Profile And Cluster Analysis Of The Genes Expressed In Human CD4～+ And CD8～+T Lymphocytes From A Patient With Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is one of the severe autoimmune diseases characterized by mutiple organ injuries in association with the persisting production of abundant autoantibodies to components of the cell nucleus. The inflammatory pathological findings in patients with SLE are those of immune complex deposition, vasculitis and vasculopathy lead to morphological changes and dysfunction with tissues and organs. Most of SLE patients die of renal failure coming from uremia with lupus nephritis.The exact aetiology and pathogenesis of SLE remains elusive. The clinical manifestations of SLE are extremely complicated, associated to a multifactorial interaction among various genetic and environmental factors. SLE is commonly known as a stem cell disease. Multiple genes contribute to the disease susceptibility. SLE also shows a strong familial aggregation with a much higher frequency among first-degree relatives of patients. Finally, various environmental or yet unknown factors are probably required to trigger the disease.The possible roles of pathogenesis with defective regulatory mechanisms from SLE patients are concentrated on more and more research works. Although the origin and the release of self-antigen are unknown, T cell is responsible for autoimmune diseases by recognizing self-antigen and instigating a response that might cause the disease. The loss of immune tolerance, increased antigenic load, excess T cell helps, imbalances of T cell suppression, defective B cell suppression, and the shifting of T helper 1 (Th1) to Th2 immune responses lead to B cell hyperactivity and the production of pathogenic autoantibodies. Th cells together with Ts cells keep a balance for stabilization of immune responses, whereas SLE might result from an imbalance of these cells' biological activities. The abnormal of genes expression of T cells could give rise to produce inappropriate cytokines under the certain condition at a certain time.In this study, the genes expressed in CD4+ and CD8+ T cells derived from a systemic lupus erythematosus patient before and after receiving chemotherapy were analyzed qualitatively and quantitatively with long serial analysis of gene expression (LongSAGE) protocol, and then the genes expression profile was established. We try to find out the relationship of the expressed genes in CD4+ and CD8+ T cells to SLE by the genes functional clustering analysis.CD4+ and CD8+ T cells were isolated from a female SLE patient. The patient complained facial erythema, transitory and migratory arthragia with her giant joints, mainly at shoulders, hipbones and knees for 2 months. She also had irregular low fever. Erythema with typical butterfly distribution showed at her face. Many abnormal laboratory findings were found. There were hemolytic anemia, thromocytopenia and leukopenia; the erythrocyte sedimentation rate was markedly elevated; level of IgG was high, while albumin was low, and C3 was much low. Protein, red blood cells, white blood cells and casts were found in the urine. Immunological examination was positive for anti-dsDNA antibody, and antinuclear antibodies with a ratio of 1:320. Ultrasonic examination showed pericardiac effusion. The score of SLE Disease Activity Index 2000(SLEDAI-2K) was 29. After chemotherapy with administrations of cyclophosphamide, hydroxychloroquine and cortiscosteroids for 5 months, the patient's facial erythema and arthragia disappeared. All laboratory abnormal findings returned to be normal references and SLEDAI-2K decreased to zero. These showed that the activity of SLE with the patient was retrieved into an inactive stage. Peripheral blood mononuclear cells (PBMC) were extracted from the patient at the first hospitalization and the disease stabilization respectively. Anti-CD4-FITC and Anti-CD8-FITC, combining with anti-FITC microbeads, were applied for islolation of CD4 + andCD8+ T cells.Total RNA was isolated from CD4+ and CD8+ T cells respectively only with the cells' purity was more than 95% by flow cytometry examination. The RNA samples were amplified with CD4, CD8 and CD 19 primers by RT-PCR. Isolated RNA from CD4+ T cells only produced CD4 bands, and was negative for CD8 and CD 19 amplification; RNA samples from CD8+ T cells were positive for CD8 amplification without any amplification for CD4 and CD 19 products. These showed that the RNA samples were derived from homogeneous cells and would keep the characteristics of the certain cells for construction of LongSAGE libraries, and the genes expression profile could represent the portrait ofcellular molecules.LongSAGE library was constructed from RNA corresponding to 106 cells. The mRNA binds to biotylated oligo-dT magnetic beads. The cDNA synthesized on the beads containing mRNA using Superscript? II Reverse Transcriptase, following the second-strand DNA synthesis with using E. coli DNA polymerase and E. coli DNA ligase. After verifying the cDNA synthesis integrity with PCR amplifications with primer-sets of GAPDH and EF (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and elongation factor-1 a (EF)), the DNA sample was digested with anchoring enzyme Nla III, and the cleavage product was divided equally into two new tubes. Adapter A and Adapter B ligated to cDNA, then the tags were released with digestion of tagging enzyme Mme I. The tags linked together to establish the 130bp ditags, which contains fragmants of expressed genes. Scale-up PCR tubes were amplified to obtain adequate 130bp ditags and the latter was purified from polyacrylamide gel. 130bp ditags were digested with Ma III again to produce 34bp ditags. The 34bp ditags were interlinked to yield concatemers. The concatemers were cloned into pZErO?-l vector. By transforming competent E. coli cells with vectors, the transformants were screened with PCR to evaluate the transforming efficiency. Finally, the positive resistant transformants for the presence of insert were ready to sequence. The expressed tags were extracted from the raw sequencing files and put into database. Therefore, four LongSAGE libraries were constructed from CD4+ and CD8+ T cells isolated from the SLE patient.During the construction of LongSAGE libraries, four protocol procedures had been modified so that the libraries were achieved successfully. There were: (1) the micro-beads placed on a magnetic stand for double time as the kit protocol's recommendation, and the beads' lose were decreased; (2) the amount of adaptors was cut down and the 130 bp ditags production were higher than that of the protocol, and the 130 bp ditags became more specific; (3) the polyacrylamide gel pieces were eluted twices, and the recycled purifying DNA was increased; (4) the eluted DNA solution from gel was extracted by phenol/chloroform to prevent the DNA degradation from nuclease, and more concatemers were obtained.14,130 tags were obtained from T4-1(CD4+ T cells isolated from the active state of the SLE patient) cell LongSAGE library with 6,162 unique tags, and 16,147 tags in T4-1(CD4+ T cells isolated from the inactive state of the SLE patient) cells LongSAGE library with6,960 unique tags. T8-1 LongSAGE library produced 9,259 unique tags in a total number of 36,510 tags. A total number of 34,887 tags in T8-2 library with 16,202 unique tags were also obtained, but about 53 percent of them were novel tags and were more than the other libraries. The distribution of expressed genes in T8-2 LongSAGE library showed a great deal of low-aboundance expressed genes with the diversity of complexity. These indicated that CD8+ T cell could produce more factors to elaborately regulate the immune environment in the inactive state of SLE. However, in the active state of SLE, the genes expressed in CD4+ and CD8+ T cells were lack of enough complexity and could not keep the balance of adaptive immune and autoimmune.Gene functional clustering analysis showed that many genes in an abnormal expression were relative to SLE. We noted that the expression of Hb a , 3 and 6 in CD4+ T cells of the patient could contribute the mechanism of pathogenesis with SLE. There were many genes expressed in the abnormality of cellular signal transduction with CD4+ and CD8+ T cells. Autoimmune could result from certain expressed genes in an improper way with increased or decreased mount of molecules in the immune system.Construction of genes expression profile in CD4+ and CD8+ T cells with SLE could play a role in the understanding of mechanisms of SLE. Abnormal factors or low and over expressed molecules in CD4+ and CD8+ T cells could disturb the immune regulation, then promote the outcome of SLE.