Associations Of Erythrocyte Immune Molecules, T Lymphocyte Subgroups And Single Nucleotide Polymorphisms Of Related Genes With Systemic Lupus Erythematosus And Hepatocellular Carcinoma

In 1981, Siegel raised the theory of red-cell immune system which broke the bound of blood cells' functions. Another theory of hematogenic immune reaction route was raised by Guo in 2004, the main idea of which is that 85% of immune complexes in blood are adhered by erythrocytes, and then transported to phagocytes or endothelial system to discard, or presented to T lymphocytes to kill. It is to say that red-cell immunity is the main immunological reaction route. Erythrocytes have many immune functions, among which immune adherence function of complement receptor type 1(CR1) and chemokine receptor function of Duffy antigen/receptor for chemokines(DARC) are paid more attention to, while CD59 can inhibit the cytolytic activity of human complement system and mediate signal transmission between erythrocytes and T lymphocytes. Otherwise, it is well known that CD4⁺ T lymphocytes with helper function and CD8⁺ T lymphocytes with suppressor and cytotoxic function perform special immune function together.The morbidity of systemic lupus erythematosus(SLE) in China occupies the first place in the world, and therapy of it is a difficult problem for the world. While both the morbidity and the mortality of liver cancer are high in China. Some researches in practice or theoretical presumptions suggest that the levels of CR1, DARC and CD59 on erythrocytes and T lymphocyte subgroups change in patients with SLE or hepatocellular carcinoma(HCC), but there are no detailed studies about them and also standard methods for measuring erythrocyte immune molecules. Otherwise, associations of genes for these molecules with hereditary susceptibility to diseases are not reported. In this research, the associations of erythrocyte immune molecules, T lymphocyte subgroups and single nucleotide polymorphisms(SNP) of related genes with SLE and HCC were studied, and the main contents, methods and results of which were as follows:Firstly, establishiment and optimization of method for measuring the levels of CR1, CD59 and DARC on erythrocytes. The molecules were measured by flow cytometry with direct fluorescent antibody technique, and then reliabilities of data analysis methods and effects of decoagulants(dipotassium ethylenediamine tetraacetic acid, heparin lithium and natrium citricum), sample deposit times and antibodies quantities on results were studied. Results: The percentage of CR1-positive erythrocytes was greatly affected by the choice of Ml boundary, and also it couldn't reflect individual differences in DARC and CD59. The geometric mean fluorescence intensity(GMFI) of CR1(CD59) increased with the elevation of FL2(FL1) voltage, while the geometric mean fluorescence intensity ratio(GMFIR) wasn't influenced by it. There were no statistically significant differences in GMFIR among decoagulants or sample deposit times(P>0.05). 7, 7 and 3 microliters of antibodies was respectively optimal for measuring CR1, CD59 and DARC on 5×10⁵ human erythrocytes.Secondly, establishment and optimization of method for detecting SNPs of candidate genes. Pairwise tagger method was used to choice tag SNPs of candidate genes. After multiplex primers were designed and synthesized, candidate tag SNPs of genomic DNA of 91 healthy persons were detected by multiplex PCR-fluorescent labeled single base extension-tag microarray hybridization genotyping technique, and individual genotypes were determined by software of SNPstream^(?) genotyping system. SNPs with genotyping success rate more than 90% were selected to continuous study. The coincidence rates of SNPs' genotyping results were calculated after repetitive experiments on 46 DNA samples were did. Results: 38 tag SNPs were choiced from CR1, DARC, CD59, CD4, CD8A and CD8B gene, and