Early Rheumatoid Arthritis, Cold And Heat Syndromes Genome Informatics Preliminary Study

AimsTo explore the differences of the gene expression profiles of CD4~+ T lymphocytes in peripheral blood between the normal control populations and the patients with rheumatoid arthritis (RA), and between the heat and cold syndrome patients with rheumatoid arthritis using cDNA microarray technology.Methods33 RA patients were differentiated into hot and cold syndrome group according to corresponding syndrome standards. Peripheral blood CD4~+ T lymphocytes from the RA patients and normal control populations were isolated and collected using Ficoll-Hypaque gradient centrifugation method. The mRNA of all the CD4~+T lymphocytes samples were extracted and identified, and then were labeled and hybridized to microarrays containing approximately 24000 cDNA spots. The gene expression data were analyzed by t-test, analysis of variance (ANOVA), and hierarchical clustering analysis methods using genepix 4100, Cluster and TreeView software (Stanford University) and with the help of GeneSifter microarray data analysis system to find the differences of gene expression profiles between the normal populations and RA patients, and between the RA patients with hot and cold syndrome. The genes with differential expression were validated by Realtime-PCR.Results1 Hierarchical clustering analysis showed there were 149 differential expression genes between the normal control group and RA patients group, 77 up-regulated genes and 72 down-regulated genes were found in RA patients group compared with the normal control group, (fold change > or = 1.5;P < or = 0.05). We used GeneSifter software to do some pathway analysis of these genes, and found that they involved into many biological pathways, including MAPK signaling pathway, Jak-STAT signaling pathway, Purine metabolism, Regulation of actincytosketeton, cell cycle, acylglycerol metabolism, Notch signaling pathway, and so on. Among those differently expressed genes, some genes with relation to inflammatory reaction and zymogenesis like interferon, TNF, and protein phosphorylase were up-regulated, and on the other hand some immunological suppression genes like Protein phosphatase inhibitor, MHC-I like factors, IL-2 receptors were down-regulated.2 The results also showed that the gene expression profile in RA patients with heat syndrome was clearly distinct from those with cold syndrome, 27 up-regulated genes and 15 down-regulated genes were found in RA patients with heat syndrome compared with cold syndrome. GeneSifter software was used to do some pathway analysis of these differently expressed genes. And those genes are found related to some biological pathways like biosynthesis of bile acid, biosynthesis pathway 2 of fat acid, fat acid metabolism, degradation process of leucine and Isoleucine, metabolism pathway of lactamine and aspartic acid, Calcium signaling pathway, MAPK signaling pathway, Regulation of actin cytosketeton, etc.3 SI00, calcium binding protein A8 (calgranulin A), was verified to have significant difference between the normal control group and RA patients using RT-PCR.4 There are two genes, Homo sapiens cDNA FLJ1143 5 fis, clone HEMBA1001208 and Nit protein 2, we find that they were both expressed differently, between the normal control populations and the patients with rheumatoid arthritis (RA), and between the heat and cold syndrome patients with rheumatoid arthritis using cDNA microarray technology.Conclusion1 There were 149 differential expression genes were found between the normal control group and RA patients group, among these genes, some genes which have positive correlation with inflammatory reaction were up-regulated and other inhibiting body immunity genes were down-regulated, suggest that theinflammatory reaction of the body was ascending. The detection result using RT-PCR was unanimous with the result by the data analysis using microarray technology.2 42 genes were found differentially expressed between the heat syndrome patients and the cold syndrome patients in the RA patients group, and these expressed differently genes related to some energy metabolism and signal transduction changes.3 Not all the differential expressed genes between the normal control and RA patients were coincide with the differential expressed genes between the heat syndrome patients and the cold syndrome patients with rheumatoid arthritis. Their expressed differences have their own gene science basis.