创伤后脓毒症遗传易感性的流行病学和临床关联性研究

发布时间：2018-08-22 21:14

【摘要】：背景目前,创伤是世界范围内的一个较为严重的公共健康问题,也是青年人致死的主要原因。随着治疗手段和护理技术的不断发展,严重创伤病人也逐渐能够在创伤中生存下来。然而创伤后并发症仍然是住院病人致死的主要原因。因此,在第一时间预测并阻止创伤并发症的发生和发展将会是治疗创伤的一项有效措施。众所周知,脓毒症以及多器官功能障碍综合征(MODS)是较为常见且严重的创伤并发症。当机体遇到微生物侵袭后所发生的超过机体承受能力的炎症反应是脓毒症发生的根本原因。众多炎症因子的产生和释放也将会促进血管内凝血和多器官功能障碍的发生。这种过度的炎症反应发生的机制或许能为我们探索脓毒症以及多器官功能障碍的相关危险因素提供有价值的线索。白介素6 (interleukin-6,IL-6)是一种由内皮细胞、单核细胞以及成纤维细胞等产生的细胞因子。它能够刺激B、T等淋巴细胞,而且可以诱导发热。目前的一些研究证实IL-6在细菌入侵机体时所发生的炎症反应中扮演者重要的角色。Toll样受体2 (Toll-like Receptor 2, TLR2)是TLR家族中的一员,它能够识别一系列的细菌脂蛋白分子。有研究认为TLR2蛋白分子是抵抗感染的最原始屏障中的一员。截止目前,越来越多的学者试图探索IL-6和TLR2两个基因上的单核苷酸多态性与脓毒症发生风险的临床关联性。在这些常见的SNP位点中,IL-6-174G/C和TLR2 Arg753Gln两个多态性位点被广泛研究。然而,目前的结果却存在较大的争议。因此,我们在论文的第一部分通过meta分析进一步探索IL-6-174G/C和TLR2 Arg753Gln两个位点的多态性对脓毒症发生和发展的影响。过氧化物酶体增殖物激活受体(Peroxisome Proliferator-Activated Receptors, PPARs)是一类核受体超家族的转录因子。它们在调节代谢、细胞的增殖和分化以及免疫反应等过程中发挥着重要的作用。目前为止,在人类中共发现了 PPAR家族的三种亚型,即PPARα、PPARβ/δ以及PPARγ。除了关于PPAR家族成员在代谢方面重要作用的报道之外,人们开始越来越多地探索该家族成员在炎性疾病的发生和发展中的作用。这其中包括炎性肠病、肿瘤和蛋白尿性肾病等较为常见的炎性疾病。目前的研究显示这类受体能够对NF-κB的下游的炎性目的基因进行转录调节,这或许是该家族成员发挥抗炎作用的一种较为常见的机制。而且PPAR家族成员的配体能够对炎症反应发生部位的白细胞聚集过程产生抑制作用。因此,探索PPAR家族成员在脓毒症和多器官功能障碍发生发展中的潜在作用或许对我们进一步明确该疾病的病理生理以及治疗起到重要的作用。近期,越来越多的研究发现单核苷酸多态性(SNP)对创伤后不同患者的炎症反应差异有着重要的作用。虽然现阶段不少学者已经对PPAR家族基因在炎性疾病中的作用展开一定的研究,但是针对该家族成员的等位基因突变对创伤后脓毒症以及MODS的影响却少有探究。基于以上信息,我们在论文的第二部分通过在PPAR家族成员的全基因范围内挑选SNP位点,继而探索其对创伤后脓毒症和多器官功能障碍的影响。材料和方法1. meta分析我们首先通过PubMed, Embase和Web of Knowledge三个数据库对已发表文献进行检索。两位实验人员按照相应的标准独立对检索的文献展开筛查,并完成对相关数据的提取工作。由于目前人们对脓毒症的遗传模型还不确定,所以我们采用等位基因模型(B vs.A)、共显性模型(BBvs.AA)、隐性模型(BBvs.AB+AA)以及显性模型(BB+AB vs.AA)分别进行meta分析。我们用比值比和95%置信区间来评价相应的SNP位点和脓毒症发生风险的相关性。Z检验用于评价合并后的比值比。除此之外,我们又分别进行了亚组分析、异质性分析、敏感性分析以及发表偏倚的检测来进一步评价相关SNP位点与脓毒症的风险。2. PPAR家族基因多态性与创伤后脓毒症发生风险的临床关联性研究我们研究中共纳入734例中国创伤病人,所有患者均为生活在重庆的汉族人。基因的研究区域包含转录起始位点上游延伸3kb区域、终止密码下游延伸3kb以及基因的所有外显子和内含子。通过登录HapMap数据库的网站,我们获得上述研究区域内的所有SNP位点信息。利用Haploview软件对PPARα、PPARβ和PPARy基因研究区域内的所有SNP位点分别构建单倍型。共计挑选出9个标签SNP位点。我们使用改进的多重高温连接酶检测反应(iMLDR)技术对所收集的样本DNA展开基因分型工作。另外,我们采用酶联免疫反应的方法对经过LPS刺激后的外周血白细胞的肿瘤坏死因子α表达水平进行检测。结果1. meta分析我们的结果显示共有20个研究和7个研究分别探索了 IL-6-174G/C位点的基因多态性与脓毒症的发生和死亡的相关性。Meta分析显示目前在四种遗传模型下,并没有直接的证据证明IL-6-174G/C位点的突变与脓毒症的发生和死亡存在明显的相关性。同样的,亚组分析结果也显示二者之间没有必然的相关性存在。虽然我们在隐性模型下发现IL-6-174G/C位点的突变与脓毒症的死亡率有统计学相关性,但是经过Bonferroni校正后这种关联性消失。在TLR2Arg753Gln位点的基因多态性和脓毒症的发生风险的meta分析中我们共纳入了 12项研究,包含898名脓毒症病人和1517名对照。结果显示在等位基因模型和显性模型下,TLR2Arg753Gln位点的多态性和脓毒症的发生风险存在明显的关联。另外,亚组分析与总的分析结果一致,TLR2Arg753Gln位点的多态性能够影响脓毒症的发生。2. PPAR家族基因多态性与创伤后脓毒症发生风险的临床关联性研究研究中的734名病人均存活48小时以上。随访发现共有300人发生脓毒症,其中138人(46%)出现病原菌血培养阳性。374人(51%)发生器官功能障碍,其中116人发生两个或两个以上器官功能障碍。统计发现呼吸道感染是最常见的感染类型。在所有PPAR家族的SNP位点中我们筛选出9个标签SNP位点,分别为rs135551、rs5769178、rs4253711、rs4823613、rs6902123、rs2016520、rs4684846、rs10865710 和 rs1822825。除rs2016520位点位于5'UTR位置和rs10865710位点位于Exon A2位置以外,其余位点均位于内含子区域内。在我们的研究人群中,这9个标签SNP位点的最小等位基因频率(MAF)分别为 7.6% (rs135551)、15% (rs5769178)、14.4% (rs4253711)、23.2%(rs4823613)、3.1% (rs6902123)、30.4% (rs2016520)、45.8% (rs4684846)、34.6% (rs10865710)和45.5% (rs1822825)。所有位点的基因型在研究人群中的分布均满足哈代-温伯格平衡。进一步统计分析显示,我们所研究的每一个SNP位点的不同基因型的人群在性别、年龄和ISS评分上都没有明显的差异。