青藤碱对同种异体胰岛刺激淋巴细胞的免疫抑制作用

目的研究青藤碱(SIN)对同种异体胰岛刺激淋巴细胞的免疫抑制作用以及对胰岛活性和功能的影响。 方法以Wistar大鼠胰岛作为刺激原，Lewis大鼠脾淋巴细胞作为反应细胞体外共同培养，分为4组：SIN组为SIN＋胰岛＋淋巴细胞共培养；他克莫司(Tac)组为Tac＋胰岛＋淋巴细胞共培养；阴性对照组为胰岛＋淋巴细胞共培养；空白对照组为单纯淋巴细胞培养。四甲基噻唑蓝法检测各组淋巴细胞增殖情况，ELISA法检测上清液细胞因子(IFN-γ、IL-2、IL-10)浓度以及胰岛素分泌量，吖啶橙－碘化丙啶染色检测胰岛细胞活性。采用单因素方差分析比较4组混合淋巴细胞培养后OD值、细胞增殖率、上清液细胞因子浓度、胰岛素分泌量及胰岛素刺激指数等指标，采用LSD法进行组间两两比较。 结果混合淋巴细胞培养第3天，阴性对照组、SIN组、Tac组和空白对照组OD值分别为0.524±0.048、0.438±0.032、0.429±0.037、0.327±0.026；SIN组OD值低于阴性对照组、高于空白对照组，差异均有统计学意义(P均<0.05)，但与Tac组相比差异无统计学意义(P>0.05)。计算淋巴细胞增殖率SIN组和Tac组分别为(33.9±2.8)%、(33.2±3.0)%，均低于阴性对照组(P均<0.05)，但SIN组和Tac组差异无统计学意义(P>0.05)。SIN组上清液IFN-γ、IL-2浓度分别为(67±6)、(322±21) pg/mL，均低于阴性对照组，高于空白对照组(P均<0.05)，但与Tac组差异无统计学意义(P>0.05)；IL-10浓度为(76±4) pg/mL，高于其他3组，差异均有统计学意义(P均<0.05)。SIN组高糖及低糖刺激下胰岛素分泌量分别为(14.7±2.1)、(8.3±1.2) mU/L，计算胰岛素刺激指数为1.73±0.24，胰岛细胞存活率为(82.5±4.7)%，均优于Tac组和阴性对照组(P均<0.05)。 结论SIN对同种异体胰岛刺激的淋巴细胞具有免疫抑制作用，同时可减轻胰岛免疫损伤；与他克莫司相比，能更好地保持胰岛细胞活性及分泌功能。

The immunosuppressive effect of sinomenine on lymphocytes stimulated by allogeneic islets

ObjectiveTo investigate the immunosuppressive effect of sinomenine (SIN) on the lymphocytes stimulated by allogeneic islets and the influence on activity and function of islets. MethodsIsolated islets from Wistar rats as the stimulus and lymphocytes from spleen of Lewis rats as the reactive cells were cocultured in vitro, and then divided into 4 groups: SIN group with SIN, islets and lymphocytes cocultured; Tac (tacrolimus) group with Tac, islets and lymphocytes cocultured; negative control group with islets and lymphocytes cocultured; blank control group with lymphocyte cultured alone.The concentration of interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-10 and insulin in the supernatant were detected by enzyme-linked immunosorbent assay, the proliferation of lymphocytes was measured by methyl thiazolyl tetrazolium assay, and the activity of islet cells was tested by acridine orange and propidium iodide staining. ResultsOn the 3th days after cocultured, the proliferation rates of lymphocytes in the SIN group and Tac group were (33.9±2.8)% and (33.2±3.0)% respectively, both lower than that in the negative control group (P all <0.05), but there was no statistical difference between the SIN group and Tac group (P>0.05). The concentration of IFN-γ and IL-2 in the SIN group were (67±6) and (322±21) pg/mL respectively, both lower than those in the negative control group and higher than those in the blank control group (P all <0.05), but no statistical difference compared with the Tac group (P>0.05). The supernatant concentration of IL-10 in the SIN group was (76±4) pg/mL, and higher than those in the other 3 groups (P all <0.05). The insulin stimulation index and activity of islets in the SIN group were (1.73±0.24) and (82.5±4.7)% respectively, both superior to those in the Tac group and the negative control group (P all <0.05). ConclusionsSIN not only had immunosuppressive effect on lymphocytes stimulated by allogeneic islets, but also could alleviate the immune injury to the islets. What′s more, it could maintain the activity and insulin secretion function of islet cells better compared with Tac.