Immunoregulatory Effects Of Thalidomide On Systemic Lupus Erythematosus (SLE) And Rheumatoid Arthritis

Objective:To detect the immunoregulatory effect of thalidomide on peripheral blood T-lymphocytes of systemic lupus erythematosus patients,Fibroblast-like synoviocytes of SD rats and Thl7 cells of RA patients.Methods:The experiment is divided into three parts(1)Part one:the experimental part with SLE patients:isolated the mononuclear cells from the peripheral blood of SLE patients,incubate the cells to 24 hole cell culture flask.The cells were kept under the conditions of 37℃,5%CO₂ for 2h.Then discard the adherent cells,collected cell suspension.Re-suspended the cells with DMEM that contained 10%FBS.Sub-groups: the control group,DMSO group,according to the concentration,Thd groups are divided into five:2.5,25,100,300,500μg/ml,after 72h's incubation these T cells' proliferation was deteted by MTT while after 24h's incubation the apoptosis,CD28 expression by CD3⁺T lymphocyte and CD152 expression by CD8⁺T lymphocyte were analyzed by flow cytometry(FCM).The mRNA expression of IL-6,IL-10 and TNF-αwere measured by RT-PCR after 72h.(2) Part two:the experimental part with Fibroblast-like synoviocytes of SD rats.Selection of normal SD rats,the fibroblast-like synoviocytes(FLS) are separated and cultured by collagenase and trypsin digestion way.When the cells were subcultured to the third generation,thalidomide was added and stayed for 24 hours. Sub-groups:the control group,DMSO group,according to the concentration,Thd groups are divided into five:300,500,800μg/ml.The proliferation was deteted by MTT while apoptosis were analyzed by flow cytometry.(3)Part three:the experimental part with RA patients.sub-group:accordance with the disease activity score(DAS) the patients are divided into three groups:RA disease activity group,RA stability group and the healthy control group.Mononuclear cells were separated from the peripheral blood of the RA patients.CD4⁺T cells which were negatively selected by immunomagnetic beads will be stimulated by PMA/Ion.At the same time an intervention of thalidomide(final concentration:500μg/ml) will be kept in the system for 6 hours.After the next steps of fixing,anti-reflective coating and intracellular staining,the level of IL-17 will be detected by FCM.Results:(1) In vitro 5001ag/ml thalidomide inhibited the expression of CD3+CD28,however,it promoted the early apoptosis;100μg/ml～500μg/ml thalidomide inhibited the proliferation,while promoted the expression of CD8+CD152.500μg/ml thalidomide inhibited the mRNA expression of IL-6,2.5μg/ml～500μg/ml thalidomide inhibited IL-10,TNF-αmRNA expression.(2) Thalidomide can inhibited the proliferation of FLS.It can also induce the apoptosis of FLS.Both these effects are concentration-dependent.(3) after the intervention of Thd,the intracellular expression of IL-17 by CD4⁺T cells from the peripheral blood of RA disease activity group,RA stability group and the healthy control group are obviously decreased,the difference was statistically significant.Conclusions:Thd have shown immunosuppressive effects on peripheral blood T lymphocytes of SLE patients,Fibroblast-like synoviocytes of SD rats and the intracellular expression of IL-17 by CD4⁺T cells from the peripheral blood of RA patients.It may reverse the immune imbalance of patients with autoimmune disease,making it possible to achieve therapeutic purposes.