| Objective:(1) To explore the Uyghur epidemiology and risk factors of the thromboangiitis obliterans(TAO) in Xinjiang Awat area.(2) Screening the susceptibility single-nucleotide polymorphisms(SNPS) and find the possible biological process and pathways of Uyghur thromboangiitis obliterans.(3) Combined the risk factors and the second part result in vitro validation. HUVEC under cotinine treatment, TLR-4/My D88 mediated immune inflammatory pathways and control mechanism of thromboangiitis obliterans. Methods: Part One: A cross section study was carried on Xinjiang Uyghur resident in Awat Akesu area. Combined with current epidemiological investigation questionnaire, physical examination, blood test and contrast-enhanced ultrasound examination results. Analysis the common vascular disease risk characteristics with individual human behavior habits and determine serum levels of cotinine, interleukin-6, 17(IL-6, IL-17), and the TNF alpha. Using ELISA to determine the concentration of IL-17, Using chemiluminescence to determination the concentration of TNF alpha and IL-6. A single factor conditional logistic regression analysis is used by the types of obtained information, also is adopted to improve the single factor analysis. Then put the preliminary screening which closely associated with the thromboangiitis obliterans factors, into the multi-factor logistic regression model for multiple factors analysis. Part Two: A method of case-control study was adopted, 80 Uyghur patients were chosen as cases, the age ranged from 20~47; 80 from the uygur normal population which has the same genetic background as control according to the ratio of 1∶1 comparison. Genotypes using Affymetrix Genome-Wide Human SNP Array 6.0 chip to analysises the susceptibility SNP under Uyghur population. Through the analysis of the quality control and finally determined the Uyghur population susceptibility SNP of the disease. The enrichment analysis for susceptible SNPs related gene and upstream or downstream has discoveryed the differences in Gene. Then try to find the related genic function and their cell signal pathway. Part Three: HUVEC in vitro, recovery, passage, cell identification, detection,reaching to steady status. Carry through MTT experiment, using cotinine to treat HUVEC, Grope for the cotinine react concentrations. Cells can be divided into normal control and cotinine 1 ug and 100 ug/ml transfection group. Using QPCR to detect HUVEC TLR4 proteins of cells and cell surface protein expression under cotinine processing. ELISA method were used to detect TNF alpha in cell cultures and IL-6 concentration change under cotinine treatment; Using western blot method to detect the nf-kappa B and I kappa B predominate-alpha changes in protein levels. Comprehensive analysis TLR-4 expression and TLR-4/My D88/NF-κB Inflammatory signaling pathway activation under cotinine treatment. Results: Part one:(1) Xinjiang aksu awat region TAO related epidemiological survey was successfully accomplished; The TAO prevalence rate of 8.7% in this Awat region, all of which were Uyghur men.(2) Income(P<0.05), the amount of smoking(X2=13.48, P<0.05), the history of disease(P<0.05), the degree of tolerance to cold(X2=941.30, P<0.05), the history of limb ischemia(X2=769.67, P<0.05), migrating phlebitis(X2=523.11, P<0.05), the history of intermittent claudication(X2=489.89, P<0.05), Limb pain and gangrene(X2=331.48, P<0.05), dorsalis pedis pulse(P<0.05), Serum levels of cotinine(Z=16.17, P<0.05), Serum levels of IL-6(Z=15.60, P<0.05), Serum levels of IL-17(Z=12.76, P<0.05), Serum levels of TNF-α(Z=11.57, P<0.05) the difference was statistically significant compare with the control group, it is the factor of the influence.(3) After screening the discovery after multiariable logistic regression analysis serum levels of cotinine(X2=14.45, P<0.05, OR=20.25, 95%CI: 3.96-145.38) was obvious relevance with the disease, it is a risk factor of the disease. Part Two:(1) After strict quality control, 484730 SNPs were analysised in 70 cases and 64 controls by using Cochran-Armitage trend test to access the genotype-phenotype association. Both the cases and control samples match well in this database.(2) The candidated SNP mainly enriched in cell adhesion, biological adhesion, response to molecule of fungal origin, regulation of cellular component size, cell-cell adhesion, intracellular signaling cascade, positive regulation of tumor necrosis factor production, pattern recognition receptor signaling pathway, activation of innate immune response, innate immune response-activating signal transduction. Part Three(1) The cotinine has no significant effect on HUVEC cell proliferation, 1000 ug/ml for the cell inhibitory effect may be due to the too much drug concentration, causes the cytotoxicity.(2) The cotinine had no significant effect on migration of HUVEC.(3) Cotinine can significantly inhibit TLR-4 m RNA expression.(4) Cotinine had no significant influence of TLR-4 protein expression.(5) Cotinine processing, the proportion of TLR-4 on HUVEC cells membrane in 3h and 24h increased significantly. The cotinine actives the TLR-4 mediated inflammatory signaling pathways by increasing the ratio TLR-4 on the membrane.(6) Cotinine can significantly reduce the IκB-α predominate, make the exptession of NF-κB signal pathway active.(7) Cotinine has a significant increase in extracellular TNF alpha and secretion of IL-6. Conclusion: Part one:(1) Different ethnic and gender may have different prevalence rate. The prevalence rate of TAO in Uighur was higher than that in han people, more men than women.(2) The prevalence rate of TAO is closely related with serum levels of cotinine, serum levels of IL-6, serum levels of IL-17, serum levels of TNF-α. The income, the amount of smoking, the history of disease, the degree of tolerance to cold, the history of limb ischemia, migrating phlebitis, the history of intermittent claudication, Limb pain and gangrene, dorsalis pedis pulse, are the influence factors of TAO. TAO were related with cotinine metabolic, endothelial injury, inflammatory cytokines release, estrogen levels, these are the components of autoimmune inflammatory disease. Part Two:(1) We have performed a GWAS syudy for TAO in Uyghur population. Our study identifies 26 susceptibility SNP. In particular, it highlighted potential pathogenic overlap between TAO and immune inflammation, emphasized that the adhesion factor and inflammatory factor both play an important role in disease and also provided theoretical basis for further pathogenesis. But it needs further repeated verification.(2) The enrichment analysis for TAO susceptible SNP was conducted by bioinformatics analysis tool one important pathway and two important factors(a. adhension factor b.inflammation factor IL-17) were found associated with TAO, which provided vital clue for the further functional study of TAO. Part three:(1) Cotinine increases the TLR-4 RNA expression in HUVEC membrane surface.(2) Cotinine activates the inflammation related signaling pathway in HUVEC.(3) Cotinine activates TLR-4/My D88/NF-κB signaling pathways of inflammation in HUVEC. |
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