【作者】 顾玮；

【导师】 蒋建新；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 研究背景:严重创伤是青年人首要死亡原因,已成为世界第三大常见死亡原因。在第一时间获救的严重创伤患者往往在伤后并发脓毒症和多器官衰竭等严重并发症,并最终死亡。创伤后并发症发病率高、死亡率高、治疗费用高的“三高现象”,对人类健康和社会经济发展已构成巨大的负面影响,因而是目前全球范围内危重病医学领域内亟待解决的重大科学问题。虽然目前脓毒症的治疗方法众多,但是脓毒症的治疗水平并无明显的提高。其关键问题在于目前脓毒症的治疗都是在脓毒症发生后。由于缺乏能早期判断脓毒症风险性的预警诊断方法,因而难以在脓毒症发生前或发生即刻进行预防或早期治疗。细胞因子是脓毒症病理过程中重要的效应分子,直接反映机体的免疫应答类型;而模式识别受体是脓毒症发生早期,机体识别病原菌分子,并做出迅速免疫应答的第一道防线。因此,本研究拟从细胞因子和模式识别受体入手,选择一些脓毒症相关的重要基因及多态性位点,通过生物信息学预测、体外功能验证和临床关联研究,明确其功能意义和临床预警诊断价值,从而探寻脓毒症早期诊断方法,以达到减少或减轻危重病人并发脓毒症,进一步提高临床危重病人的救治水平的目的。材料与方法:1.样本:本研究共收集656例样本,分别采自348例健康志愿者和308例严重创伤患者。所有人均为居住在重庆的中国汉族人,相互间无血缘关系。348例健康志愿者中男性219例,女性129例,平均年龄为25岁(18-53岁),均为志愿献血者。308例严重创伤患者样本(男性242例、女性46例),于2005年1月至2008年1月间从大坪医院和重庆市急救中心收集。患者纳入标准为:(1)创伤后24小时内;(2)ISS评分≥16分或AIS评分≥3分;(3)年龄≥16岁且≤70岁,性别不限;(4)存活时间超过1周。其平均年龄38.5岁,平均创伤严重程度评分(Injury severity score,ISS)为25.5分。2.基因测序与生物信息学分析:为筛查中国汉族人群中IL-6基因启动子区SNPs分布情况,取27例中国汉族健康人DNA样本,对IL-6基因启动子区-2996~+53bp区域(参考序列Y00081.1)进行双向直接测序。将测序结果构建重叠群,进行多重比对,寻找SNPs位点。利用在线生物信息学软件(http://125.itba.mi.cnr.it/genebin/wwwrepeat.pl和http://motif.genome.ad.jp/)对测序发现的-572C/G进行分析,观察其是位于重复序列内,还是位于重要的表达调节部位或其附近,以及碱基改变是否会影响转录因子结合位点变化,从理论上初步分析其生物学意义。3.报告基因实验:为进一步明确-572C/G对IL-6启动子活性的影响,用PCR方法以野生型DNA样本为模版,扩增IL-6启动子-1728～+36bp片段,构建pGL3BH-CC质粒。通过定点突变,构建pGL3BH-GG质粒。将两种质粒转染至U-937和K-562细胞系中,通过化学发光检测仪观察碱基改变对启动子活性的影响。4.基因芯片制备:为建立高通量基因分型技术平台,根据选择的9个细胞因子和13个SNP位点,分别设计一对PCR扩增引物和两条杂交探针。按设计的芯片矩阵将探针点制在芯片上,并与多重PCR产物杂交。待玻片晾干后用ScanArray 3000扫描仪扫描,ImaGene软件分析扫描结果。通过改变PCR条件、杂交温度优化实验条件。5.基因分型:分别采用RFLP-PCR(CD14/-159、-1145,MD-2/-1625、IL-6/-572)和SNP芯片(13个细胞因子SNPs)进行基因分型,芯片分型结果用ScanArray 3000扫描仪扫描,ImaGene软件分析。基因分型结果均通过随机抽取10例样本测序确认。6.细胞因子表达水平检测:取严重创伤患者入院后第一天全血,100ng/ml LPS刺激4小时后,离心收集血浆。用ELISA方法检测血浆细胞因子表达水平。7.临床关联研究:脓毒症诊断标准:(1)有病原菌培养证据;(2)体温高于38.5℃或低于36.5℃;(3)白细胞计数高于10×109/L或低于4×109/L。根据患者呼吸功能(氧合指数)、肾功能(血浆肌酐浓度)、肝功能(血浆胆红素浓度)、心血管功能(血压调整后心率)、凝血功能(血小板计数)和中枢神经系统(格拉斯哥评分)等变化计数器官功能障碍评分。创伤ISS评分采用简明损伤定级标准(1998版)。8.统计分析:各SNP的等位基因型频率由基因计数获得。H-W平衡采用卡方检验计算。组间荧光素酶活性通过单因素方差分析比较。多态性与MODS评分、细胞因子表达水平间的关系采用单因素方差方法统计。等位计量效应采用线性回归分析统计,并用年龄、性别及ISS评分校正。显性及隐性遗传模式下各基因型与脓毒症发生率之间的关系采用卡方检验方法统计。等位计量效应采用多重Logister回归分析计算,并对年龄、性别及ISS评分校正。检验效能通过在线软件Power and Sample Size Program software (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize)计算。结果:1.通过对中国汉族重庆人群IL-6启动子区SNP筛查,发现中国汉族人群中IL-6启动子区仅含有-572C/G多态性位点,发生频率为23.8%。未发现在西方人群中普遍存在的-597和-174位点多态性。生物信息学分析显示IL-6 -572位点不位于重复序列内,且可能引起转录因子结合位点改变。通过报告基因实验和血浆IL-6表达水平检测,发现含-572C的质粒转录活性明显高于-572G质粒,携带-572G个体血浆IL-6表达水平明显低于-572C个体,且呈等位剂量效应关系。临床关联研究证实携带IL-6/-572G突变等位基因的创伤病人脓毒症发生率明显低于IL-6/-572C携带者。-572G位点与创伤病人的MODS评分降低有一定关系,但各基因型组间无明显差异。2.研制出检测13个细胞因子SNP(IL-1α-889C/T,IL-1β-1470G/C、-511C/T、-31C/T,IL-4 -589T/C,IL-6 -572C/G,IL-8 -251T/A,IL-10 -1082A/G、-819T/C、-592A/C,TNF-α-308G/A,TNF-β252G/A,IFN-γ874A/T)的SNP芯片,重复性达到96%以上,准确率达到100%。通过分析各基因型与细胞因子水平、脓毒症发生率和MODS评分间关系,筛选出8个与三者关系最为密切的多态性位点(IL-1β-1470G/C、-511C/T、-31C/T, IL-4 -589T/C, IL-6 -572C/G, IL-8 -252T/A, IL-10 -819T/C, TNF-α-308G/A)。各患者所含这8个SNPs中高反应性基因型的数量越多,则其脓毒症发生率越高,同时MODS评分也越高。3.通过对105例严重创伤患者进行基因分型,发现CD14/-1145、-159和MD-2/-1625多态性发生频率分别为42.9%、47.6%和22.4%,均为高频SNP位点,且与健康人群中发生频率相似。CD14/-1145A、-159C携带者创伤病人MODS评分和脓毒症发生率低于野生型等位基因携带者,且两个位点具有协同效应。同时,证实MD2/-1625G突变等位基因携带者创伤病人MOD评分和脓毒症发生率明显高于野生型等位基因携带者。结论:1.中国汉族人群中IL-6基因启动子区仅存在-572C/G多态性位点,未发现西方人群中存在的-597和-174多态性位点。IL-6/-572C→G变异能明显降低启动子活性、抑制IL-6基因转录,为功能性多态性位点。2.成功研制出检测9个细胞因子基因、13个SNPs位点的SNP芯片。3.利用SNP芯片,筛选出能预测严重创伤患者并发症风险性的8个细胞因子多态性位点(IL-1β-1470G/C、-511C/T、-31C/T, IL-4 -589T/C, IL-6 -572C/G, IL-8 -252T/A, IL-10 -819T/C, TNF-α-308G/A),以及各多态性位点间协同效应与并发症风险程度的关系。4.利用临床关联研究,进一步明确模式识别受体CD14 -159、-1145和MD-2 -1625对于判断严重创伤患者并发症风险性也具有重要意义。更多还原