multiplex PCR primers for them were validated in simplex PCR. There were 8 SNPs with genotyping success rate less than 90%, and in 30 SNPs with genotyping success rate more than 90%, 3 of them, rs2707212, rs3829972 and rs10200219, had lower coincidence rates, while the rates of other 27 SNPs were more than 93.5%, and their total rate was 98.4%.Thirdly, characteristics of erythrocyte immune molecules, T lymphocyte subgroups and SNPs of related genes in healthy persons. The levels of CR1, DARC and CD59 on erythrocytes and T lymphocyte subgroups, and 30 SNPs from 6 genes were detected. SNPs in Hardy-Weinberg equilibrium(HWE) were selected to continuous study. Haploview V4.1 software was used in linkage disequilibrium analysis of SNPs, haplotype block construction with four-gamete test, haplotype frequencies calculation and gender association study. Correlations among erythrocyte immune molecules, T lymphocyte subgroups, gender, age and genotypes were analyzed with Pearson's correlation or Spearman's correlation, and main affection factors of them were analyzed with general linear model for univariate and multifactors. Results: There were no statistically correlations among CR1, DARC and CD59 levels, and percentages of CD4⁺ and CD8⁺ T lymphocytes(P>0.05). 3 SNPs including rs2707212, rs3829972 and rs10200219 were not in HWE, while genotypes and alleles of other 27 SNPs in HWE were not associated with gender(P>0.05). 6 haplotype blocks were constructed, and all the haplotypes in them didn't have associations with gender(P>0.05). CR1-GMFIR was negatively correlated with rs4844600(GG/GA/AA), rs9429945(CC/CT/TT) and rs11118167(TT/TC/CC)(P<0.05, P<0.01 and P<0.01, respectively). DARC-GMFIR was correlated with gender(female/male) positively and rs863002(CC/CT/TT) negatively(P<0.05). Percentage of CD4⁺ T lymphocytes was positively correlated with rs11064404(TT/TC/CC)(P<0.05), and percentage of CD8⁺ T lymphocytes was negatively correlated with age(P＜0.05). The above factors were main affection factors of corresponding indexes except rs4844600.Fourthly, effects of stress factors on erythrocyte and T lymphocyte immune. CR1 and CD59 on erythrocytes stored at 4℃, CR1, CD59 on erythrocytes and percentages of CD4⁺ and CD8⁺ T cells from samples collected at pre- and post-operation with mild or profound hypothermal cardiopulmonary bypass(CPB) were measured. Results: There were no significantly statistical differences in CR1-GMFIR among groups of different storage times(P>0.05). CD59-GMFIR decreased gradually in 9 weeks, and there were statistically significant differences among groups(P＜0.01). RBC counts, CR1-GMFIR, CD59-GMFIR, percentages of CD3⁺ and CD4⁺ T cells, and CD4⁺ T/CD8⁺ T ratio of patients with both profound and mild hypothermal CPB had decreasing tendencies from pre-CPB to time of minimum body temperature or post-CPB or 1 day after operation, and then began to turn back, while lymphocyte counts and percentage of CD8⁺ T cells increased from pre-CPB to post-CPB, plunged at 1 day after operation, and then turned back. In profound hypothermal CPB, erythrocyte immune was impaired slightly and could recover quickly, while T lymphocyte immune was damaged severely and couldn't recover to the level at pre-CPB until 7 days after operation. There were contrary changes in erythrocyte and T lymphocyte immune in mild hypothermal CPB. The extent of erythrocyte immune damage by 3 multiple stress factors from least to most was storing at 4℃, profound hypothermal CPB and mild hypothermal CPB.Fifthly, associations of erythrocyte immune molecules, T lymphocyte subgroups and SNPs of related genes with SLE. The levels of CR1, DARC and CD59 on erythrocyte and T lymphocyte subgroups, and 27 SNPs from 6 genes were detected. The differences in erythrocyte immune molecules and T lymphocyte subgroups levels, genotypes, alleles and haplotypes whose frequencies were more than 5%, among groups were compared