维吾尔族饲鸽者肺Th1/Th2失衡的相关分子生物学机制研究

Pigeon breeder's disease (PBD) belongs to the hypersensitivity pneumonitis (HP). The latest research reported that the cause of PBD is connected with the imbalance between Th1 and Th2 cells,although the mechanism is not clear yet. Studies have found that the imbalance between Th1 and Th2 cells is involved with the relevant factors of the SNP. It is also found that other imbalance diseases between Th1 and Th2 cells such as connective tissue disease have connection with the DNA Methylation. After doing some research of the patients with HP in Xinjiang, we found that most patients with PBD were Uygur because of the habit that Uygur enjoy keeping doves, so we carried on a further research and found that there were twenty Uygur patients with PBD suffering from the imbalance of immune factors of Th1 and Th2 cells. Therefore, we propose a hypothesis that single nucleotide polymorphisms and DNA methylation may be relevant to the imbalance of the Th1/Th2 in Uygur patients with PBD. Our study regards Uygur patients with PBD as the research object to detect the related immunological indexes of Th1/Th2, the Th1/Th2 cytokines type, single nucleotide polymorphism and abnormal methylation genes. We Implement demethylation for the abnormal high methylation genes with the help of methyltransferases inhibitors and motivate methylation processing for the abnormal low methylation genes with the help of HCY. We intend to clarify the molecular biology mechanism about the imbalance of Th1/Th2 in Uygur patients with PBD in order to provide a new approach to genetic diagnosis and therapy.

饲鸽者肺（PBD）属过敏性肺炎（HP），研究认为PBD发病与Th1/Th2失衡有关，其机制尚不清楚，有研究发现Th1/Th2失衡与相应因子的单核苷酸多态性有关，也有研究发现结缔组织病等其它Th1/Th2失衡类疾病与DNA甲基化关系密切。课题组对新疆HP患者研究发现维吾尔族PBD病人较多，进一步研究发现20例维吾尔族PBD患者存在Th1/Th2相关免疫因子失衡。因此提出假说： 单核苷酸多态性及DNA甲基化与维吾尔族PBD患者Th1/Th2失衡机制可能有关。本研究以维吾尔族PBD患者为研究对象，检测Th1、Th2相关免疫学指标，检测Th1、Th2型细胞因子单核苷酸多态性及异常甲基化基因，对异常高甲基化的基因采用甲基转移酶抑制剂去甲基化处理，对异常低甲基化基因采用同型半胱氨酸促甲基化处理，阐明维吾尔族PBD患者Th1/Th2失衡的相关分子生物学机制，为基因诊断、治疗提供新思路。

本课题研究对象为新疆维吾尔族饲养鸽子患病者为病例组，未患病者为阴性对照组及同期、同一地区健康体检者为正常对照组；对新疆南疆地区维吾尔族饲养鸽子人群进行问卷调查，建立首个新疆维吾尔族饲鸽者肺(PBL)的资源库，采用病例对照研究方法，采用ELISA法检测三组血清及BALF中Th1/Th2失衡相关因子的表达水平；采用流式细胞仪检测外周血CD4+CD25+FOX3+Treg细胞比例和T淋巴细胞亚群；采用MassARRAY技术对Th1/Th2失衡相关的96个基因位点进行多态性分析；通过全基因组进行甲基化初筛发现6个异常甲基化基因后扩大样本进行验证；研究发现：1.PBL患者的发病与性别、环境、抗原暴露时间相关；2.维吾尔族PBL患者血清及BALF中IL-2、MIP-1a、IL-8表达水平存在差异；3.维吾尔族PBL患者体内CD4+CD25+FoxP3+Treg细胞的表达下调通过影响Th1/Th2失衡而在PBL发病中起作用，4.IL-2、MIP-1a、TNF-a、IL-4基因位点存在多态性，且与PBL的易感性相关；IL-2(rs2069762)携带杂合子GT基因型；IL-4(rs2070874)携带纯合子TT可能是维吾尔族PBL的危险因素；5.未发现HLA-DQA1、HLA-DRB5基因的异常甲基化与PBL的易感性相关；6. HLA-A基因CpG5 位点基因存在异常高甲基化状态，通过影响HLA-A基因转录、翻译过程导致维吾尔族PBL的发生；7.未发现 ADAMS12异常甲基化参与维吾尔族PBL的发病及纤维化过程。8.Notch4基因异常高甲基化状态通过炎症反应、信号通路等参与PBL肺纤维化的进程；9. Smad3基因异常低甲基化通过提高其mRNA的表达参与了PBL发生肺纤维化进程。本研究首次构建了以维吾尔族为主体建立了新疆地区PBL资源库。明确了环境，遗传因素对维吾尔族PBL的影响，首次证实了CD4+CD25+Foxp3+Treg细胞水平表达比例异常影响了PBL的发病机制；证实了IL-2、MIP-1a、TNF-a、IL-4是维吾尔族PBL的易感基因； HLA-A基因甲基化与维吾尔族PBL的易感性有关，Notch4、Smad3基因影响了PBL肺纤维化的进程；研究结果为今后各民族患者的诊断及个体化治疗提供了基因层面的依据。