论文标题:血小板活化因子乙酰水解酶活性及基因单核苷酸多态性与银屑病的关系
The Relationship of Platelet Activating Factor Acetylhydrolase Activity and Single Nucleotide Polymorphism in Its Gene with Psoriasis
论文作者 林大东
论文导师 顾军,论文学位 硕士,论文专业 皮肤病与性病学
论文单位 第二军医大学,点击次数 121,论文页数 58页File Size638k
2004-05-01论文网 http://www.lw23.com/lunwen_117963817/ 汉族; PAF; PAF-AH; 银屑病; 活性; 基因 ; SNPs
Chinese,PAF,PAF-AH,psoriasis,Activity ,Gene mutation,SNPs
血小板活化因子(PAF)是由多种炎症细胞(嗜中性白细胞、巨噬细胞及酸性粒细胞等)、血小板及血管内皮细胞等产生的一种高生理活性磷脂。PAF通过与细胞膜表面的受体结合而发挥效应,已证实PAF不仅在生殖、胚胎发育、血压等方面具有调节功能,同时又是强效的炎症介质,在许多疾病如:哮喘、败血症性休克、自身免疫性疾病、变态反应性疾病、移植排斥发病过程中扮演重要角色,在动物实验中,PAF受体拮抗剂能有效地改善病变的过程。PAF在生物体内以动态形式存在,细胞受刺激后生成PAF,随之很快被代谢清除。Barr在1980年首先报道血浆中存在着一种灭活PAF的物质,即PAF乙酰水解酶(PAF-AH)。近年来的研究表明,PAF-AH在控制PAF的释放及降解,维持体内PAF的生理平衡中起着重要的作用。PAF-AH可以水解PAF 的sn-2位上的乙酰基而使PAF失活。PAF-AH根据存在形式分为两种类型即:血浆型(pPAH-AH)及细胞型(cPAF-AH),其中后者有三种同工酶Ⅰa, Ⅰb及Ⅱ型。PAF-AHⅠb与人类LIS-1基因产物同源(99%),LIS-1基因突变可导致MDL,即一种大脑皮层脑回缺失性疾病,已证实这与脑内cPAF-AH活性降低、PAF异常升高有关。1988 年,Miwa报导816名健康日本成人中32人无血浆PAF-AH活性,健康儿童中的比率为2/211,有严重哮喘的儿童此酶缺乏概率高于正常儿童,提示PAF-AH与炎症反应相关。自1995年成功地克隆PAF-AH后,人们对其进行了深入的研究,PAF-AH基因位于人类第6条染色体,由12个外显子组成,分布在45Kb的核苷酸中,该序列编码了441个氨基酸。点突变研究表明,273丝氨酸、296天冬氨酸及351组氨酸是PAF-AH活性所必须的。令人感兴趣的是此基因正好位于哮喘及异位性皮炎的高敏感区, Stafforini等首先报道日本人群中存在279及281位点单核苷酸多态性(SNPs),此位点的突变导致了PAF-AH活性的降低,与哮喘发生密切相关。随机抽查发现日本人群中Val279Phe突变杂合子为27%,纯合子为4%。哮喘病人明显升高,而异位性皮炎病人多有哮喘病史。Henderson在鼠的哮喘模型中,用卵白蛋白致敏后再次给予抗原,引发肺中大量酸性粒细胞等炎症细胞浸润,分泌粘液堵塞气道导致气管的超敏反应,给予重组PAF-AH对减轻晚期肺部炎症有明显效应,提示PAF-AH对气道过敏反应的病人可能有治疗作用。近来报道在日本人群中Val279Phe与休克及非家族性肥厚性心肌病亦有关。PAF-AH的279及281位点的 SNPs解释了多数日本人PAF-AH酶缺陷的原因。然而,Kruse报道表明在北美及欧洲人群中并不存在279及281 SNPs,而92、198及379位点存在影响PAF-AH酶动力学的SNPs,且后两者与哮喘及异位性疾病相关。提示不同种族和人群的哮喘及异位性皮炎与PAF-AH SPNs位点相关性不同。银屑病是皮肤病中的常见病、多发病,PAF在其病理机制中占有重要的地位,明确中国汉族人群PAF-AH与银屑病的关系,对疾病的发病机制及防治有重要意义。第一部分 银屑病患者的PAF-AH活性检测及其临床意义目的:检测不同病期银屑病患者的外周血中血浆PAF-AH活性的变化,分析其与银屑病的相关性,探索PAF-AH在银屑病发病机制中的作用。方法:采用酶水解底物显色法测定48名(进展期、静止期和退行期各16名)银屑病患者和44名健康对照者血浆中的PAF-AH活性。结果:银屑病患者的血浆PAF-AH活性为24.93±3.72nmol/min/mL;健康对照者血浆PAF-AH活性为28.51±4.91nmol/min/mL,两组比较差异具有有显著性(P<0.01)。试剂盒所提供的PAF-AH标准品活性为41.65 nmol/min/mL。银屑病患者中进展期组的血浆PAF-AH活性为22.53±3.35nmol/min/mL;静止期组为25.83±2.76nmol/min/mL;退行期组为26.43±3.88nmol/min/mL,ANOVA/snk分析显示进展期组的酶活性明显低于后二者,静止期组和退行期组的差异不具有显著性。结论:银屑病患者的血浆PAF-AH活性降低导致血小板活化因子水解减少,间接的促进了炎症反应的发生,PAF-AH活性不足可能是银屑病发病的内源性因素之一。进展期银屑病患者的酶活性低于静止期和退行期,符合各期的炎症状态,酶活性的水平可能与银屑病的病情活动性有关,其活性水平在进行病情及预后判断时可能具有一定的参考意义。抑制PAF产生的药物以及强效PAF拮抗剂在银屑病治疗方面可能具有一定的前景。第二部分 PAF-AH基因Val279Phe突变与银屑病的相关性研究目的:研究PAF-AH基因Val279Phe突变与银屑病之间的相关性,及其对PAF-AH活性的影响,初步探讨PAF-AH基因SNPs在银屑病发病机制中的作用。方法:应用等位基因特异性多聚酶链式反应技术分析银屑病患者及健康对照者PAF-AH基因Val279Phe位点的多态性,并对含有突变等位基因的多态性片断进行DNA测序分析。结果:所有研究对象中发现3例Val279Phe突变型杂合子,其中2例银屑病患者,1例健康对照者,未发现突变型纯合子;突变等位基因频率为1.32%,杂合子占2.63%,明显低于日本人群(27%)。全体研究对象的G等位基因频率为0.99,T突变等位基因的频率为0.01,与日本人群中的分布有较大差别。T等位基因在银屑病组中的频率为1.67%,在健康对照组为0.93%,两组比较差异不具有显著性(P>0.1)。结论:PAF-AH基因的Val279Phe突变与银屑病没有明显的相关关系,可能不是银屑病患者血浆PAF-AH活性降低的主要原因。
Platelet-activating factor (PAF,1-O-alkyl-2-acetylsn-glycero-3-phosphocholine) is a biologically active phospholipid with diverse potent biological effects. It is associated with the pathology of several human diseases, particularly allergy and inflammation, affecting the respiratory, vascular, digestive and reproductive systems. Its accumulation is tightly regulated at the synthetic and degradative levels to avoid the inappropriately high accumulation of PAF observed in many diseases,such as systemic lupus erythematosus ,allergic diseases and septic shock and so on.PAF is degraded to inactive products by hydrolysis of the acetyl group at the sn -2 