【Abstract】 Background:Major trauma is the leading cause of death in young adults and the third most common cause of death overall. Victims of severe injuries who survive the initial hours are likely to develop sepsis and sepsis-associated multiple organ dysfunction syndrome (MODS), which remains a worldwide problem that is the leading cause of intensive care unit mortality. The pathogenesis underlying sepsis and MODS has been demonstrated to be overwhelming immune inflammatory response, which might be preventable and controllable if being effectively managed at the early stage. To this end, it might be pivotal to find an effective way for evaluation of the risk for the sepsis and MODS in major trauma patients.Cytokines are critical determinants for the magnitude of immune inflammatory response, which might profoundly affect the inflammatory response to trauma and predispose trauma patients to susceptibility or resistance to sepsis and MODS. While pattern recognition receptors (PRRs), which have been shown to be elevated after injury or hemorrhagic shock, are considered as important mechanism for that injury primes the innate immune system and leads to overwhelming proinflammatory response when bacteria and their products enter into body. Therefore, SNPs of some important cytokines and PRRs were selected. Their functionality and clinical relevance were analyzed through bioinformatic studies, in vitro functional studies and clinical association studies.Materials and Methods:1. Study population. A total of 656 unrelated adult Chinese, comprising 348 healthy blood donors and 308 patients with major trauma, were recruited in this study. All of them are Han Chinese and live in the Chongqing district. The 348 healthy blood donors consisted of 219 men and 129 women, with a median age of 25 yrs (range, 18–53 yrs). The 308 trauma patients, 242 men and 46 women, were consecutively admitted to the Department of Trauma Surgery in the Daping Hospital and the Chongqing Emergency Medical Center, Chongqing, China, between January 1, 2005, and January 1, 2008. They were enrolled in the study if they met the following criteria: 1) between 16 and 70 yrs of age, 2) expected Injury Severity Score of 16, and 3) probability of survival of 1 wk (sepsis or multiple organ dysfunction usually occur 1 wk after trauma). Their average age was 38.5 and median ISS was 25.5.2. Sequencing of the IL-6 Promoter and bioinformatic analysis. According to distribution of IL-6 promoter SNPs in the International HapMap Project SNP database, a 3-kb sequence from position -2996 to +53 of the IL-6 gene was sequenced for discovery of SNPs (refer to GenBank, Accession