observed the changes of six major kinds of fecal bacteria at five time points (before CCL4 administration,4 weeks after administration,liver cirrhosis,7 days and 30 days after the liver transplant) by using the real-time fluorescence quantitative PCR. The results showed that the quantities of Bacteroides genus,Clostridium and Enterobacter spp were deccreasing in liver cirrhosis group comparing with the control group,However,they gradually increased in liver transplantation group and reached normal level in one month after liver transplantation group.Bifidobacterium increased in the process of liver cirrhosis and began to decrease significantly 7 days after liver transplantation,but it had partial recovery after 30 days.The changes of Lactic acid bacteria and enterococci were not obvious in liver cirrhosis process.3.We analysed the intestinal molecular flora structure at the 4 time point including CCL4 before delivery,liver cirrhosis,7 and 30 days after liver transplantation by using PCR-DGGE method based on 16S rDNA V3 area.The results showed that there was small difference of intestinal molecular flora structure between individuals in the control group.In the contrary,there was significant difference in the liver cirrhosis group.Moreover,the intestinal molecular flora structure significantly changed in the group of 30 days after liver transplantation comparing with the control group. Interestingly,the smallest difference was seen in the groups of 7 and 30 days post transplantation groups.4.Application of group-specific PCR-DGGE technology,further analysis of intestinal predominant bacteria such as Clostridium genera,the genus Bifidobacterium and Bacteroides.The study found that:(1) The intestinal bacteria molecular structure had small individual differences in Clostridium genera group,the genus Bifidobacterium group and Bacteroides,group CCL4 before delivery.(2) Compared to the before CCL4 administration group,the fundamental changes of Bacteroides structure have taken place in liver cirrhosis group and 30 days after the liver transplant group.Of which 30 days after transplant was the farthest away from the normal group But it had posed a similar bacteria spectrum between liver cirrhosis groups and the seven days after transplant groups.There were 16 species of Bacteroide to have largest contributions to the divide the groups.Furthermore,most of them could not be cultured. (3) The Clostridium molecular ecology according to the PCA indicated:individual distribution of dispersion and large individual differences inner the liver cirrhosis group and 30 days group.The Clostridium composition before cirrhosis was obviously different from after cirrhosis,Furthermore,the difference between groups was greater than the inter-group differences.Clostridium basic ecological structure of the molecule returned to the normal levels 7 days after operation.However,Clostridium bacteria composition after 30 days transplantion was totally different from the normal group,and there was the farthest distance between the two groups.There were 12 species of Clostridium to have largest contributions to the divide the groups.Moreover,most of them could not be cultured.(4) Bifidobacterium DGGE fingerprinting was carried out principal component analysis,the structure of the composition of Bifidobacterium relatively simple,Even though the intestinal Bifidobacterium molecular structure had small individual differences and formed as cluster CCL4 before delivery.But at the time of liver cirrhosis,it should not be clustered,distributing in all quadrants.It can collect together when 7 days and 30 days after liver transplantation again,which showed that the composition of Bifidobacterium can restore in acute phase rejection,and then keep stability for at least 30 days.5.The dynamic changes law of the serum metabolic spectrum(18 substances) were explored in total of 22 rats before and after liver cirrhosis or liver transplantation using the ~1H NMR combined with multivariate statistical analysis.The results showed that: lactic acid,high-density lipoprotein,unsaturated fatty acid,betaine increased significantly,and the branched-chain amino acids such as glutamate,glutamine,valine, etc;O-acetyl glycoprotein,N-acetyl-glycoprotein,glucose;low-density lipoprotein and very low density lipoprotein,fatty acid;acid decreased when Cirrhosis formed.With the implantation of liver,the abnormal metabolites gradually restored,but the metabolite recovery time varies.Further analysis by the use of PCA:The metabolic spectrum was completely separated in cirrhosis model group,the normal control group and 7 days after liver transplantation group.They distributed in different regions,showed that their metabolism in a completely different composition.But there is some overlap in the metabolism spectrum between 30 days after liver transplantation and normal group. This showed that there is a part of the restoration of metabolic spectrum at this time. ConclusionsIn this study,the intestinal molecular flora and serum metabonomic spectrum were carried out in different times of liver cirrhosis and liver transplantation,using by systemic biology method.It discovered that the different categories were in the different disease status under the six major intestinal bacteria and spectrum of metabolic changes. At the same time,it was confirmed that the existence of an imbalance of intestinal flora in the liver transplantation rat.It will help to restore the structure of normal flora,to protect the intestinal mucosal barrier,to reduce bacterial translocation and transplant infections by using the probiotics(including probiotics,etc.)更多还原