eIF4E、MMP-9与寻常型银屑病的相关性研究

发布时间：2018-05-13 02:19

  本文选题：银屑病 + 免疫组织化学　； 参考：《山东大学》2011年博士论文

【摘要】：银屑病(psoriasis, PS)是一种常见红斑、鳞屑性皮肤病,在不同种族、区域中具有不同发病率。目前认为该病是在一定遗传因素背景下,由环境因素诱发的,由免疫介导的一种慢性、反复性、炎症性皮肤病。T细胞免疫功能异常是银屑病免疫功能紊乱的核心,贯穿疾病发生、发展全过程。感染、受潮、外伤、精神压力等,是诱发或加重银屑病的重要环境因素。银屑病属多基因遗传病,易感基因数目较多,目前已发现了多个与银屑病相关的易感位点(PSORS1-PSORS9),其中位于6p21的PSORS1和位于17q25的PSORS2是最主要的易感基因位点。随着全基因组关联分析方法(genome-wide association studies, GWAS)的出现,更多新的位点被发现。 角质形成细胞的增殖分化异常是银屑病发生发展中的重要特征。真核细胞翻译起始因子4E(eukaryotic translation initiation factor 4E, eIF4E)作为最有效的调控因子,在真核细胞蛋白质合成翻译水平上控制着蛋白质合成的速率,与细胞的转化、凋亡耐受、创伤的愈合和寿命的延长密切相关。在正常生理状态下,形成有限的具有活性的eIF4E,用于维持细胞正常生命活动所需mRNA的翻译表达；当机体存在肿瘤时,eIF4E基因表达增高,其下游各种癌基因的表达增强。有研究证明银屑病皮损中磷酸化的eIF4E及eIF4E结合蛋白(eIF4E binding proteins,4E-BP)的蛋白水平和mRNA表达水平均较非皮损区高,说明eIF4E在银屑病皮损中可能存在异常表达,参与疾病的病理过程。基质金属蛋白酶(matrix metalloproteinases, MMPs)作为一类蛋白水解酶,具有结构相似、功能复杂的特点,在细胞与细胞外基质之间参与细胞表面、细胞外基质蛋白的降解过程,是细胞微环境的重要调节因子,在细胞增殖、迁移、分化、老化、凋亡和宿主防御方面发挥重要作用。MMP-9是MMPs家族成员之一,属eIF4E下游的作用因子。MMP-9与银屑病的关系国内外已有报道。 本研究利用免疫组化、逆转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)技术对寻常型银屑病皮损中eIF4E和MMP-9进行检测,探讨eIF4E和MMP-9与寻常型银屑病皮损中的表达情况。另选择散发寻常型银屑病病例,利用第三代遗传标志单核苷酸多态性(single nucleotide polymorphisms,SNPs)基因分型技术,选择eIF4E和MMP-9基因中的3个SNP位点进行病例对照研究,旨在探讨eIF4E和MMP-9是否与银屑病具有相关性,寻找该区域是否存在银屑病易感基因。 第一部分eIF4E和MMP-9在寻常型银屑病皮损中的表达研究目的：探讨eIF4E和MMP-9在寻常型银屑病皮损中的表达情况及其与疾病的关系。 研究方法： 1.选择40例寻常型银屑病患者(男21例,女19例),皮肤活检取下皮损分成两半,一半制成银屑病组织蜡块(共40块),另一半置液氮冰冻；选择30例正常皮肤,制成正常皮肤组织蜡块30块及相同组织液氮冰冻。采用PV-9000免疫组织化学法检测eIF4E和MMP-9在银屑病皮损及正常组织中表达情况。统计学分析采用Wilcoxon秩和检验,对eIF4E和MMP-9在银屑病皮损与正常皮损中的差异表达进行对比分析,P值0.05为差异有统计学意义；采用Spearman相关性检验分析eIF4E与MMP-9的相关性,P值0.05为差异有统计学意义。 2.利用RT-PCR法检测40例寻常型银屑病皮损组织和30例正常皮肤组织标本中eIF4E和MMP-9的表达。采用图像分析技术定量分析其在正常皮肤组织中与寻常型银屑病皮损组织中的表达差异性。 