No. Y00081.1). Human genetic DNA samples were collected from 27 unrelated, healthy Han Chinese subjects. Overlapping primer sets were designed on the basis of size and overlap of polymerase chain reaction amplicons by use of Primer 3.0. SNPs were found through conting construction and multiple assemble. Then, IL-6 -572C/G was analyzed using online bioinformatics tools (http://125.itba.mi.cnr. it/genebin/wwwrepeat.pl and http://motif.genome.ad.jp/) to predict its potential functions.3. Report assay. In order to further validate the effect of -572C/G on IL-6 promoter activities, the wildtype IL-6 gene promoter, a sequence of -1728~+36bp, was amplificated. pGL3BH-CC plasmid was constructed and Site-Directed mutated to pGL3BH-GG plasmid. After transfection of the both plasmids into U-937 and K-562 cell lines, luciferase activity was measured using the Luciferase Assay System.4. Preparation of allele-specific oligonucleotide array. Allele-specific oligonucleotides(ASOs) were designed with Oligo 6 and synthesized using an Expedite 8909 nucleic acid synthesizer with a 5-terminal (CH2)6–NH2 modification and purified by perfusion chromatography on a BioCAD Sprint system. The ASOs were diluted to 100μmol/L with 3×SSC and were spotted on aldehyde coated glass slides using a Cartesian Pixsys 7500. The slides were stored at -4°C until use. Before use, the slides were washed in 0.2% SDS for 3 min and pure water for 2 min, and dried in the air.5. Genotyping. Genotyping was performed through RFLP-PCR (CD14/-159, -1145, MD-2/-1625 and IL-6/-572) and SNP array (13 cytokine gene polymorphisms) respectively. The results were confirmed by resequencing random ten samples. 6. Plasma cytokines’level assay. The whole blood collected from the healthy blood donors and trauma patients within 24 hrs after admission were mixed 1:1 with Roswell Park Memorial Institute 1640 culture medium, and incubated with 100ng/ml lipopolysaccharide in a sample mixer at 37°C for 4 hrs. The cytokines’levels in the supernatants were determined with enzyme-linked immunosorbent assay according to the manufacturer’s instructions.7. Clinical evaluation. The patients with major trauma were prospectively monitored after admission by physicians who did not know the genotypes. Sepsis was defined if patients met all the following criteria: clinical evidence of infection, body temperature of 38.5°C or 36.5°C, and leukocyte count of 10×109/L or 4×109/L. When patients were diagnosed with sepsis, assessments of respiratory (PaO2/FIO2 ratio), hepatic (serum bilirubin), renal (serum creatinine), cardiovascular (pressure-adjusted heart rate), hematologic (platelet count), and central nervous (Glasgow Coma Scale) systems were made and calculated multiple organ dysfunction scores. ISS was performed according to the abbreviated injury scale 1998.8. Statistical analysis. Allele frequencies for each SNP were determined by gene counting. Genotype distribution was tested for departure from Hardy-Weinberg equilibrium using chisquare analyses. Luciferase activities were compared using one-way analysis of variance. The association between