本文選題：銀屑病 + 免疫組織化學(xué)�。� 參考：《山東大學(xué)》2011年博士論文

【摘要】：銀屑病(psoriasis, PS)是一種常見紅斑、鱗屑性皮膚病,在不同種族、區(qū)域中具有不同發(fā)病率。目前認(rèn)為該病是在一定遺傳因素背景下,由環(huán)境因素誘發(fā)的,由免疫介導(dǎo)的一種慢性、反復(fù)性、炎癥性皮膚病。T細(xì)胞免疫功能異常是銀屑病免疫功能紊亂的核心,貫穿疾病發(fā)生、發(fā)展全過程。感染、受潮、外傷、精神壓力等,是誘發(fā)或加重銀屑病的重要環(huán)境因素。銀屑病屬多基因遺傳病,易感基因數(shù)目較多,目前已發(fā)現(xiàn)了多個與銀屑病相關(guān)的易感位點(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點。隨著全基因組關(guān)聯(lián)分析方法(genome-wide association studies, GWAS)的出現(xiàn),更多新的位點被發(fā)現(xiàn)。 角質(zhì)形成細(xì)胞的增殖分化異常是銀屑病發(fā)生發(fā)展中的重要特征。真核細(xì)胞翻譯起始因子4E(eukaryotic translation initiation factor 4E, eIF4E)作為最有效的調(diào)控因子,在真核細(xì)胞蛋白質(zhì)合成翻譯水平上控制著蛋白質(zhì)合成的速率,與細(xì)胞的轉(zhuǎn)化、凋亡耐受、創(chuàng)傷的愈合和壽命的延長密切相關(guān)。在正常生理狀態(tài)下,形成有限的具有活性的eIF4E,用于維持細(xì)胞正常生命活動所需mRNA的翻譯表達(dá)；當(dāng)機(jī)體存在腫瘤時,eIF4E基因表達(dá)增高,其下游各種癌基因的表達(dá)增強(qiáng)。有研究證明銀屑病皮損中磷酸化的eIF4E及eIF4E結(jié)合蛋白(eIF4E binding proteins,4E-BP)的蛋白水平和mRNA表達(dá)水平均較非皮損區(qū)高,說明eIF4E在銀屑病皮損中可能存在異常表達(dá),參與疾病的病理過程�；|(zhì)金屬蛋白酶(matrix metalloproteinases, MMPs)作為一類蛋白水解酶,具有結(jié)構(gòu)相似、功能復(fù)雜的特點,在細(xì)胞與細(xì)胞外基質(zhì)之間參與細(xì)胞表面、細(xì)胞外基質(zhì)蛋白的降解過程,是細(xì)胞微環(huán)境的重要調(diào)節(jié)因子,在細(xì)胞增殖、遷移、分化、老化、凋亡和宿主防御方面發(fā)揮重要作用。MMP-9是MMPs家族成員之一,屬eIF4E下游的作用因子。MMP-9與銀屑病的關(guān)系國內(nèi)外已有報道。 本研究利用免疫組化、逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(reverse transcription polymerase chain reaction,RT-PCR)技術(shù)對尋常型銀屑病皮損中eIF4E和MMP-9進(jìn)行檢測,探討eIF4E和MMP-9與尋常型銀屑病皮損中的表達(dá)情況。另選擇散發(fā)尋常型銀屑病病例,利用第三代遺傳標(biāo)志單核苷酸多態(tài)性(single nucleotide polymorphisms,SNPs)基因分型技術(shù),選擇eIF4E和MMP-9基因中的3個SNP位點進(jìn)行病例對照研究,旨在探討eIF4E和MMP-9是否與銀屑病具有相關(guān)性,尋找該區(qū)域是否存在銀屑病易感基因。 第一部分eIF4E和MMP-9在尋常型銀屑病皮損中的表達(dá)研究目的：探討eIF4E和MMP-9在尋常型銀屑病皮損中的表達(dá)情況及其與疾病的關(guān)系。 研究方法： 1.選擇40例尋常型銀屑病患者(男21例,女19例),皮膚活檢取下皮損分成兩半,一半制成銀屑病組織蠟塊(共40塊),另一半置液氮冰凍；選擇30例正常皮膚,制成正常皮膚組織蠟塊30塊及相同組織液氮冰凍。采用PV-9000免疫組織化學(xué)法檢測eIF4E和MMP-9在銀屑病皮損及正常組織中表達(dá)情況。統(tǒng)計學(xué)分析采用Wilcoxon秩和檢驗,對eIF4E和MMP-9在銀屑病皮損與正常皮損中的差異表達(dá)進(jìn)行對比分析,P值0.05為差異有統(tǒng)計學(xué)意義；采用Spearman相關(guān)性檢驗分析eIF4E與MMP-9的相關(guān)性,P值0.05為差異有統(tǒng)計學(xué)意義。 2.利用RT-PCR法檢測40例尋常型銀屑病皮損組織和30例正常皮膚組織標(biāo)本中eIF4E和MMP-9的表達(dá)。采用圖像分析技術(shù)定量分析其在正常皮膚組織中與尋常型銀屑病皮損組織中的表達(dá)差異性。 