结果： 1.免疫组化结果显示：正常皮肤角质形成细胞中未见eIF4E表达,银屑病皮损中eIF4E以弱阳性和阳性为主。MMP-9在正常皮肤与银屑病皮损中均见表达,但银屑病皮损中以阳性、强阳性为主。eIF4E在银屑病皮损中的阳性表达与发病年龄相关,与病程、临床类型、临床分期、PASI评分无相关性；MMP-9在银屑病皮损中的阳性表达与发病年龄、病程、临床类型、临床分期、PAS I评分无相关性。 2. Spearman等级相关性分析显示：银屑病皮损中eIF4E的表达与MMP-9的表达水平无相关性(r=0.05,P0.05)。 3.RT-PCR结果显示：银屑病皮损组和正常皮肤组扩增目的片段后,eIF4E cDNA的吸光度为1.39±0.13,正常皮肤表达为0,表达存在差异,具有统计学意义(P0.005)；正常皮肤中MMP-9 cDNA的吸光度值为2.00±0.11,银屑病皮损中MMP-9 cDNA的吸光度值为6.71±0.68,MMP-9在正常对照组、寻常型银屑病皮损中的表达存在差异,具有统计学意义(P0.005)。 结论：eIF4E和MMP-9在基因转录水平及蛋白质水平上在银屑病皮损中均表达阳性,且均高于正常皮肤组织,两者可能参与银屑病的病理过程。 第二部分eIF4E和MMP-9基因与汉族寻常型银屑病的关联分析 研究目的：探讨eIF4E和MMP-9基因内的单核苷酸多态性与寻常型银屑病的关系,以期揭示上述基因是否为银屑病的易感基因。 研究方法：采用基于群体的病例对照关联分析方法,收集188例银屑病患者(男性110例,女性78例)及280例健康对照人群临床资料及外周血标本,常规方法提取外周血基因组DNA。利用Taqman分型方法对MMP-9基因内的2个SNP位点rs4810482及rs3918254以及eIF4E基因内的SNP位点rs11723037进行基因分型,对基因型频率及等位基因频率采用PLINK软件进行统计学分析；利用Haploview4.2软件对MMP-9两个位点进行连锁不平衡分析；采用生物信息学手段分析rs4810482位点所在序列(NT_011362.10:g.14830642TC),采用MatInspector软件预测转录因子结合位点。 结果： 1.利用PLINK1.07进行Hardy-weinberg平衡检验,所有三个位点的基因型频率与等位基因频率分布均符合遗传平衡； 2.位于MMP-9基因上游调控区域的rs4810482等位基因T在银屑病组中的频率显著低于对照组(OR=1.49,95%CI=1.12-1.99, p=0.006),在隐性与显性遗传模型分析中,此差异仍然具有统计学意义。生物信息学分析表明,该位点可能改变了转录因子的结合位点； 3.利用Haploview4.2软件对MMP-9两个位点进行连锁不平衡分析的结果表明,两位点之间处于较弱的连锁不平衡(r2=0.06) 4.在检测的其它两个位点中,rs11723037(eIF4E)和rs3918254 (MMP-9)位点的基因型频率与等位基因频率分布在银屑病组与对照组中差异均无统计学意义。 结论 1.位于MMP-9基因上游调控区域的rs4810482与银屑病具有显著相关性,MMP-9可能是银屑病的一个易感基因。 2. eIF4E基因与银屑病无相关性。
[Abstract]:psoriasis ( psoriasis , ps ) is a common red spot , squamous dermatosis , and has different incidence in different races and regions . It is considered that the disease is the core of immune function disorder of psoriasis . It is an important environmental factor to induce or aggravate psoriasis .

