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Objective To study the optimum determination conditions for lymphocytic proliferation by CCK-8 method in mice.Methods To study the different influence factors of spleen cell proliferation experiment stimulated by mitogen concanavalin A (ConA) or lipopolysaccharide (LPS),including cell preparation method,lymphocytic density,FBS and stimulating agent concentration in culture medium,and stimulating immediately or 24 h after preparing cell,with cross design or two factor completely randomized design.Results Spleen lymphocytic proliferation rate of preparation method by light suppression was higher than that of the light grind.The appropriate concentration of spleen cells was 5 × 106/mL.The proliferation rate has no significant difference after being stimulated for 48 or 72 h by ConA (2,5,or 1 0 μg/mL) or LPS (10,20,or 50 μg/mL) under 10％,15％,or 20％ FBS concentration in culture medium.The proliferation rate of stimulating immediately after preparing cell was higher than that of 24 h after preparing cell.Conclusion The optimum conditions of Balb/C mouse spleen cell proliferation assay stimulated by ConA and LPS are as follows:preparation of spleen cells with light pressure,spleen cell concentration of 5 × 106/mL,direct stimulation with 2-10 μg/mL ConA or 10-50 μg/mL LPS in the day of preparation.
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Objective To observe the pathological characteristics of thyroiditis induced by iodine excess and thyroglobulin (Tg) immunization and to explore the mechanism of thyroiditis induced by iodine excess. Methods NOD mice were used for intaking 0.05% Nal water and(or) Tg immunization. Morphologic change in thyroid and apoptosis were observed. The levels of serum TT4, TSH, thyroglobulin antibody (TgAb) and thyroid peroxidase antibody (TPOAb) were measured. Responding to Tg, lymphocytic proliferation of lymph node and spleen, interleukin-4(IL-4)and γ-interferon(IFN-γ) levels in culture medium of splenocytes were detected. Real-time PCR Was used to detect mRNA expressions of IL-4, IFN-γ, chemokine ligand 10 (CXCL10) and intercellular adhesion molecular-1(ICAM-1) in thyroid. Results Distended thyroid follicles,colloid accumulation, intense lymphocytic infiltration and disorganization were seen in thyroid of iodine excess group, along with increased apoptosis of thyroid cells(34.66～ 2.78 vs 5.11±0.62 ,P<0.01). The levels of TT4 were lowered while TSH raised ,but no production of thyroid-specific autoantibodies was revealed. Lymph node and spleen cells showed positive respornse under stimulation of Tg. The level of IFN-γ[(1. 272±0.049 vs 1. 139±0. 025)ng/L,P<0. 01] was raised in culture medium of splenocytes but not IL-4. The expression of IFN-γ, CXCLI0 and ICAM-1 mRNA were increased in thyroid. But in Tg group, some lymphocytes were scattered in thyroid, autoantibodies emerged ,and the level of IL-4 was increased in cuhure medium of splenocytes[(18. 508±0. 113 vs 13. 368±0. 016)ng/L, P<0. 01]. ledine excess combined with Tg enhanced these inflammatory reaction. Conclusion Iodine excess induced thyroiditis in NOD mice. The process seems to be Th1 response dominant organ-specific autoimmune diseases. Iodine excess and Tg immunizatiou play a synergistic role in inducing experimental autoimmune thyroiditis.
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Objective To explore the immunosuppressive effect of XGD 1 and its mechanism. Methods Different concentrations of XGD 1 were added to PHA or ConA induced human peripheral blood T lymphocyte.Seventy two hours later modified MTT assay was employed to test the effect of XGD 1 on T cell proliferation. Flowcytometry was used to examine the effect of XGD 1 on the expression of IL 2 receptor(IL 2R) on the surface of T cells individually at 48 h and 72 h.And the effects of XGD 1 combined with cyclosporine A(CsA) on the proliferation of T lymphocytes and the expression of IL 2R were also investigated. Results In the concentration range of 0.2～25 ?g/ml,XGD 1 exerted marked inhibitory effect on PHA or ConA induced T cell proliferation,which was proportional to dose. Flowcytometry showed that XGD 1 inhibited the expression of IL 2R,and the percentage of IL 2R? positive cells after stimulation of PHA decreased from 47.67% to 25.03% in the presence of XGD 1 (1 ?g/ml).XGD 1 and CsA had synergism in inhibition of T cell proliferation and IL 2R expression. Conclusion The study suggests that XGD 1 has immunosuppressive effect. The suppressive effect of XGD 1 on T cell proliferation is most probably mediated by decreasing IL 2R expression.
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Objective To manufacture rapamycin (RPM)-a new type immunosuppressant made in our country and investigate its immunosuppressive mechanism.Methods The effect of RPM on mouse splenocyte (Tc) proliferation induced by ConA,splenocyte (Bc) proliferation induced by LPS,DTH induced by DNCB and rat GVHR were studied.Results RPM obviously restrained the proliferation of Tc with IC50=1 nmol/L,significantly lower than IC50(10 nmol/L) of CsA (P＜0.05).RPM evidently repressed the proliferation of Bc with IC50=1 nmol/L significantly lower than IC50＞10 nmol/L of CsA(P＜0.05).RPM inhibited murine DTH to DNCB with ED50:1.8 mg/kg,significantly lower than ED50＞30mg/kg of CsA(P＜0.05).RPM controlled rat's GVHR with ED50=3 mg/kg,significantly lower than ED50≥30 mg/kg of CsA(P＜0.05).Conclusions RPM can inhibit lymphocytic proliferation and DTH,GVHR more markedly than CsA and they have combined efforts.
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Objective: To study difference of the immune response between oral administration and intraperitoneal injection of total polysaccharide of Sijunzi decoction (SJTP) in mice. Methods: The immunosuppressed mice model was established by intraperitoneal injection of cyclophosphamide (CY). The effects of SJTP on the weight of immune organs,hemolysin response and delayed type hypersensitivity (DTH) were observed,and the influences on normal mouse spleen lymphocytic proliferation of mice were also studied. Results:SJTP can remarkably counteract the immunosuppression induced by CY, decrease the weight loss immune organs,increase the hemolysin response and DTH,and promote the proliferation of lymphocytes.The effect of oral administration is superior to intrapertoneal injection.Conclusion:SJTP can improve the humoral and cellalar immunity of mice.The immune activities resulting from oral administration of SJTP have some special characteristics,and its possible mechanism is also discussed.