FOXP3基因多态性与斑秃的关联性研究

发布时间：2018-04-28 00:12

本文选题：斑秃 + FOXP3基因　；参考：《第三军医大学》2010年硕士论文

【摘要】： 研究背景和目的: 斑秃(alopecia areata ,AA)是一种累及头皮的慢性炎症性疾病,它以突发性、非瘢痕性毛发斑片状脱落为特点,也可以广泛脱落并融合成片。不同年龄和不同性别均可发生。其病因和发病机理尚未完全明了,多认为与遗传易感性、免疫功能失调、神经精神因素及环境因素有关,然而有研究证实斑秃是一种器官特异性的自身免疫性疾病,T淋巴细胞在斑秃的发生发展中起了重要的作用。早在20世纪50年代就有研究认为斑秃的发病有自身免疫机制的参与,后来大量的研究也都支持这一观点。研究证实斑秃的毛囊功能障碍是由T细胞介导的免疫功能紊乱所致。其可能的机制是在遗传和环境因素的基础上,毛囊自身抗原暴露,产生自身抗体,活化了自体反应的淋巴细胞,启动了以TH1细胞因子反应模式为主的免疫反应。有学者在伴有斑秃的重症联合免疫缺陷老鼠模型研究中发现斑秃是一种T淋巴细胞介导的自身免疫性疾病,自身抗体活化自身反应性淋巴细胞进而诱导了斑秃的发生。许多类似的试验进一步证实了CD4、CD8 T细胞在斑秃的发生中起了关键的作用,尤其是CD8 T细胞。但CD4+CD25+T细胞的显著减少在斑秃的发生中所起的作用也不容忽视。有研究发现斑秃皮损中毛囊间淋巴细胞浸润以CD4+T细胞为主,伴随CD8+T细胞浸润,共同参与了斑秃的发病过程。有研究者在C3H/HeJ模型研究中更进一步证实了CD8+T细胞可能是斑秃发病环节中的关键因子,CD4+CD25-T细胞可能与弥漫性、系统性脱发有关,而CD4+CD25+T细胞则是重要的调节因子。此外,大量临床和实验室数据都显示,除T细胞外,细胞因子也是造成斑秃的关键因素之一。斑秃毛囊局部淋巴细胞浸润往往同时有细胞因子表达增多,某些细胞因子的增多有助于淋巴细胞进一步活化与浸润。目前研究较多的主要是IL-1、IL-2、α-TNF、IFNγ、IL-4、IL-10等。国外一些学者已证实FOXP3作为免疫抑制因子,在人类FOXP3基因可表达于CD4+CD25+T细胞和CD8+CD25-T细胞,且在CD4+CD25+T细胞中的表达明显高于CD8+CD25-T细胞,而在小鼠则仅特异性表达于CD4+CD25+T细胞。Foxp3在Treg细胞上特异性表达,且其蛋白表达对于Treg的成熟发育和其抑制功能均具有重要意义。研究表明,在人类和小鼠患自身免疫性疾病时缺乏的Foxp3转录因子,该因子能够被CD4+T调控细胞特异的表达。此外,使用逆转录病毒转化Foxp3基因进入未激活的T细胞使之成为与自然产生的CD4+调控T细胞具有相类似的功能。FOXP3不仅对于胸腺内调节性T细胞的发育是必须的,还可将外源性FOXP3表达于CD4+ CD25-T细胞使其获得很多调节性T细胞的标志,从而使FOXP3在CD4+CD25-T细胞和CD8+T细胞中过量表达赋予这些细胞某些抑制功能。同时FOXP3还参与调节细胞因子的表达,FOXP3基因可抑制细胞因子IL-2、IL-4、IFN-γ等的表达。而这些调节性T细胞和细胞因子在斑秃的发生、发展过程中又起着极其重要的作用。由此推测,FOXP3基因表达状态有可能参与斑秃的发病机制。本研究在中国重庆地区汉族人群中检测斑秃患者及健康对照FOXP3基因多态性位点rs3761547和rs3761548的基因型分布,对其基因多态性进行研究,探讨FOXP3基因多态性与斑秃易感性的关系。主要技术方法: 收集240例斑秃患者的外周血样标本,同时收集相同年龄阶段、地域、种族的248例健康者外周血样标本作为对照,以血液基因组DNA提取试剂盒提取DNA,结合HapMap网站中汉族人群资料,选取rs3761547和rs3761548共2个单核苷酸多态性(single nucleotide polymorphism,SNP)位点,采用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)的方法进行基因分型,并对已完成基因分型的DNA测序以验证试验的准确性。统计学分析单核苷酸多态性。结果: 1、与正常对照比较,斑秃患者组FOXP3基因rs3761548位点的基因型分布有差异,具有统计学意义(p=0.015); 2、rs3761547位点的基因型分布在正常对照组与斑秃患者组之间没有差异(p=0.12)。 3、非条件Logistic回归分析显示,rs3761548位点的CC基因型相对于AA和AC基因型来说,对斑秃的发病具有保护效应(adjusted OR:0.69;95% CI:0.48-0.98)。 4、单倍型分析结果显示,与对照组相比较,斑秃患者单倍型GA和单倍型GC的分布存在显著差异(P0.0001)。 5、非条件Logistic回归分析显示,GA单倍型是斑秃发病的危险因子(OR:3.2010;95% CI:2.1538-4.7573),而GC单倍型为斑秃发病的保护因子(OR:0.4831;95% CI: 0.3577-0.6526)。结论: FOXP3基因多态性位点rs3761548与斑秃发病相关,可能是中国重庆地区汉族人群斑秃发病的危险因素之一,而rs3761547位点可能与斑秃的发病无关。
[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 .

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R758.7

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