發(fā)布時間：2018-08-22 21:14

【摘要】：背景目前,創(chuàng)傷是世界范圍內(nèi)的一個較為嚴重的公共健康問題,也是青年人致死的主要原因。隨著治療手段和護理技術(shù)的不斷發(fā)展,嚴重創(chuàng)傷病人也逐漸能夠在創(chuàng)傷中生存下來。然而創(chuàng)傷后并發(fā)癥仍然是住院病人致死的主要原因。因此,在第一時間預(yù)測并阻止創(chuàng)傷并發(fā)癥的發(fā)生和發(fā)展將會是治療創(chuàng)傷的一項有效措施。眾所周知,膿毒癥以及多器官功能障礙綜合征(MODS)是較為常見且嚴重的創(chuàng)傷并發(fā)癥。當(dāng)機體遇到微生物侵襲后所發(fā)生的超過機體承受能力的炎癥反應(yīng)是膿毒癥發(fā)生的根本原因。眾多炎癥因子的產(chǎn)生和釋放也將會促進血管內(nèi)凝血和多器官功能障礙的發(fā)生。這種過度的炎癥反應(yīng)發(fā)生的機制或許能為我們探索膿毒癥以及多器官功能障礙的相關(guān)危險因素提供有價值的線索。白介素6 (interleukin-6,IL-6)是一種由內(nèi)皮細胞、單核細胞以及成纖維細胞等產(chǎn)生的細胞因子。它能夠刺激B、T等淋巴細胞,而且可以誘導(dǎo)發(fā)熱。目前的一些研究證實IL-6在細菌入侵機體時所發(fā)生的炎癥反應(yīng)中扮演者重要的角色。Toll樣受體2 (Toll-like Receptor 2, TLR2)是TLR家族中的一員,它能夠識別一系列的細菌脂蛋白分子。有研究認為TLR2蛋白分子是抵抗感染的最原始屏障中的一員。截止目前,越來越多的學(xué)者試圖探索IL-6和TLR2兩個基因上的單核苷酸多態(tài)性與膿毒癥發(fā)生風(fēng)險的臨床關(guān)聯(lián)性。在這些常見的SNP位點中,IL-6-174G/C和TLR2 Arg753Gln兩個多態(tài)性位點被廣泛研究。然而,目前的結(jié)果卻存在較大的爭議。因此,我們在論文的第一部分通過meta分析進一步探索IL-6-174G/C和TLR2 Arg753Gln兩個位點的多態(tài)性對膿毒癥發(fā)生和發(fā)展的影響。過氧化物酶體增殖物激活受體(Peroxisome Proliferator-Activated Receptors, PPARs)是一類核受體超家族的轉(zhuǎn)錄因子。它們在調(diào)節(jié)代謝、細胞的增殖和分化以及免疫反應(yīng)等過程中發(fā)揮著重要的作用。目前為止,在人類中共發(fā)現(xiàn)了 PPAR家族的三種亞型,即PPARα、PPARβ/δ以及PPARγ。除了關(guān)于PPAR家族成員在代謝方面重要作用的報道之外,人們開始越來越多地探索該家族成員在炎性疾病的發(fā)生和發(fā)展中的作用。這其中包括炎性腸病、腫瘤和蛋白尿性腎病等較為常見的炎性疾病。目前的研究顯示這類受體能夠?qū)F-κB的下游的炎性目的基因進行轉(zhuǎn)錄調(diào)節(jié),這或許是該家族成員發(fā)揮抗炎作用的一種較為常見的機制。而且PPAR家族成員的配體能夠?qū)ρ装Y反應(yīng)發(fā)生部位的白細胞聚集過程產(chǎn)生抑制作用。因此,探索PPAR家族成員在膿毒癥和多器官功能障礙發(fā)生發(fā)展中的潛在作用或許對我們進一步明確該疾病的病理生理以及治療起到重要的作用。近期,越來越多的研究發(fā)現(xiàn)單核苷酸多態(tài)性(SNP)對創(chuàng)傷后不同患者的炎癥反應(yīng)差異有著重要的作用。雖然現(xiàn)階段不少學(xué)者已經(jīng)對PPAR家族基因在炎性疾病中的作用展開一定的研究,但是針對該家族成員的等位基因突變對創(chuàng)傷后膿毒癥以及MODS的影響卻少有探究。基于以上信息,我們在論文的第二部分通過在PPAR家族成員的全基因范圍內(nèi)挑選SNP位點,繼而探索其對創(chuàng)傷后膿毒癥和多器官功能障礙的影響。材料和方法1. meta分析我們首先通過PubMed, Embase和Web of Knowledge三個數(shù)據(jù)庫對已發(fā)表文獻進行檢索。