The proliferation and differentiation of keratinocytes is an important feature in the development of psoriasis . The eukaryotic translation initiation factor 4E ( eIF4E ) , as the most effective regulatory factor , controls the rate of protein synthesis at the level of translation initiation factor 4E ( eukaryotic translation initiation factor 4E , eIF4E ) .
The expression of eIF4E binding proteins ( 4E - BP ) and eIF4E binding proteins ( 4E - BP ) in the lesions of psoriasis are higher than those of non - skin lesions . It is shown that eIF4E plays an important role in the pathogenesis of psoriasis .

In this study , eIF4E and MMP - 9 were detected by immunohistochemistry , reverse transcription polymerase chain reaction ( RT - PCR ) , and the expression of eIF4E and MMP - 9 in skin lesions of psoriasis vulgaris were investigated .

Objective : To investigate the expression of eIF4E and MMP - 9 in the lesions of psoriasis vulgaris and its relationship with disease .

Study method :

1 . 40 patients with psoriasis vulgaris ( 21 male and 19 female ) were selected , skin biopsy was divided into two halves , half of which was made into psoriasis tissue wax block ( 40 blocks ) and the other half was frozen with liquid nitrogen ;
The expression of eIF4E and MMP - 9 in skin lesions and normal tissues of psoriasis was detected by PV - 9000 immunohistochemical method .
The correlation between eIF4E and MMP - 9 was analyzed by the Pearson correlation test , and the difference of P value 0.05 was statistically significant .

2 . The expression of eIF4E and MMP - 9 in 40 normal skin tissue specimens and 30 normal skin tissues were detected by RT - PCR . The difference of expression of eIF4E and MMP - 9 in normal skin tissue was analyzed by image analysis technique .

Results :

1 . Immunohistochemical results showed that the expression of eIF4E was not seen in normal skin keratinocytes , and eIF4E was mainly weak positive and positive in the skin lesions of psoriasis . MMP - 9 was positive in normal skin and psoriasis lesions , but the positive expression of eIF4E in the lesions of psoriasis was related to the age , and there was no correlation with the course , clinical type , clinical stage and PASI score .
The positive expression of MMP - 9 in the lesions of psoriasis was not related to the age , course , clinical type , clinical stage and PAS I score .

2.The expression of eIF4E was not correlated with the expression level of MMP - 9 in the lesions of psoriasis ( r = 0.05 , P0.05 ) .

3 . The results of RT - PCR showed that the absorbance of eIF4E cDNA was 1 . 39 卤 0.13 , and that of normal skin was 1 . 39 卤 0 . 13 .
The absorbance of MMP - 9 cDNA in normal skin was 2.00 卤 0.11 , and the absorbance of MMP - 9 cDNA was 6.71 卤 0.68 and MMP - 9 was significantly different in normal controls .

Conclusion : Both eIF4E and MMP - 9 are positive in both gene transcription level and protein level in psoriasis lesions , and both are higher than normal skin tissues . Both may participate in the pathological process of psoriasis .

Association between the second part eIF4E and MMP - 9 gene and psoriasis vulgaris

Objective : To investigate the relationship between single nucleotide polymorphism and psoriasis vulgaris in eIF4E and MMP - 9 genes , with a view to revealing whether the above genes are susceptible genes of psoriasis .

Methods : The genomic DNA of peripheral blood was collected from 188 patients with psoriasis ( 110 male , 78 female ) and 280 healthy controls by using group - based case - control correlation analysis method .
The two sites of MMP - 9 were analyzed by Haploview 4.2 software .
The sequence of rs4810482 locus ( NT _ 011362 . 10 : g.148 30642TC ) was analyzed by bioinformatics , and the transcription factor binding site was predicted by MatInspector software .

Results :

1 . The Hardy - weinberg equilibrium test was carried out using PLINK1 . 07 , and the genotype frequency and allele frequency distribution of all three sites were consistent with the genetic equilibrium ;


2 . The frequency of rs4810482 allele T located in the upstream regulatory region of MMP - 9 gene was significantly lower in the psoriasis group than in the control group ( OR = 1 . 49 , 95 % CI = 1.12 - 1.99 , p = 0.006 ) .


3 . The results of linkage disequilibrium analysis of two sites of MMP - 9 using the Haploview 4.2 software show that the linkage disequilibrium between the two sites is weak ( r2 = 0.06 ) .

4 . In the other two sites tested , rs11723037 ( eIF4E ) and rs3918254 ( MMP - 9 ) loci had no significant difference in genotype frequency and allele frequency between the psoriasis group and the control group .

Conclusion

1 . rs4810482 , located in the upstream regulatory region of the MMP - 9 gene , has a significant correlation with psoriasis and MMP - 9 may be a susceptible gene of psoriasis .

2 . There was no correlation between eIF4E gene and psoriasis .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R758.63

【参考文献】

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