蠕形螨病与病原致病相关基因和宿主易感基因的相关性研究

It is known that the pathogenicity of Demodex had been debated for a long time and its pathogenic mechanism is still uncertain. Under this background, the applicants have confirmed the strong association between Demodex infestation and rosacea/blepharitis (OR=7.57 and 4.89) by meta-analysis in the prophase. Therefore, we proposed the hypothesis that the development of demodicidosis might be related to genotypic differences of stock trees in etiology and differences of predisposing genes in hosts. In the present project, a case control study will be conducted. Firstly, in order to find the virulence gene related with the development of demodicidosis, SSH technique will be applied to screen the differential expression gene of Demodex from the patients in case and control group. Secondly, for the sake of screening and identifying the predisposing genes for demodicidosis, searching and finding new alleles in Chinese, PCR-SSP technique will be employed to complete genotyping of HLA-A and -B alleles (HLA class I) of hosts. And PCR-SBT technique will be used to sequence the HLA-DR alleles (HLA class Ⅱ) which are significantly related to diseases. Thirdly, to analyze the relationship between immune state of hosts and density of Demodex infection/HLA alleles, the molecular CD and activity of white blood cells will be tested by immunodetection methods. In a word, the object of this project is aiming at revealing the correlation between demodicidosis and pathopoiesis related gene in etiology/predisposing genes in hosts, and illuminating the molecular pathogenesis of demodicidosis, which will provide experimental data and new thinking for clinical diagnosis as well as prevention and cure for demodicidosis, and promote our research on pathogenicity of Demodex at the leading position in the world.

在蠕形螨致病性长期存在争议和致病机制不明的背景下，申请者前期meta分析已确认蠕形螨与酒渣鼻和睑缘炎强关联（OR=7.57，4.89），提出蠕形螨病发生与病原种株基因型差异和宿主易感基因差异相关联的假设。本项目采用病例对照研究，通过SSH技术对病例组和对照组的蠕形螨进行差异表达基因筛选，找出与蠕形螨病发生有关的致病基因；采用PCR-SSP技术对宿主HLA-I类A,C位点进行基因分型，并采用PCR-SBT技术对与疾病密切相关的HLA-Ⅱ 类DR位点基因序列进行测序，筛选鉴定中国人对蠕形螨病的易感基因，查找发现新的等位基因；免疫检测CD分子以及白细胞活性，分析宿主免疫状态与蠕形螨感染密度以及HLA基因的关系。旨在揭示蠕形螨病与病原致病相关基因和宿主易感基因的相关性，阐明蠕形螨病的分子发病机制，为蠕形螨病的临床诊断和防治提供新的实验依据和新思路，使我国在蠕形螨致病性研究领域走在世界前列。

在蠕形螨致病性长期存在争议、柯霍氏法则无法直接验证的背景下，前期采用meta分析和横断面调查，间接确认蠕形螨感染与酒渣鼻等多种面部皮肤病显著关联，尤其是毛囊蠕形螨（D.f）。论文的发表引起了路透社、MedwireNews和中国教育网等20余家媒体报道和转载。但蠕形螨个体极小、虫源获取困难，导致DNA/RNA水平研究滞后或空白，阻碍了分子发病机制的研究。本项目取得以下开拓性进展：一是发现chelex-100是提取单只蠕形螨gDNA的好方法，实现了人蠕形螨四种表型和寄生在人、犬、羊的四种蠕形螨分子鉴定与亲缘关系研究，为D.f和皮脂蠕形螨(D.b)种群致病差异研究创造了条件。二是选择个体大有报道的兔痒螨进行预实验，创建的Azanno法结合液氮研磨是提取螨类RNA和构建cDNA文库快速、简便、重复性好的有效方法。Azanno法优于OMEGA法和trizol法，螨数由文献700mg逐步减少到68mg、200只（约7mg），最终减少到10只，Pso C2引物检测能够满足cDNA建库要求。三是选择个体较大的犬疥螨预实验建立了RNA提取、转录组测序、功能注释与验证方法，为个体极小的蠕形螨提供关键技术支撑。四是上述基础上，首次实现了D.f和D.b的RNA提取、cDNA文库构建、转录组测序和功能注释；筛选出D.f和D.b上调或下调log2≥10的Unigene 95条，CDS验证9条，表达量验证3条，原核表达验证1条；建立的高通量测序方法筛选差异基因，既优于申请书SSH技术，也为致病基因筛选奠定了基础。五是增加了基于DGGE技术对D.f和D.b体内细菌与面部皮肤病关联性的研究，初步阐明蠕形螨体内细菌在面部皮肤病发生中的作用。最后，结合本项目研究成果和国内外文献，以“蠕形螨病：一种新现的螨源性皮肤病”为题对其发现过程、典型病例、致病机制、临床诊断和治疗等现状进行了全面介绍，旨在提高国内皮肤科医生对新现蠕形螨病的认识和重视，减少临床误诊，消除螨虫危害。本项目发表论文20篇，SCI论文17篇，向GenBank投递序列357条，授权国家实用专利1项，参加会议交流5次，学术讲座4次，陕西台科普宣讲2次。随着成果发表，我们在国内外螨虫研究领域的学术影响力快速提升。