position, to produce the biologically inactive products lyso-PAF and acetate. This reaction is catalyzed by PAF acetylhydrolase, a calcium-independent phospholipase A2 specific for hydrolysis of phospholipids containing short and/or oxidized chains at the sn -2 position of the glycerol backbone. There are two type of PAF-AH,namely plasma PAF-AH and cellular PAF-AH .The latter have three isoenzymes: type Ⅰa ,typeⅠb and type Ⅱ. The Ⅰb subunit is 99% homologous to a product of the LIS1 gene, mutations of which cause type I lissencephaly.The activity of PAF-AH was first described in 1980. Subsequently, Blank and colleagues reported that this enzyme is widely distributed in mammalian tissues and blood and that it is specific for deacetylation of phospholipids. Miwa et al. reported that 4% of the Japanese population is deficient in PAF acetylhydrolase activity in plasma, and that the prevalence of deficiency of the enzyme is higher in children with severe asthma. It suggested that PAF-AH plays an important role in the inflammatory reaction. The gene for the plasmatic PAF-AH has been mapped to chromosome 6p21.2-p12, consisting of 12 exons,originally assigned as the human leukocyte antigen region. The predicted amino acid sequence of the plasma form of PAF-AH contains a Gly-Xaa-Ser- Xaa-Gly motif characteristic of lipases and esterases. The serine residue in this conserved motif is essential for activity and it explains the ability of serine esterase inhibitors to abolish enzymatic activity. It is interesting that PAF-AH gene is in the region which is high sensitive to asthma and atopic dermatitis. To determine the molecular basis of inherited PAF-AH deficiency, Stafforini et al amplified all coding exons in the PAF acetylhydrolase gene using DNA isolated from subjects with normal and deficient levels of PAF-AH activity. They identified two point mutation, Val279Phe and Gln281Arg near the active site of PAF-AH that is related to deficency of plasma activity in Japanese subjects. This mutation, Val279Phe, as a heterozygous trait is present in 27%,as well as a homozygote trait in 4%, in the Japanese population. The mutant alle is higher in the patients with asthma than that in the health population. In a murine asthma model, Henderson examined the anti-inflammatory activities of recombinant human (rPAF-AH). In this model, mice sensitized to ovalbumin by i.p. and intranasal (i.n.) routes are challenged with the allergen by i.n. administration. The ovalbumin challenge elicits an eosinophil infiltration into the lungs with widespread mucus occlusion of the airways and results in bronchial hyperreactivity. The administration of rPAF-AH had a marked effect on late-phase pulmonary inflammation, which included a significant reduction in airway eosinophil infiltration, mucus hypersecretion, and airway hyperreactivity in response to methacholine challenge. These studies demonstrate that elevating plasma levels of PAF-AH through the administration of rPAF-AH is effective in blocking the late-phase pulmonary inflammation that occurs in this murine allergen-challenge asthma model. These results suggest that rPAF-AH may have therapeutic effects in patients with allergic airway inflammation. In recent,it is reported that Val279Phe SNPs is associated with shock and nonfamilial hypertrophic cardiomyopathy. Two loss-of-function mutations of PAF-AH, Val279Phe and Gln281Arg, associate
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