PPARγ基因rs10865710位点携带G等位基因的病人与携带C等位基因的病人相比会明显提高脓毒症的发生率和MODS评分(隐性模型下该位点突变与脓毒症发生风险的相关性P=0.002,与多器官功能障碍评分的相关性P=0.041;显性模型下位点突变与脓毒症发生风险的相关性P=0.046)。对该位点在等位基因模型下进行回归分析时我们发现该位点与脓毒症的发生风险仍有明显的相关性(P=0.001)。而其他八个位点在我们的研究中并未发现与创伤后脓毒症和MODS评分具有明显的相关性。进一步分析显示,在60例创伤病人的标本(rs10865710位点基因型为GG、GC和CC的样本各20例)中,rs10865710位点的突变与LPS诱导外周白细胞后肿瘤坏死因子α的表达水平有明显的相关性。携带G等位基因的病人肿瘤坏死因子α的表达水平明显高于携带C等位基因的病人(显性模型下P=0.041,隐性模型下P=0.027)。结论本文第一部分结果显示TLR2 Arg753Gln位点的多态性可以显著影响脓毒症的发生风险。因此,该位点或许能够用于脓毒症发生风险的预警诊断。然而,meta分析结果表明IL-6-174 G/C位点的多态性对脓毒症的发生率和死亡率并没有明显影响。我们需要大样本的研究来进一步证实我们的结论。第二部分中,我们在PPAR家族三个成员基因的研究区域内共挑选出9个SNP位点。结果显示在中国汉族人中PPARy基因rs10865710位点的突变与创伤后脓毒症和MODS的风险具有明显的相关性。进一步研究发现该位点突变与LPS诱导的外周白细胞的肿瘤坏死因子α的表达水平有很强的关联性。因此,该位点今后或许可以用来对创伤后病人进行脓毒症和多器官功能障碍的风险评估。
[Abstract]:Background At present, trauma is a serious public health problem worldwide and a major cause of death among young people. With the development of treatment and nursing techniques, severe trauma patients can gradually survive trauma. However, post-traumatic complications are still the main cause of death among hospitalized patients. It is well known that sepsis and multiple organ dysfunction syndrome (MODS) are common and serious traumatic complications. Inflammatory reactions that exceed the body's tolerance occur when the organism encounters microbial invasion. Interleukin-6 (interleukin-6) may provide valuable clues to explore the risk factors associated with sepsis and multiple organ dysfunction. Leukemin-6 (IL-6) is a cytokine produced by endothelial cells, monocytes and fibroblasts. It can stimulate lymphocytes such as B and T and induce fever. Recent studies have confirmed that IL-6 plays an important role in the inflammatory response to bacterial invasion. Toll-like Receptor 2 (Toll-like Receptor 2) TLR2, a member of the TLR family, is capable of recognizing a range of bacterial lipoprotein molecules. Studies have suggested that TLR2 is one of the most primitive barriers against infection. Among these common SNP loci, IL-6-174G/C and TLR2 Arg753Gln have been extensively studied. However, the results are controversial. Therefore, in the first part of this paper, we further explore the effects of IL-6-174G/C and TLR2 Arg753Gln polymorphisms on the occurrence and development of sepsis by meta-analysis. Peroxisome Proliferator-Activated Receptors (PPARs) are transcription factors of a nuclear receptor superfamily. They play an important role in regulating metabolism, cell proliferation and differentiation, and immune response. Up to now, three subtypes of PPARs have been found in humans. In addition to reports on the important role of PPAR family members in metabolism, more and more people are exploring the role of PPAR family members in the development and progression of inflammatory diseases, including inflammatory bowel disease, tumor and proteinuria nephropathy. It is suggested that these receptors can transcribe and regulate the downstream inflammatory target genes of NF-kappa B, which may be a common mechanism for the anti-inflammatory effect of members of the PPAR family. Moreover, ligands of PPAR family