【作者】 王巍巍；

【导师】 宋诗铎； 祁伟； 王玉宝； 刘德梦；

【作者基本信息】 天津医科大学 ， 内科学， 2010， 硕士

【摘要】 目的：脓毒症是危重患者主要死亡原因之一,给临床治疗带来严峻挑战。近些年来,研究表明,血小板活化因子乙酰水解酶(PAF acetylhydrolases, PAF-AH,又称为PLA2G7)能够特异性降解重要促炎因子——血小板活化因子(Platelet activating factor, PAF),在脓毒症中具有潜在的保护作用。PAF-AH基因单核苷酸多态性可能影响其酶的活性,与多种炎症性疾病及其预后相关。目前国内外PAF-AH基因多态性与脓毒症是否相关的报道不多。本实验采用分子生物学方法对脓毒症患者PAF-AH基因Ala379Val和Val279Phe位点基因多态性进行分析,探讨其多态性与脓毒症易感性及预后的关系。方法：1、标本收集：脓毒症组来源于2008年10月至2009年8月天津两家3级甲等医院收治的脓毒症患者66例。依据诊断标准将入选的脓毒症患者分成普通脓毒症组和严重脓毒症组；根据住院28天转归的不同分为生存组和死亡组。对照组来自门诊健康体检人群,共68例。2、采用PCR-RFLP技术,扩增PAF-AH基因的第11和第9外显子,然后分别应用限制性内切酶Fnu4hⅠ、AclⅠ消化PCR产物,通过电泳酶切图谱直接判断碱基的变异,进行SNP基因分型。同时对含有等位基因多态性的片段进行DNA测序。分析66例不同程度的脓毒症患者及68名健康对照者PAF-AH基因Ala379Val、Val279Phe位点的多态性,用统计学方法计算SNP的基因型及等位基因的分布频率。结果：1、PAF-AH基因Ala379Val位点3种基因型中,脓毒症组66例中仅1例纯合子Val/Val型,19例杂合子Val/Ala型,46例纯合子Ala/Ala型；对照组68例中2例Val/Val型,22例Val/Ala型,44例Ala/Ala型。Ala379Val基因型及等位基因频率分布在对照组和脓毒症组之间、在普通脓毒症组和严重脓毒症组及对照组之间、在存活组和死亡组及对照组之间尚无统计学意义。2、PAF-AH基因Val279Phe位点3种基因型中,脓毒症组66例中全部为纯合子Val/Val型,对照组68例中仅1例纯合子Phe/Phe型,其余全部为纯合子Val/Val型,均未发现杂合子Val/Phe型。Val279Phe基因型及等位基因频率分布在对照组和脓毒症组之间、在普通脓毒症组和严重脓毒症组及对照组之间、在存活组和死亡组及对照组之间尚无统计学意义。结论：PAF-AH基因Ala379Val、Val279Phe位点多态性与脓毒症易感性、预后及病情严重程度可能无明显相关。更多还原

【Abstract】 Objective:Sepsis was one of the leading causes of death in critically ill patients; pose serious challenges to the clinical treatment. In recent years, studies had shown that platelet activating factor acetylhydrolase (PAF acetylhydrolases, PAF-AH, also known as PLA2G7) to specific degradation of platelet activating factor which was an important pro-inflammatory cytokines. In septic disease PAF-AH had a potentially protective effect. PAF-AH gene single nucleotide polymorphisms could affect its enzyme activity, with a variety of inflammatory disease and its prognosis. However, currently the relation of sepsis and PAF-AH gene polymorphism was not clarified in domestic and abroad. In this study we used molecular biology methods to analysis PAF-AH gene Ala379Val and Val279Phe polymorphisms in patients with sepsis and explored the relations between polymorphisms and sepsis susceptibility and prognosis.Methods:1. Specimen collection:66 patients of sepsis group were selected from October 2008 to August 2009 in Tianjin two Third Grade Class hospitals admitted with sepsis. Based on diagnostic criteria of sepsis patients were divided into mild group and severe sepsis group; according to 28-day turn-over patients were divided into survival group and death group.68 cases of control group come from the healthy out-patient medical groups.2. By PCR-RFLP technique, PAF-AH gene exon 11 and 9 was amplified. Then PCR products were separately digested by restriction endonuclease Fnu4h I and Acl I. SNP variation and genotyping were directly determined by gel electrophoresis patterns. At the same time, PCR products containing allele gene polymorphism were sent to sequence analysis. PAF-AH gene Ala379Val and Val279Phe polymorphisms were analyzed in 66 cases of varying degrees of sepsis patients and 68 healthy controls. Then SNP genotype and allele frequency distribution were computed.Results:1. All the samples of the PAF-AH gene in the Val379Ala site had three kinds of genotypes:in 66 cases of sepsis group there were 1 homozygous Val/Val type, 19 heterozygous Val/Ala type,46 homozygous Ala/Ala type; in 68 cases of control group there were 2 Val/Val type,22 Val/Ala type,44 Ala/Ala type. The Val379Ala genotypic distribution and allele gene frequency in the patients with sepsis was not significantly different from those in the healthy controls; no statistically significant difference was observed among the survival group、the death group and the control group; no statistically significant difference was observed among the mild sepsis group, severe sepsis group and control group.2. PAF-AH gene of Val279Phe polymorphism could have three kinds of genotypes:All 66 patients in the sepsis group were the homozygous Val/Val type; control group 68 cases, only one case was homozygous Phe/Phe type, and the others were homozygous Val/Val type, not found heterozygous Val/Phe type. The Val279Phe genotypic distribution and allele gene frequency in the patients with sepsis was not significantly different from those in the healthy controls; no statistically significant difference was observed among the survival group、the death group and the control group; no statistically significant difference was observed among the mild sepsis group, severe sepsis group and control group.Conclusion:No associations were found between PAF-AH gene Val379Ala and Val279Phe polymorphisms and sepsis susceptibility, prognosis and severity.更多还原