本文選題：斑禿 + FOXP3基因��； 參考：《第三軍醫(yī)大學》2010年碩士論文

【摘要】： 研究背景和目的: 斑禿(alopecia areata ,AA)是一種累及頭皮的慢性炎癥性疾病,它以突發(fā)性、非瘢痕性毛發(fā)斑片狀脫落為特點,也可以廣泛脫落并融合成片。不同年齡和不同性別均可發(fā)生。其病因和發(fā)病機理尚未完全明了,多認為與遺傳易感性、免疫功能失調(diào)、神經(jīng)精神因素及環(huán)境因素有關(guān),然而有研究證實斑禿是一種器官特異性的自身免疫性疾病,T淋巴細胞在斑禿的發(fā)生發(fā)展中起了重要的作用。 早在20世紀50年代就有研究認為斑禿的發(fā)病有自身免疫機制的參與,后來大量的研究也都支持這一觀點。研究證實斑禿的毛囊功能障礙是由T細胞介導的免疫功能紊亂所致。其可能的機制是在遺傳和環(huán)境因素的基礎(chǔ)上,毛囊自身抗原暴露,產(chǎn)生自身抗體,活化了自體反應的淋巴細胞,啟動了以TH1細胞因子反應模式為主的免疫反應。 有學者在伴有斑禿的重癥聯(lián)合免疫缺陷老鼠模型研究中發(fā)現(xiàn)斑禿是一種T淋巴細胞介導的自身免疫性疾病,自身抗體活化自身反應性淋巴細胞進而誘導了斑禿的發(fā)生。許多類似的試驗進一步證實了CD4、CD8 T細胞在斑禿的發(fā)生中起了關(guān)鍵的作用,尤其是CD8 T細胞。但CD4+CD25+T細胞的顯著減少在斑禿的發(fā)生中所起的作用也不容忽視。 有研究發(fā)現(xiàn)斑禿皮損中毛囊間淋巴細胞浸潤以CD4+T細胞為主,伴隨CD8+T細胞浸潤,共同參與了斑禿的發(fā)病過程。有研究者在C3H/HeJ模型研究中更進一步證實了CD8+T細胞可能是斑禿發(fā)病環(huán)節(jié)中的關(guān)鍵因子,CD4+CD25-T細胞可能與彌漫性、系統(tǒng)性脫發(fā)有關(guān),而CD4+CD25+T細胞則是重要的調(diào)節(jié)因子。 此外,大量臨床和實驗室數(shù)據(jù)都顯示,除T細胞外,細胞因子也是造成斑禿的關(guān)鍵因素之一。斑禿毛囊局部淋巴細胞浸潤往往同時有細胞因子表達增多,某些細胞因子的增多有助于淋巴細胞進一步活化與浸潤。目前研究較多的主要是IL-1、IL-2、α-TNF、IFNγ、IL-4、IL-10等。 國外一些學者已證實FOXP3作為免疫抑制因子,在人類FOXP3基因可表達于CD4+CD25+T細胞和CD8+CD25-T細胞,且在CD4+CD25+T細胞中的表達明顯高于CD8+CD25-T細胞,而在小鼠則僅特異性表達于CD4+CD25+T細胞。Foxp3在Treg細胞上特異性表達,且其蛋白表達對于Treg的成熟發(fā)育和其抑制功能均具有重要意義。研究表明,在人類和小鼠患自身免疫性疾病時缺乏的Foxp3轉(zhuǎn)錄因子,該因子能夠被CD4+T調(diào)控細胞特異的表達。此外,使用逆轉(zhuǎn)錄病毒轉(zhuǎn)化Foxp3基因進入未激活的T細胞使之成為與自然產(chǎn)生的CD4+調(diào)控T細胞具有相類似的功能。FOXP3不僅對于胸腺內(nèi)調(diào)節(jié)性T細胞的發(fā)育是必須的,還可將外源性FOXP3表達于CD4+ CD25-T細胞使其獲得很多調(diào)節(jié)性T細胞的標志,從而使FOXP3在CD4+CD25-T細胞和CD8+T細胞中過量表達賦予這些細胞某些抑制功能。同時FOXP3還參與調(diào)節(jié)細胞因子的表達,FOXP3基因可抑制細胞因子IL-2、IL-4、IFN-γ等的表達。而這些調(diào)節(jié)性T細胞和細胞因子在斑禿的發(fā)生、發(fā)展過程中又起著極其重要的作用。 由此推測,FOXP3基因表達狀態(tài)有可能參與斑禿的發(fā)病機制。本研究在中國重慶地區(qū)漢族人群中檢測斑禿患者及健康對照FOXP3基因多態(tài)性位點rs3761547和rs3761548的基因型分布,對其基因多態(tài)性進行研究,探討FOXP3基因多態(tài)性與斑禿易感性的關(guān)系。 主要技術(shù)方法: 收集240例斑禿患者的外周血樣標本,同時收集相同年齡階段、地域、種族的248例健康者外周血樣標本作為對照,以血液基因組DNA提取試劑盒提取DNA,結(jié)合HapMap網(wǎng)站中漢族人群資料,選取rs3761547和rs3761548共2個單核苷酸多態(tài)性(single nucleotide polymorphism,SNP)位點,采用聚合酶鏈反應-限制性片段長度多態(tài)性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)的方法進行基因分型,并對已完成基因分型的DNA測序以驗證試驗的準確性。統(tǒng)計學分析單核苷酸多態(tài)性。 結(jié)果: 1、與正常對照比較,斑禿患者組FOXP3基因rs3761548位點的基因型分布有差異,具有統(tǒng)計學意義(p=0.015); 2、rs3761547位點的基因型分布在正常對照組與斑禿患者組之間沒有差異(p=0.12)。 3、非條件Logistic回歸分析顯示,rs3761548位點的CC基因型相對于AA和AC基因型來說,對斑禿的發(fā)病具有保護效應(adjusted OR:0.69;95% CI:0.48-0.98)。 4、單倍型分析結(jié)果顯示,與對照組相比較,斑禿患者單倍型GA和單倍型GC的分布存在顯著差異(P0.0001)。 5、非條件Logistic回歸分析顯示,GA單倍型是斑禿發(fā)病的危險因子(OR:3.2010;95% CI:2.1538-4.7573),而GC單倍型為斑禿發(fā)病的保護因子(OR:0.4831;95% CI: 0.3577-0.6526)。 結(jié)論: FOXP3基因多態(tài)性位點rs3761548與斑禿發(fā)病相關(guān),可能是中國重慶地區(qū)漢族人群斑禿發(fā)病的危險因素之一,而rs3761547位點可能與斑禿的發(fā)病無關(guān)。
[Abstract]:Background and purpose of the study :