兩位實驗人員按照相應(yīng)的標準獨立對檢索的文獻展開篩查,并完成對相關(guān)數(shù)據(jù)的提取工作。由于目前人們對膿毒癥的遺傳模型還不確定,所以我們采用等位基因模型(B vs.A)、共顯性模型(BBvs.AA)、隱性模型(BBvs.AB+AA)以及顯性模型(BB+AB vs.AA)分別進行meta分析。我們用比值比和95%置信區(qū)間來評價相應(yīng)的SNP位點和膿毒癥發(fā)生風(fēng)險的相關(guān)性。Z檢驗用于評價合并后的比值比。除此之外,我們又分別進行了亞組分析、異質(zhì)性分析、敏感性分析以及發(fā)表偏倚的檢測來進一步評價相關(guān)SNP位點與膿毒癥的風(fēng)險。2. PPAR家族基因多態(tài)性與創(chuàng)傷后膿毒癥發(fā)生風(fēng)險的臨床關(guān)聯(lián)性研究我們研究中共納入734例中國創(chuàng)傷病人,所有患者均為生活在重慶的漢族人�；虻难芯繀^(qū)域包含轉(zhuǎn)錄起始位點上游延伸3kb區(qū)域、終止密碼下游延伸3kb以及基因的所有外顯子和內(nèi)含子。通過登錄HapMap數(shù)據(jù)庫的網(wǎng)站,我們獲得上述研究區(qū)域內(nèi)的所有SNP位點信息。利用Haploview軟件對PPARα、PPARβ和PPARy基因研究區(qū)域內(nèi)的所有SNP位點分別構(gòu)建單倍型。共計挑選出9個標簽SNP位點。我們使用改進的多重高溫連接酶檢測反應(yīng)(iMLDR)技術(shù)對所收集的樣本DNA展開基因分型工作。另外,我們采用酶聯(lián)免疫反應(yīng)的方法對經(jīng)過LPS刺激后的外周血白細胞的腫瘤壞死因子α表達水平進行檢測。結(jié)果1. meta分析我們的結(jié)果顯示共有20個研究和7個研究分別探索了 IL-6-174G/C位點的基因多態(tài)性與膿毒癥的發(fā)生和死亡的相關(guān)性。Meta分析顯示目前在四種遺傳模型下,并沒有直接的證據(jù)證明IL-6-174G/C位點的突變與膿毒癥的發(fā)生和死亡存在明顯的相關(guān)性。同樣的,亞組分析結(jié)果也顯示二者之間沒有必然的相關(guān)性存在。雖然我們在隱性模型下發(fā)現(xiàn)IL-6-174G/C位點的突變與膿毒癥的死亡率有統(tǒng)計學(xué)相關(guān)性,但是經(jīng)過Bonferroni校正后這種關(guān)聯(lián)性消失。在TLR2Arg753Gln位點的基因多態(tài)性和膿毒癥的發(fā)生風(fēng)險的meta分析中我們共納入了 12項研究,包含898名膿毒癥病人和1517名對照。結(jié)果顯示在等位基因模型和顯性模型下,TLR2Arg753Gln位點的多態(tài)性和膿毒癥的發(fā)生風(fēng)險存在明顯的關(guān)聯(lián)。另外,亞組分析與總的分析結(jié)果一致,TLR2Arg753Gln位點的多態(tài)性能夠影響膿毒癥的發(fā)生。2. PPAR家族基因多態(tài)性與創(chuàng)傷后膿毒癥發(fā)生風(fēng)險的臨床關(guān)聯(lián)性研究研究中的734名病人均存活48小時以上。隨訪發(fā)現(xiàn)共有300人發(fā)生膿毒癥,其中138人(46%)出現(xiàn)病原菌血培養(yǎng)陽性。374人(51%)發(fā)生器官功能障礙,其中116人發(fā)生兩個或兩個以上器官功能障礙。統(tǒng)計發(fā)現(xiàn)呼吸道感染是最常見的感染類型。在所有PPAR家族的SNP位點中我們篩選出9個標簽SNP位點,分別為rs135551、rs5769178、rs4253711、rs4823613、rs6902123、rs2016520、rs4684846、rs10865710 和 rs1822825。除rs2016520位點位于5'UTR位置和rs10865710位點位于Exon A2位置以外,其余位點均位于內(nèi)含子區(qū)域內(nèi)。在我們的研究人群中,這9個標簽SNP位點的最小等位基因頻率(MAF)分別為 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)。