members can inhibit the leukocyte aggregation process at inflammatory sites. The potential role of sepsis and multiple organ dysfunction may play an important role in further understanding the pathophysiology and treatment of sepsis. Recently, more and more studies have found that single nucleotide polymorphism (SNP) plays an important role in differentiating inflammatory responses in different post-traumatic patients. Scholars have studied the role of PPAR family genes in inflammatory diseases, but little has been done to investigate the effects of allele mutations on post-traumatic sepsis and MODS. Material and Methods 1. Meta-analysis was used to retrieve the published literature through PubMed, Embase and Web of Knowledge databases. Two experimenters independently screened the retrieved literature according to the corresponding criteria and completed the relevant data extraction. Because the genetic model of sepsis is still uncertain, we used the allele model (B vs. A), the co-dominant model (BB vs. AA), the recessive model (BB vs. AB + AA) and the dominant model (BB + AB vs. AA) for meta-analysis. We used the ratio and 95% confidence interval to evaluate the corresponding SNP loci and sepsis occurrence. In addition, subgroup analysis, heterogeneity analysis, sensitivity analysis, and publication bias tests were performed to further evaluate the association between SNP loci and sepsis risk. 2. Clinical association between PPAR family genetic polymorphisms and the risk of post-traumatic sepsis. In this study, 734 Chinese trauma patients were enrolled. All of them were Han Chinese living in Chongqing. The gene study area included an extension of 3 KB upstream of the transcriptional initiation site, a termination of 3 KB downstream of the codon, and all the exons and introns of the gene. Haploview software was used to construct haplotypes of all the SNP loci in the study area of PPAR alpha, PPAR beta and PPAR y genes. A total of nine labeled SNP loci were selected. The expression of TNF-alpha in peripheral blood leukocytes stimulated by LPS was detected by ELISA. Results 1. Meta-analysis showed that 20 studies and 7 studies explored the association of IL-6-174G/C gene polymorphism with the occurrence and death of sepsis. Ta analysis showed that there was no direct evidence of a significant correlation between IL-6-174G/C mutation and the occurrence and death of sepsis in the four genetic models. Similarly, subgroup analysis showed that there was no necessary correlation between IL-6-174G/C mutation and sepsis. There was a statistically significant association with sepsis mortality, but the association disappeared after Bonferroni's correction. In the meta-analysis of TLR2Arg753Gln gene polymorphism and sepsis risk, we included 12 studies, including 898 sepsis patients and 1517 controls. The results showed allele model and dominance. The polymorphism of TLR2Arg753Gln locus was significantly associated with the risk of sepsis. In addition, the subgroup analysis was consistent with the overall analysis. The polymorphism of TLR2Arg753Gln locus could influence the occurrence of sepsis. 