缺血性脑卒中及其中医证候与TOLL样受体信号通路遗传变异关联研究

The disease burden of ischemic stroke (IS) is so heavy. To date the etiology and pathogenesis of IS is not fully known and now it is still the focus of research. In this study we will adopt the modern biological technology to explore the molecular biology mark of traditional Chinese medicine (TCM) syndrome, which will have a positive significance for revealing the objective material basis of syndrome to further promote the objectification and normalization of TCM syndrome differentiation and typing. We have screened out the important biological pathway, Toll-like receptor signaling pathways and important genes, which have an effect on ischemic stroke with gene set enrichment analysis method. TaqMan SNP genotyping assays will be used in our study to genotype 18 single nucleotide polymorphisms(SNPs) in 10 key genes on Toll-like receptor signaling pathways in 900 patients with IS (300 wind phlegm with blood stasis type, 300 wind phlegm with fire hyperactivity type, 300 qi deficiency with blood stasis type) and 900 controls and detect peripheral blood gene expression and inflammatory factor level such as TNF-a in 30 patients with IS and 30 controls to verify genetic risk variation that have an influence on IS and TCM syndrome susceptibility, elaborate the genetic mechanism of IS from the molecular genetics, and reveal molecular genetics basis of ischemic stroke of different TCM syndromes from gene-disease-syndrome related perspective, which will provide a reference for objective diagnosis of TCM syndrome and more specific individual therapy.

缺血性脑卒中（简称IS）疾病负担沉重，其病因和发病机制尚未完全阐明，仍是研究热点。采用现代生物学技术来探寻中医证候的分子生物学标志，对揭示证的客观物质基础进而促进中医辨证分型的客观化和规范化具有积极意义。我们已采用基因组富集法筛选出了影响缺血性脑卒中的重要生物通路- - TOLL样受体信号通路及重要基因。本研究拟在900例IS患者（风痰火亢、风痰瘀阻、气虚血瘀证各300例）和900例正常对照中，运用TaqMan基因分型技术对Toll样受体信号通路10个关键基因的18个SNPs进行基因分型，并测定IS患者和正常对照各30例外周血基因表达和TNF-α等炎性因子水平，从而探明影响IS及其中医证候易感性的遗传风险变异，从分子遗传学上阐述IS的遗传机制，并且从基因-疾病-证候相关角度揭示不同中医证候缺血性脑卒中的分子遗传学基础，从而为中医证候的客观化诊断以及更有针对性地个体化治疗提供参考依据。

缺血性脑卒中（简称IS）疾病负担沉重，然而其病因和发病机制尚未完全阐明。本研究探讨了TOLL样受体信号通路上的10个重要基因的17个SNPs与中风风痰瘀阻、气虚血瘀、风痰火亢证的相关性, 以探明影响中风证候易感性的风险遗传变异，从分子遗传学上揭示IS不同中医证候的分子遗传学基础。采用病例-对照的研究方法，本研究共纳入中风风痰瘀阻证311例、气虚血瘀证284例、风痰火亢证300例、正常对照905例，运用实时荧光定量PCR（RT-PCR）测定mRNA表达水平，Sequenom MassARRAY iPLEX进行基因分型，ELISA测定炎性细胞因子血清水平。研究结果显示：1）遗传关联研究发现，TOLL样受体信号通路上的TLR5、TLR7、TLR8、TRAF6多态性与缺血性中风风痰瘀阻证，以及TLR7、MYD88多态性与风痰火亢证均显著关联（均P<0.05），TLR4、TRAF6、MAP2K4多态性与风痰火亢证, 及MYD88、TRAF6、TLR8、AP-1、MAPK1多态性与风痰瘀阻证、气虚血瘀证患者的血脂显著关联（均P<0.05）。2）通过基因表达测定研究，发现中风风痰瘀阻证、气虚血瘀证、风痰火亢证患者的TLR5、MYD88，风痰火亢证患者的MAPK1 mRNA表达水平均显著低于对照组（均P<0.05），而风痰瘀阻证、气虚血瘀证、风痰火亢证患者的TRAF6、CHUK，及风痰瘀阻证、气虚血瘀证的TLR7基因mRNA表达水平则均显著高于对照组（均P<0.05）。3）通过对细胞因子含量的测定，发现中风风痰瘀阻证、气虚血瘀证患者的IL-1b、IL-6、IL-8、TNF-a和风痰火亢证患者的IL-6、IL-8、TNF-a血清水平显著高于对照组（均P<0.05），且携带不同基因型的AP-1、TRAF6多态性在中风风痰瘀阻证，TLR4、TLR8、MYD88、TRAF6、CHUK多态性在中风气虚血瘀证，与TLR5、MYD88、TRAF6、MAPK1多态性在风痰火亢证中的血清炎性细胞因子水平具有显著差异（均P<0.05）。以上研究结果表明，TOLL样受体信号通路上关键基因的遗传变异可能参与缺血性中风风痰瘀阻证、风痰火亢证、气虚血瘀证的发生机制。