所有位點的基因型在研究人群中的分布均滿足哈代-溫伯格平衡。進一步統(tǒng)計分析顯示,我們所研究的每一個SNP位點的不同基因型的人群在性別、年齡和ISS評分上都沒有明顯的差異。PPARγ基因rs10865710位點攜帶G等位基因的病人與攜帶C等位基因的病人相比會明顯提高膿毒癥的發(fā)生率和MODS評分(隱性模型下該位點突變與膿毒癥發(fā)生風(fēng)險的相關(guān)性P=0.002,與多器官功能障礙評分的相關(guān)性P=0.041;顯性模型下位點突變與膿毒癥發(fā)生風(fēng)險的相關(guān)性P=0.046)。對該位點在等位基因模型下進行回歸分析時我們發(fā)現(xiàn)該位點與膿毒癥的發(fā)生風(fēng)險仍有明顯的相關(guān)性(P=0.001)。而其他八個位點在我們的研究中并未發(fā)現(xiàn)與創(chuàng)傷后膿毒癥和MODS評分具有明顯的相關(guān)性。進一步分析顯示,在60例創(chuàng)傷病人的標本(rs10865710位點基因型為GG、GC和CC的樣本各20例)中,rs10865710位點的突變與LPS誘導(dǎo)外周白細胞后腫瘤壞死因子α的表達水平有明顯的相關(guān)性。攜帶G等位基因的病人腫瘤壞死因子α的表達水平明顯高于攜帶C等位基因的病人(顯性模型下P=0.041,隱性模型下P=0.027)。結(jié)論本文第一部分結(jié)果顯示TLR2 Arg753Gln位點的多態(tài)性可以顯著影響膿毒癥的發(fā)生風(fēng)險。因此,該位點或許能夠用于膿毒癥發(fā)生風(fēng)險的預(yù)警診斷。然而,meta分析結(jié)果表明IL-6-174 G/C位點的多態(tài)性對膿毒癥的發(fā)生率和死亡率并沒有明顯影響。我們需要大樣本的研究來進一步證實我們的結(jié)論。第二部分中,我們在PPAR家族三個成員基因的研究區(qū)域內(nèi)共挑選出9個SNP位點。結(jié)果顯示在中國漢族人中PPARy基因rs10865710位點的突變與創(chuàng)傷后膿毒癥和MODS的風(fēng)險具有明顯的相關(guān)性。進一步研究發(fā)現(xiàn)該位點突變與LPS誘導(dǎo)的外周白細胞的腫瘤壞死因子α的表達水平有很強的關(guān)聯(lián)性。因此,該位點今后或許可以用來對創(chuàng)傷后病人進行膿毒癥和多器官功能障礙的風(fēng)險評估。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R641;R459.7

【參考文獻】

相關(guān)期刊論文 前2條

1 Sawako Kuruma;Naoto Egawa;Masanao Kurata;Goro Honda;Terumi Kamisawa;Junko Ueda;Hiroshi Ishii;Makoto Ueno;Haruhisa Nakao;Mitsuru Mori;Keitaro Matsuo;Satoyo Hosono;Shinichi Ohkawa;Kenji Wakai;Kozue Nakamura;Akiko Tamakoshi;Masanori Nojima;Mami Takahashi;Kazuaki Shimada;Takeshi Nishiyama;Shogo Kikuchi;Yingsong Lin;;case-control study of diabetes-related genetic variants and pancreatic cancer risk in Japan[J];World Journal of Gastroenterology;2014年46期

2 Yi Ding;Zhi-Rong Guo;Ming Wu;Qiu Chen;Hao Yu;Wen-Shu Luo;;Gene-Gene Interaction between PPARd and PPARy Is Associated with Abdominal Obesity in a Chinese Population[J];遺傳學(xué)報;2012年12期

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本文編號：2198316