and odds ratios(OR) and 95% confidence intervals(CI) were calculated. Results: CR1-GMFIR, DARC-GMFIR and CD4⁺ T/CD8⁺ T ratio in SLE group were lower than those in control group(P＜0.01, P<0.05 and P<0.01, respectively), while percentages of CD3⁺ and CD8⁺ T lymphocytes were higher than those in control group(P＜0.01). There were significant associations between 8 SNPs, 3 haplotypes and SLE(P<0.01 or P<0.05). When compared with noncarriers, the risks of developing SLE of carriers of CR1-rs4844600/GG genotype(OR:8.672, 95% CI:3.864-19.462) and G allele(OR:7.419, 95% CI:3.425-16.073), CR1-rs3818361/CC genotype(OR: 1.872, 95% CI: 1.113-3.149) and C allele(OR: 1.575, 95% CI: 1.067-2.325), CR1-rs11118167/TT genotype(OR:2.083, 95% CI: 1.065-4.071) and T allele(OR: 1.941, 95% CI: 1.050-3.588), CD59-rs11585AT genotype(OR:2.294, 95% CI: 1.226-4.293) and T allele(OR: 1.975, 95% CP.1.351-2.888), CD59-rs12576440/AG genotype(OR:25.673, 95% CI:10.440-63.137) and G allele (OR:32.571, 95% CI:13.916-76.235), CD4-rs1055141/CT genotype(OR:2.280, 95% CI: 1.261-4.123) and T allele(OR: 2.210, 95% CI: 1.293-3.778), CD8B-rs13400210/TT genotype(OR:8.661, 95% CI: 1.066-70.378) and T allele (OR:8.389, 95% CI: 1.041-67.613), CD8B-rs4832054/ AG genotype(OR:2.046, 95% CI: 1.055-3.967), CR1-rs11118167-rs3818361-rsl7048010/TCT haplotype (OR.1.863, 95% CI:1.288-2.693), CD59-rs831629-rs12576440-rs1738548-rs2231454/TGTG haplotype (OR:12.663, 95% CI: 4.959-32.336) and CD4-rs11064410-rs1055141-rs3213426-rs3213427/ATAT haplotype (OR:2.478, 95% CI: 1.116-5.505) were increased.Sixthly, associations of erythrocyte immune molecules, T lymphocyte subgroups and SNPs of related genes with HCC. Methods were identical with case control study of SLE. Results: CR1-GMFIR, DARC-GMFIR, CD59-GMFIR and percentages of CD3⁺ and CD4⁺ T lymphocytes in HCC group were lower than those in control group(P<0.05 for DARC and P<0.01 for others). There were significant associations between 5 SNPs and HCC(P<0.01 or P＜0.05). When compared with noncarriers, the risks of developing HCC of carriers of CR1-rs4844600/GG genotype (OR:2.458, 95% CI: 1.357-4.451) and G allele (OR:1.945, 95% CI:1.183-3.199), CR1-rs9429945/CC genotype (OR: 1.761, 95% CI: 1.006-3.084), DARC-rs863002/CC genotype (OR:2.325, 95% CI:0.990-5.462) and C allele (OR:2.191, 95% CI:0.960-5.002), CD8A-rs3020729/CT genotype (OR:1.936, 95% CL1.046-3.584) and CD8B-rs4832054/GG genotype (OR:2.354, 95% CI: 1.002-5.533) were increased.In conclusions, methods for measuring CR1, CD59 and DARC on erythrocytes by flow cytometry with direct fluorescent antibody technique and detecting tag SNPs of candidate genes by multiplex PCR-fluorescent labeled single base extension-tag microarray hybridization genotyping technique are established and optimized successfully. Levels of CR1, DARC, CD59 and T lymphocyte subgroups, correlations among them and the main affection factors of them in healthy persons are clear, and 27 tag SNPs from 6 genes are selected to continuous study, part of which are in 6 haplotype blocks, and there are no significant differences in genotypes, alleles and haplotypes of SNPs between genders. Stress factors can impair erythrocyte and T lymphocyte immune functions. CR1-GMFIR of suspending erythrocytes stored at 4℃is slightly impaired, while CD59-GMFIR is severely affected. Profound hypothermal CPB impairs erythrocyte immune slightly, while mild hypothermal CPB does severely. But changes in T lymphocyte immune shows that profound hypothermal CPB leads to immunosuppression, while mild hypothermal CPB induces inflammatory reaction. The extent of erythrocyte immune damage by 3 multiple stress factors from least to most is storing at 4℃, profound hypothermal CPB and mild hypothermal CPB. In case control study, erythrocyte and T lymphocyte immune functions decrease in patients with SLE or HCC. 8 SNPs and 3 haplotypes are associated with SLE, and 5 SNPs are associated with HCC.