新型膦酸酯类化合物DHBMGP2的免疫抑制作用

摘要

目的:从细胞和整体动物水平研究新型膦酸酯类化合物DHBMGP2免疫抑制活性.方法:分离正常BALB/c小鼠脾淋巴细胞,与DHBMGP2共培养72h,用[3H]TdR掺入法测定淋巴细胞增殖反应;染料排斥法检测24h细胞存活率,观察淋巴细胞毒性.雄性BALB/c小鼠用二硝基氯苯(DNCB)皮肤致敏2次(间隔1周),制备小鼠迟发型超敏反应模型,初次致敏当天开始ig给药,每天1次,连续10d,第11天处死动物测定耳肿胀度.雌性BALB/c小鼠足跖sc注射鸡卵清蛋白(OVA)免疫2次(间隔2周),制备致敏模型,首次免疫当天开始ig给药,每天1次,连续4周,每周眼球后静脉丛采血,ELISA法检测血清OVA特异性IgG,IgG和IgG2a杭体水平,[3H]TdR掺入法检测OVA杭原特异性T细胞反应.结果:DHBMGP2 1-100μmol·L-1体外应用可明显抑制小鼠脾淋巴细胞增殖反应,对脾淋巴细胞24h存活率无明显影响.DHBMGP2 20,40和80mg·kg-1可以减轻DTH模型小鼠耳肿胀度,耳肿胀度由模型组的(8.1±3.4)mg分别降低为(5.2±2.1),(5.1±1.0)和(5.4±1.3)mg,抑制其迟发型超敏反应.ig给予DHBMGP2 40和80mg·kg-1可以明显抑制OVA致敏小鼠杭原特异性T细胞反应,[3H]TdR掺入值由模型组的(975±46)cpm分别降低为(769±30)和(601±45)cpm,但对血清OVA特异性杭体水平无明显影响.结论:DHBMGP2体内外应用对细胞免疫反应具有抑制作用,对体液免疫反应无明显影响.%OBJECTIVE To investigate the immunosuppressive activity of the novel phosphonate compound DHBMGP2 in vitro and in vivo. METHODS In vitro, splenocytes from BALB/c mice was co-cultured with DHBMGP2 for 72 h, splenocyte proliferation was mearsured by [ 3H] thymidine uptake assay, and the 24 h survival rate was determined using trypan blue rejection assay.Dinitrochlorobenzene (DNCB)-induced delayed type hypersensitivity (DTH) models and ovalbumin (OVA)-immunized mouse model in BALB/c mice were used to assess immunosuppressive property of DHBMGP2 in vivo. Male BALB/c mice were sensitized with 5％ DNCB on the first day and then challenged by DNCB on the eighth day to induce the DTH model. Vehicle or DHBMGP2 20, 40 or 80 mg· kg-1 was administered ig on the first day, then once a day, for a total of 10 d. Mice were sacrificed to measure the ear swelling on the eleventh day. Female BALB/c mice were immunized subcutaneously with OVA on the first and fourteenth day to establish an OVA-immunized mice model.From the first day of immunization on mice were ig administered with vehicle or DHBMGP2 20, 40,and 80 mg· kg- 1, once a day, for 4 weeks. Blood was collected from the veniplex behind the eyeball using glass capillary once a week, and antigen-specific antibodies in serum were measured with ELISA. The mice were sacrificed at the end of 4 weeks of drug administration. Splenocyte suspension was prepared sterilized and OVA-specific T-cell responses were measured by [ 3H ] thymidine incorporation assay. RESULTS DHBMGP2 significantly suppressed concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated splenocyte proliferation in vitro, but had little effect on 24 h survival of the splenocytes. In the DTH model, DHBMGP2 20, 40 and 80 mg·kg-1 significantly decreased ear swelling from 8.1 ± 3.4 to 5.2 ± 2.1, 5.1 ± 1.0 and (5.4 ± 1.3 ) mg. After treatment with DHBMGP2 40 and 80 mg·kg-1, OVA-specific T-cell responses in OVA-immunized mice were significantly suppressed, and the [ 3H ] thymidine uptake was decreased from 975 ± 46 to 769 ± 30 and (601 ±45 )cpm. But OVA-specific antibody levels in serum were not reduced by DHBMGP2.CONCLUSION DHBMGP2 shows immunosuppressive activity on cellular immune response in vitro and in vivo, but it has little effect on humoral immune response.