Preliminary Study On The Mechanism Of TNFAIP3 Gene In Peripheral Blood B Lymphocytes Of SLE Patients

Background Systemic lupus erythematosus (SLE) is an autoimmune disease which can cause inflammation of multi-system and injuries of multi-organ characterized by the abnormal immune function of T and B cells, producing many kinds of autoantibody(for instance, antinuclear antibodies, antidsDNA antibodies and antiphospholipid antibody), causing inflammatory injuries from orrhomeningitis, arthritis, vasculitis, glomerulonephritis. Its pathological lesions involve several tissues and organs including skin, mucous membrane, heart, kidney, liver, blood system and nervous system. Up to now, the pathogenesis of SLE has not been clarified by any research. However, many researches have stated that SLE is presently related to multiple factors, such as environment (e.g. strong sunshine exposure), hormone, virus infection, immune, inheritance, medicine and so forth. Nevertheless, the previous research indicated that SLE is a polygenic disease inheriting mainly through non-Mendelian inheritance, with inheritance factor playing a major role in the induction factor of SLE. In recent years, candidate genes approach and Genome-wide association study have confirmed that TNFAIP3 is the susceptible gene of SLE in Chinese Han population. TNFAIP3 can inhibit Toll-like receptor (TLR), activation of Nuclear factor kappa B induced by Tumor necrosis factor (TNF) as a receptor and programmed cell death mediated by TNF, and inhibit ultimately the strength of NF-κB, terminate the Proinflammatory with the mediation of NF-κB and thereby inhibit B cells overactive and apoptosis defect, which may play a crucial role in occurrence and development of SLE and other autoimmune diseases.Objectives 1. To construct the retroviral recombinant expression vector and to prove it can well expressed in mammalian cells.2. Respectively, express TNFAIP3 gene that in SLE patients’ and normal controls" peripheral blood B cells, and then detect its influence on B cells growth and survival and its influences on the expression of other genes such as TLR7、TLR9、BAFF-R. Through the experimental research, the potential relation of the SLE patients’B cells of TLR7、TLR9、BAFF-R and the potential mechanism of action of TNFAIP3 on the SLE disease and its clinical significance are discussed, so as to lay a good foundation for further exploration of the TNFAIP3 gene’s biological function and immune adjustment on SLE’s pathogenesis and its aggravation. Meanwhile, the research tries to provide a new diagnosing thought on the condition of SLE and a new way of treatment on SLE.Methods 1. Constructed the retroviral expression vector of TNIP1-pBABEpuro containing the gene of TNFAIP3.2.30 patients with SLE were collected and 15 normal controls. The peripheral blood lymphocytes were isolated using Ficoll-Hypaque gradient centrifugation and using immunomagnetic beads separation CD19+B cells and set aside.3.293T cells were chosen for lentivirus packaging plasmid to TNFAIP3-pBABEpuro、pBABE. The viral supernatants were collected after 48 hours culture and add it to the culture plate containing the cells of patients with SLE and the leukomonocyte B of normal controls which were separated in advance. With the supernatants changed once after 24 hours, cells were collected after infection of 48 hours. RNA was extracted from the cell B and then transcribed inversely to cDNA; Also using Real-time PCR technology to detect TNFAIP3 mRNA expression after RNA interference B cells’ TNFAIP3, to investigate TLR7 and TLR9 expression levels; by MTT experiment determine TNFAIP3 gene defect whether effect on the growth and survival of B cells.Results 1. TNFAIP3 retrovival recombinant expression vector was constructed successfully, and proved it can well expressed in mammalian cells.2. Over-expressed respectively SLE patients and normal controls B cells TNFAIP3 gene then get TNFAIP3-pBABE expression plasmids infection group than the control group pBABE, TNFAIP3 mRNA expression was significantly upregulated. (P<0.01). Not only the over-expressed SLE patients but also the contrasting TNFAIP3 gene in B cells can increase the level of mRNA and the gene increased higher in the B cells in the SLE patients.3. Not only SLE patients but only healthy control, TNFAIP3-pBABE infected group compared with pBABE control group, TLR7 mRNA expression level was significantly upregulated. (P<0.01).4. Over-expressed respectively SLE patients and normal controls B cells TNFAIP3 gene then get TNFAIP3-pBABE expression plasmids infection group than the control group pBABE, TLR9 mRNA expression was significantly upregulated. (P<0.05).5. TNFAIP3 gene of exogenous over-expression peripheral blood of B cells can increase the level of TNFAIP3 in B cells and meanwhile the expression of the gene also affects BAFF-R mRNA in B cells which are increased significantly.6. Over-expressed SLE patients and normal controls TNFAIP3 gene in B cells did not directed impact the cells growth and survival in vitro.Conclusions 1. TNFAIP3 gene abnormal expression suggestes that the gene may play an important regulation role in the pathogenesis of SLE.2. TNFAIP3 gene may be impact on the TLR mediated signaling pathway by regulating post-receptor signaling transduction, influence the expression of TLRs gene in B cells.4. TNFAIP3 gene did not affect the growth and survival of B cells in vitro.