摘要
临床流行病学资料统计显示,脓毒症是临床危重病患者的一个重要死亡原因,革兰氏阴性细菌细胞壁的主要毒性成分—LPS是导致脓毒症的主要致病因子之一。既往我们主要针对LPS受体在脓毒症发病机理中的作用开展了大量探索性研究,现已明确TLR4/CD14/MD-2是机体识别LPS的关键模式受体,其中CD14/ MD-2基因多态性在脓毒症的易感性及严重创伤预后方面发挥了重要作用。TLR4是LPS激活细胞的重要参与分子,尽管有研究表明其基因编码区(coding sequence,CDS)及3′非翻译区(untranslated region,UTR)基因多态性与脓毒症易感性有关,但其启动子区SNPs在中国人群中的分布以及在严重创伤病人预后中的作用尚不清楚。为此,本研究用生物信息学方法筛选出TLR4基因启动子区可能影响靶基因表达的SNP位点,然后以中国重庆地区汉族人群为研究对象,采用RFLP法对SNP发生及分布频率进行检测分析,通过构建SNP位点不同等位基因启动子载体,以双荧光素酶报告系统观察SNP对TLR4基因启动子活性的影响,以流式细胞仪检测不同基因型个体TLR4蛋白的表达水平,并探讨了不同等位基因个体全血对LPS刺激的反应性差异,最后通过对临床严重创伤病人标本分析进一步探讨上述SNPs与严重创伤病人预后之间的关系。主要结果与结论如下:
1.生物信息学分析显示,TLR4基因启动子区-3000 bp内有12个SNP位点,其中-2242 T→C、-1892 G→A和-1837 A→G三个SNP位点碱基变异可能影响TLR4基因启动子区与转录因子的结合活性。
2.对379名重庆地区汉族健康人群的SNP调查分析显示,TLR4基因启动子区存在-2242 T→C、-1892 G→A和-1837 A→G碱基变异,发生频率分别为43.27%、27.70%和42.75%,均为高频SNP。
3.通过PCR和定点突变技术,构建了含TLR4基因-2331 ~ +14区域启动子及相应单点及多点突变启动子质粒。化学发光分析显示,-2242 C启动子活性明显高于-2242 T启动子活性,而其它突变启动子活性无明显差异。说明-2242 T→C碱基变异可增强TLR4基因启动子活性,有可能为功能性突变。流式细胞仪检测结果显示,正常对照组中三位点三种基因型粒细胞表达TLR4均无显著性差异;LPS刺激组中-2242位点CC纯合子和TC杂合子TLR4蛋白表达水平均显著高于TT纯合子,而-1892和-1837位点则无显著性差异,进一步说明-2242 T→C碱基变异可通过影响基因启动子活性增强靶基因的表达。
4.以LPS刺激不同基因型个体全血,-2242 CC基因型个体TNFα、IL-6和IL-8产生水平均最高,TC基因型次之,而TT基因型细胞因子产生水平最低,提示-2242 T→C碱基变异能显著增强个体对内毒素的反应性。说明该SNP对于临床脓毒症及其它炎症疾病易感性的判定具有重要意义。
5.通过对153例严重创伤病人针对-2242位点基因分型及临床资料分析,进一步验证了含TLR4基因5′启动子区-2242 C等位基因的个体在严重创伤后发展成MODS及脓毒症的危险性相对较高,对脓毒症可能是一个易感因子。因此可作为严重创伤后脓毒症等炎性相关疾病易感性的一个重要指标。
Clinical epidemiology study has shown that sepsis is an important cause of death of patients with major trauma, and LPS plays a central role in the pathogenesis of sepsis. We carried out a great quantity of exploratory study on LPS receptors in the past ten years, reseach results demonstrate that TLR4/CD14/MD-2 complex is the crucial pattern recognition receptor of LPS, and CD14, MD-2 gene polymorphisms are associated with susceptivity to sepsis and prognosis of patients with major trauma. TLR4 is also a key molecule of pattern recognition receptor in LPS passthway, though its polymorphisms in the coding sequence and 3′untranslated region (UTR) are well associated with the susceptibility to sepsis, however the distribution of its promoter polymorphisms in China population, and their clinical relevance in patients with major trauma is still unclear.
In this study, we selected the potential SNPs in TLR4 gene promoter, which maybe influence TLR4 gene expression through bioinformatics analysis. Then, we genotyped the selected SNPs in Chongqing Han population by method of restriction fragment length polymorphism. To study the function of the SNPs, we constructed different allele promoter plasmids and measured TLR4 promoter activity by Dual-Luciferase Reporter assay system. We also examined different genotype carriers’TLR4 protein levels and the significance of their whole-blood leukocytes response to LPS stimulation. Finally, we investigated the possible functional significance by observing the association with the prognosis of patients with major trauma. The main results and conclusions were summarized as follows:
1. Bioinformatics analysis shows that there are twelve SNPs in the upstream 3000 bp of TLR4 promoter. Among them, -2242T→C, -1892G→A, and -1837 A→G can change the type or numbers of binding transcription factors, which indicats that the three SNPs may affect the transcription activities of TLR4 promoter.
2. Genotyping 379 cases of Chongqing Han healthy population shows that -2242T→C, -1892G→A, and -475 A→G all exist in Chinese population, with the frequencies 43.27% , 27.70%, and 42.75% respectively.
3. TLR4 gene promoter (-2331 ~ +14), and the other vectors containing a single mutant genotype of -2242 C, -1892 A, or -1837 G, a double mutant genotype of -2242 C, -1892 A, or -2242 C, -1837 G, or -1892 A, -1837 G, and a combined mutant genotype of -2242 C, -1892 A, and -1837 G are constructed through PCR and site-directed mutagenesis technique. Luciferase Reporter assay shows that -2242 C promoter has a higher activity than -2242 T promoter, it implys that–2242 T→C base variation can enhance the activity of TLR4 promoter. There are no significant difference between -1892 A promoter and -1837 G promoter activity. Fluorescence-activated cell sorter (FACS) shows -2242 CC carriers have higher TLR4 protein expression levels than -2242 TT carriers, which indicates that -2242 T→C may enhance TLR4 gene expression through changing promoter activity.
4. Measuring LPS stimulated whole-blood leukocytes, we found that -2242 CC carriers have the highest TNFα, IL-6, and IL-8 levels and -2242 TT carriers have the least. It indicates that -2242 T→C can significantly enhance reactive potency to LPS. Results show that -2242 SNP may have an important significance in sepsis assessment.
5. In view of the functional effect of the–2242 SNP shown by the above results, we further investigated the clinical relevance of this SNP in 153 patients with major trauma. The result shows that patients who possessed the–2242 C allele were more likely to experience complications with organ dysfunction and sepsis after major trauma. So the TLR4/–2242 polymorphism may be an important functional variant which might be used as a relevant risk estimate for sepsis and other complications in trauma patients.
引文
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