SNPs and MODS or plasma cytokines’levels was determined using one-way analysis of variance. We performed linear regression analysis to quantify the allele-dose effect. The association of genotypes with sepsis morbidity was determined by chi-square analysis. Odds ratios with 95% confidence intervals were calculated by multiple logistic regression analyses to estimate the relative risk of sepsis. The power was calculated by PS: Power and Sample Size Calculation software (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize). Results were considered to be significant at P<0.05. All statistical analyses were carried out using SPSS Version 11.0. Results:1. Only one variant (C-572G) was identified in IL-6 promoter in Chinese Han population, with a frequency of 27.8%. The C/G variation at position -572 could reduce transcriptional activity of the IL-6 promoter as shown in both U-937 and K-562 cell lines and was associated with IL-6 production by peripheral leukocytes in response to ex vivo lipopolysaccharide stimulation in an allele dose–dependent effect. Moreover, the -572 polymorphism was associated with lower risk of sepsis in major trauma patients.2. Nine key cytokine genes and 13 SNPs (IL-1α-889C/T,IL-1β-1470G/C、-511C/T、-31C/T,IL-4 -589T/C,IL-6 -572C/G,IL-8 -251T/A,IL-10 -1082A/G、-819T/C、-592A/C,TNF-α-308G/A , TNF-β252G/A , IFN-γ874A/T) were selected. Allele-specific oligonucleotide array was developed successfully and used for genotyping in 308 patients with major trauma. 8 SNPs (IL-1β-1470G/C、-511C/T、-31C/T, IL-4 -589T/C, IL-6 -572C/G, IL-8 -252T/A, IL-10 -819T/C, TNF-α-308G/A ) were selected out of the 13 SNPs according to their significant relationship with respective cytokine levels, sepsis morbidity and MODS scores. The number of hyper-responsive alleles of the 8 SNPs a patient has is significantly correlated with his risk of developing sepsis and MODS.3. Trauma patients carrying CD14/–1145 A or–159 C allele had lower multiple organ dysfunction score and sepsis morbidity rate when compared with those with–1145 G or–159 T allele. In addition, both polymorphisms were in strong linkage disequilibrium, and had a marked synergistic effect. While patients who possessed the MD-2/1625 G allele were more likely to experience complications with organ dysfunction and sepsis after major trauma.Conclusions:1. -572C/G is the only SNP found in IL-6 gene promoter in Chinese Han population. -597 and -174 poymorphisms, common SNPs in Caucasian population, are not exit in Chinese Han population. IL-6/-572C→G variation could reduce promoter activity and inhibit IL-6 gene transcription, showing a functional SNP.2. Allele-specific oligonucleotide array was successfully developed for genotyping 13 SNPs in 9 cytokine genes.3. Using SNP array, 8 SNPs out of the 13 SNPs (IL-1β/-1470G, IL-1β/-511C, IL-1β/-31T, IL-4/-589C, IL-6/-572C, IL-8/-252T, IL-10/-819T and TNFα/-308A) are shown to be well associated with the development of sepsis and MODS in trauma patients.4. CD14-159T/C, -1145G/A and MD-2 -1625C/G might be used as risk determinants for sepsis and MODS in trauma patients.更多还原