2. The PPAR family gene polymorphism was associated with the risk of post-traumatic sepsis in a clinical study of 734. All patients survived for more than 48 hours. A total of 300 patients developed sepsis, 138 (46%) had positive blood culture, 374 (51%) had dysfunction of generator organs, 116 of whom had two or more organ dysfunction. Respiratory tract infection was the most common type of infection. SNP sites were found in all PPAR families. Nine SNP loci were screened out, including rs135551, rs5769178, rs4253711, rs4823613, rs6902123, rs2016520, rs4684846, rs10865710, and rs1822825. Except for rs2016520 at 5'UTR and rs10865710 at Exon A2, all the other loci were located in the intron region. The minimal allele frequencies (MAF) of labeled SNP loci were 7.6% (rs135551), 15% (rs5769178), 14.4% (rs4253711), 23.2% (rs4823613), 3.1% (rs6902123), 30.4% (rs2016520), 45.8% (rs46846), 34.6% (rs10865710) and 45.5% (rs1822825) respectively. The distribution of all genotypes in the study population met Hardy-Weinberg equilibrium. Further statistical analysis showed that there were no significant differences in gender, age, and ISS scores among the different genotypes of each SNP locus studied. Patients with PPAR gamma gene rs10865710 carrying the G allele significantly increased the incidence of sepsis and MODS scores (recessive mode) compared with patients with C allele. P = 0.002, P = 0.041, P = 0.046, P = 0.046, P = 0.046, P = 0.002, P = 0.002, P = 0.041, P = 0.046, P = 0.046, P = 0.046. There was a significant correlation (P = 0.001). The other eight loci were not found to be significantly correlated with post-traumatic sepsis and MODS scores in our study. Further analysis showed that mutations at rs10865710 were associated with LPS-induced peripheral leukocytes in 60 trauma patients (20 samples with genotype GG at rs10865710, 20 samples with GC and 20 samples with CC at rs10865710). The expression level of TNF-alpha in patients with G allele was significantly higher than that in patients with C allele (P = 0.041 in dominant model and P = 0.027 in recessive model). Conclusion The results of the first part of this paper show that the polymorphism of TLR2 Arg753Gln site can be significant. However, meta-analysis showed that the polymorphism of the IL-6-174 G/C site had no significant effect on the incidence and mortality of sepsis. We need large sample studies to further confirm our conclusions. Nine SNP loci were identified in the study area of three members of the PPAR family. The results showed that the mutation of the PPARy gene rs10865710 was significantly associated with the risk of post-traumatic sepsis and MODS in Chinese Han population. Alpha expression levels are strongly correlated. Therefore, this locus may be used to assess the risk of sepsis and multiple organ dysfunction in post-traumatic patients in the future.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R641;R459.7

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