結(jié)果： 1.免疫組化結(jié)果顯示：正常皮膚角質(zhì)形成細(xì)胞中未見eIF4E表達(dá),銀屑病皮損中eIF4E以弱陽性和陽性為主。MMP-9在正常皮膚與銀屑病皮損中均見表達(dá),但銀屑病皮損中以陽性、強(qiáng)陽性為主。eIF4E在銀屑病皮損中的陽性表達(dá)與發(fā)病年齡相關(guān),與病程、臨床類型、臨床分期、PASI評分無相關(guān)性；MMP-9在銀屑病皮損中的陽性表達(dá)與發(fā)病年齡、病程、臨床類型、臨床分期、PAS I評分無相關(guān)性。 2. Spearman等級相關(guān)性分析顯示：銀屑病皮損中eIF4E的表達(dá)與MMP-9的表達(dá)水平無相關(guān)性(r=0.05,P0.05)。 3.RT-PCR結(jié)果顯示：銀屑病皮損組和正常皮膚組擴(kuò)增目的片段后,eIF4E cDNA的吸光度為1.39±0.13,正常皮膚表達(dá)為0,表達(dá)存在差異,具有統(tǒng)計學(xué)意義(P0.005)；正常皮膚中MMP-9 cDNA的吸光度值為2.00±0.11,銀屑病皮損中MMP-9 cDNA的吸光度值為6.71±0.68,MMP-9在正常對照組、尋常型銀屑病皮損中的表達(dá)存在差異,具有統(tǒng)計學(xué)意義(P0.005)。 結(jié)論：eIF4E和MMP-9在基因轉(zhuǎn)錄水平及蛋白質(zhì)水平上在銀屑病皮損中均表達(dá)陽性,且均高于正常皮膚組織,兩者可能參與銀屑病的病理過程。 第二部分eIF4E和MMP-9基因與漢族尋常型銀屑病的關(guān)聯(lián)分析 研究目的：探討eIF4E和MMP-9基因內(nèi)的單核苷酸多態(tài)性與尋常型銀屑病的關(guān)系,以期揭示上述基因是否為銀屑病的易感基因。 研究方法：采用基于群體的病例對照關(guān)聯(lián)分析方法,收集188例銀屑病患者(男性110例,女性78例)及280例健康對照人群臨床資料及外周血標(biāo)本,常規(guī)方法提取外周血基因組DNA。利用Taqman分型方法對MMP-9基因內(nèi)的2個SNP位點rs4810482及rs3918254以及eIF4E基因內(nèi)的SNP位點rs11723037進(jìn)行基因分型,對基因型頻率及等位基因頻率采用PLINK軟件進(jìn)行統(tǒng)計學(xué)分析；利用Haploview4.2軟件對MMP-9兩個位點進(jìn)行連鎖不平衡分析；采用生物信息學(xué)手段分析rs4810482位點所在序列(NT_011362.10:g.14830642TC),采用MatInspector軟件預(yù)測轉(zhuǎn)錄因子結(jié)合位點。 結(jié)果： 1.利用PLINK1.07進(jìn)行Hardy-weinberg平衡檢驗,所有三個位點的基因型頻率與等位基因頻率分布均符合遺傳平衡； 2.位于MMP-9基因上游調(diào)控區(qū)域的rs4810482等位基因T在銀屑病組中的頻率顯著低于對照組(OR=1.49,95%CI=1.12-1.99, p=0.006),在隱性與顯性遺傳模型分析中,此差異仍然具有統(tǒng)計學(xué)意義。生物信息學(xué)分析表明,該位點可能改變了轉(zhuǎn)錄因子的結(jié)合位點； 3.利用Haploview4.2軟件對MMP-9兩個位點進(jìn)行連鎖不平衡分析的結(jié)果表明,兩位點之間處于較弱的連鎖不平衡(r2=0.06) 4.在檢測的其它兩個位點中,rs11723037(eIF4E)和rs3918254 (MMP-9)位點的基因型頻率與等位基因頻率分布在銀屑病組與對照組中差異均無統(tǒng)計學(xué)意義。 結(jié)論 1.位于MMP-9基因上游調(diào)控區(qū)域的rs4810482與銀屑病具有顯著相關(guān)性,MMP-9可能是銀屑病的一個易感基因。 2. eIF4E基因與銀屑病無相關(guān)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 鹵 0.13 , and that of normal skin was 1 . 39 鹵 0 . 13 .
The absorbance of MMP - 9 cDNA in normal skin was 2.00 鹵 0.11 , and the absorbance of MMP - 9 cDNA was 6.71 鹵 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 .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R758.63

【參考文獻(xiàn)】

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本文編號：1881217