Alopecia Areata ( AA ) is a chronic inflammatory disease involving the scalp . It is characterized by sudden and non - hypertrophic scar - like shedding , and can be widely separated and fused into tablets . The etiology and pathogenesis of alopecia are not fully understood . However , it has been shown that alopecia is an organ - specific autoimmune disease , and T lymphocytes play an important role in the development of Alopecia Areata .



The study shows that the hair follicle dysfunction is caused by T cell mediated immune function disorder . The possible mechanism is to expose the antigen of hair follicle itself on the basis of genetic and environmental factors .



In the study of the mouse model of severe combined immune deficiency with alopecia , it is found that alopecia is a T - lymphocyte - mediated autoimmune disease , and the activation of autoreactive lymphocytes by its own antibody can induce the occurrence of alopecia . Many similar experiments have further confirmed that CD4 + CD8 T cells play a key role in the occurrence of alopecia , especially CD8 T cells . However , the significant reduction of CD4 + CD25 + T cells can also be neglected in the occurrence of alopecia .



In the study of C3H / HeJ model , it has been proved that CD8 + T cells may be a key factor in the pathogenesis of alopecia . CD4 + CD25 - T cells may be associated with diffuse , systemic alopecia , while CD4 + CD25 + T cells are important regulatory factors .



In addition , a large number of clinical and laboratory data show that , besides T cells , cytokines are one of the key factors causing alopecia .



Foxp3 gene can be expressed in CD4 + CD25 + T cells and CD8 + CD25 - T cells .



In this study , the polymorphism of rs3761547 and rs3761548 locus rs3761547 and rs3761548 were detected in Chinese Han population in Chongqing , China .



Main technical methods :



The peripheral blood samples of 240 patients with Alopecia Areata were collected , and 248 healthy peripheral blood samples of the same age , region and race were collected as controls . The DNA was extracted by DNA extraction kit of blood genomic DNA . rs3761547 and rs3761548 were genotyped by PCR - RFLP , and the DNA sequencing was performed to verify the accuracy of the test . The single nucleotide polymorphism was analyzed statistically .



Results :



1 . Compared with the control group , the genotype distribution of rs3761548 locus was significantly different in the patients with Alopecia Areata ( p = 0.015 ) .



2 . There was no difference between the genotype distribution of rs3761547 locus ( p = 0.12 ) .



3 . Non - conditional Logistic regression analysis showed that the CC genotype of rs3761548 locus had a protective effect on the onset of alopecia ( adjusted OR : 0.69 ; 95 % CI : 0.48 - 0.98 ) with respect to AA and AC genotypes .



4 . The results of single - fold analysis showed that the distribution of single - fold GA and haplotype GC was significantly different from the control group ( P < 0 . 0001 ) .



5 . Non - conditional Logistic regression analysis showed that GA haplotype was the risk factor ( OR : 3.2010 ; 95 % CI : 2.1538 - 4.7573 ) , while GC haplotype was the protective factor ( OR : 0.4831 ; 95 % CI : 0.3577 - 0.6526 ) .



Conclusion :



rs3761548 polymorphism site rs3761548 is associated with the onset of alopecia , which may be one of the risk factors for the outbreak of alopecia in the Han population in Chongqing , China , and the rs3761547 locus may not be related to the onset of alopecia .

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R758.7

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