| Objective: Microparticles(MPs) viewed as “subcellular structure” releasing by cells,which has been found in variety of diseases, such as autoimmune disease. A few studys showed that MPs can break immune tolerance, trigger induction and amplification of autoimmunity response. The goals of this study were to discuss analysis of influencing factors of MPs, which to set up an optimization detection platform of MPs, moreover, to investigate immune stimulation of MPs between plasma and cells culture supernatant.Methods:1. The analysis of MPs concerned quantitative detection method and influence factors(1) The blood samples of 20 systemic lupus erythematosus and 20 rheumatoid arthritis inpatients from the Second Affiliated Hospital of Soochow University, age and sex matched 20 healthy controls were selected.(2) The number of total MPs of 20 systemic lupus erythematosus in plasma was measured by flow cytometry and Bradford protein quantitative, moreover, compared correlation with them.(3) The number of total MPs of plasma treated with different anticoagulants [sodium citrate, ethylenediamine teraaceticacid-dipotassium(EDTA-K2), heparin], and non-anticoagulant was detected by flow cytometry.(4) The number of total MPs treated with different preparation methods [washed platelet-poor plasma(PPP) and directed platelet-poor plasma(PPP)] was detected by flow cytometry.(5) The number of total MPs treated with different storage temperatures(4,-20,-80℃)and time(1, 3, 7, 10, 14 days) was detected by flow cytometry.2. The immunological function of MPs in vitro(1) The immunological function of MPs in plasmaa. 28 systemic lupus erythematosus and 29 rheumatoid arthritis inpatients and outpatients from the Second Affiliated Hospital of Soochow University, age and sex matched 40 healthy controls were also collected.b. The number of total MPs, lymphocyte-derived microparticles(LDMPs),monocyte-derived microparticles(MDMPs) in systemic lupus erythematosus, rheumatoid arthritis and healthy controls was measured by flow cytometry.c. We choosed systemic lupus erythematosus, rheumatoid arthritis healthy controls three cases, respectively. Meanwhile, we collected fresh EDTA-K2 anticoagulant plasma,which maked them directed platelet-poor plasma(PPP) by centrifugating and washing,co-incubated them with PBMC for 48 hours, finally, collected and detected the level of cytokines by CBA.(2) The immunological function of MPs in cells culture supernatanta. THP-1 cells were stimulated through activation or apoptosis by appling LPS, CAM,STS, Jurkat cells were stimulated through activation or apoptosis by adoptting PMA+PHA,CAM, STS, while counted the number of MPs released by THP-1 and Jurkat cells using flow cytometry.b. Range of concentrations(10, 20 and 40μg/m L) of MDMPs derived from apoptotic THP-1 cells were co-incubated for 14 hours with Jurkat and PBMC, The CD71 expression ratio on the surface of T lymphocytes was detected by flow cytometry.c. Range of concentrations(10, 20 and 40μg/m L) of MDMPs derived from apoptotic THP-1 cells were co-incubated for 48 hours with Jurkat and PBMC.The level of cytokines in culture supernatant was measured by CBA.Results:1. The analysis of MPs concered quantitative detection method and influence factors(1) The results implied that it has a significantly correlation between flow cytometry and Bradford protein quantiy for the number of total MPs in plasma(r=0.597, P<0.01).(2) Flow cytometry showed that sodium citrate plasma has the most MPs(F=25.04,P<0.05). Compared with EDTA-K2, heparin and non-anticoagulant, it has significantly differences(all P<0.05).(3) The number of total MPs in directed PPP samples from SLE, RA and HC were allIX higher than those of washed PPP samples(all P<0.05).(4) Compared with those of fresh plasma samples in stored for 0 day, the number of total MPs decreased significantly when samples stored for 1, 3, 7 and 10 days at the temperatures of 4 ℃,-20 ℃ and-80 ℃(all P<0.05), while the number of total MPs showed no significantly differences when stored for 14 days at the temperatures of 4℃,-20℃ and-80℃. There were also no significantly differences of the number of total MPs,when samples were stored in various storage duration(1, 3, 7, 10 and 14 days) at the three different temperatures(all P>0.05).1. The immunological function of MPs in vitro(1) The immunological function of MPs in plasmaa. The results of flow cytometry founded that the number of total MPs, LDMPs,MDMPs in patient with SLE and RA were higer than HC(all P<0.05).b. Compared with control group, the MPs derived from SLE could stimulate PBMC higer production of IL-6, TNF, IFN-γ than HC(all P<0.05), the MPs derived from RA only could stimulate PBMC higer secretion of IL-6(P<0.05).(2) The immunological function of MPs in cells culture supernatanta. Compared with control group, CAM, STS could induce THP-1 to release MDMPs(all P<0.05), PMA+PHA, STS and CAM all could induce Jurkat to release LDMPs(all P<0.05).b. Compared with control group, 20μg/ml and 40μg/ml concentrations of MDMPs could significantly increase the expression of CD71 of Jurkat cells(all P<0.05). Similarly,10μg/ml, 20μg/ml and 40μg/ml concentrations of MDMPs could significantly increase the expression of CD71 of PBMC(all P<0.05).c. Compared with control group, 20μg/ml and 40μg/ml concentrations of MDMPs could promote the production of IL-6(all P<0.05); 10μg/ml, 20μg/ml and 40μg/ml concentrations of MDMPs could promote PBMC to product IL-10(all P<0.05), besides,high concentration group could stimulate the secretion of IFN-γ(P<0.05).Conclutions:(1) Identification of the number of MPs in plasma should better choose fresh EDTA-K2 anticoagulant and should not wash and centrifuge repeatedly,furththermore, the number of total MPs shows no significant difference when stored at the temperatures of 4℃,-20℃ and-80℃ in two weeks.(2) The range concentrations of MDMPs derived from apoptotic THP-1 cells can stimulate activation of T cells and expressCD71 on the surface of T cells.(3) The MPs in plasma and MDMPs derived from apoptotic THP-1 cells culture supernatant can induce secretion cytokine of Th1/Th2, such as IL-6, IL-10, TNF or IFN-γ. |
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