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【摘要】 目的 探讨人工培养冬虫夏草———百令胶囊免疫抑制作用及作用机制。方法 人外周血异基因混合淋巴细胞培养观察百令胶囊对混合淋巴细胞反应的影响;流式细胞及透视电镜检测淋巴细胞凋亡。上清液测可溶性细胞间粘附分子-1（sICAM-1）及细胞因子IL-1β、IL-2、IL-6、IL-10。结果 流式细胞DNA定量分析显示百令胶囊抑制淋巴细胞增殖，并可见凋亡峰（亚G 1 峰）、凋亡比例呈剂量依赖性。透视电镜见百令胶囊组淋巴细胞呈典型凋亡形态。百令胶囊组IL-1β、IL-6、IL-10显著减少（P<0.001，<0.05，<0.002）。百令胶囊高浓度组sICAM-1增加（P<0.01），低浓度组sICAM-1降低（P<0.01），与IL-1β、IL-10改变呈负相关（r=-0.91，-0.99;P<0.005，<0.001）。结论 人工培养冬虫夏草百令胶囊通过诱导淋巴细胞凋亡，抑制淋巴细胞增殖。其免疫抑制效应与抑制前炎症因子IL-1β、IL-6、IL-10，增加sICAM-1相关。
关键词 冬虫夏草 淋巴细胞凋亡 细胞因子 可溶性细胞间粘附分子-1
【文献标识码】A 【文章编号】 1606-8106（2004）13-1230-03
Suppressive effect of Corbrin，a drug of
cultivated Cordyceps Sinensis，onlymphocyte proliferation by inducting apoptosis and regulating cytokines and soluble intercellular
adhesion molecule-1 Wang Suya，Chen Jianghua，Zhang Jianguo，et al.
Renal Disease Center of the First Affiliated Hospital of
ZheJiang University Medical College，Hangzhou310003.
【Abstract】 Objective Corbrin capsules，a new pleiotropic immuno-regulator and a capsules of dry powder of cordyceps sinensis which extracted from cultivati on synne-matium by fluid，could induce long-term graft accepˉtance in dog allo- transplantation and were able to replace azathioprine for antirejection therapy in kidney transplantaˉtion patients with unclear mechanism of action.Methods To explore the mechanism of immuno-suppression of the drug of Corbrin capsued.Human peripheral blood lymphocyte were exposed in mixed culture to dry powder of Corbrin with RPMI1640fluid at a series final concentration of0.5mg.ml;1.0mg/ml;5.0mg/m l;10.0mg/ml;30.0mg/ml in respectively groups in contrast to the absence of the Corbrin group and to methylprednisolone group for72hours.Cell proliferative cyclewere measured by flow cytometry.While3-color flow cytometry and transmission electron miˉcroscopy were also evaluated as assay for apoptosis of lymphocyte in vitro.In-vitro-produced pro-inflammatory inˉterleukin1beta（IL-1），IL-2，IL-6，IL-10and soluble intercellular adhesion molecule-1（sICAM-1）were alˉso measured by ELISA tests.Results Lymphocyte proliferation was suppressed.The cell cycle arrested on GI phase.And there was a significant difference in the extent of apoptosis in mixed lymphocyte reaction with Corbrin in a dose dependent manner.While in-vitro-production of pro-inflammatory IL-1beta，IL-6，IL-10were significantly lower than those of blank group（P<0.001，<0.05，<0.002，respectively）and IL-2did not change.The sICAM-1were increased in group with Corbrin5.0-30.0mg/ml and decreased in group with Corbrin0.5mg/ml，P<0.01.The sICAM-1were negatively relative with IL-1beta and IL-10（r=-0.91，-0.99;P<0.005，<0.001，respectively）.Conclusion The Corbrin terminating differentiation and inducting apoptosis on activated lymphocytes in a dose dependent has been observed in vitro.The effect of anti-proliferation and immunosuppression of Corbrin may be related to suppressing of production of IL-1beta，IL-6and IL10and augmenting sICAM-1in vitro.
Key words cordyceps sinensis lymphocyte apoptosis cytokines soluble intercel
lular adhesion molecule-1
冬虫夏草具有多重免疫效应 ［1，2］ ，其免疫抑制作用及其无毒副作用的特性近年来受到器官移植界的关注，并已用于肾移植临床 ［3，4］ 。
然而对其相关免疫抑制药理机制仍有待深入研究。本课题以人外周血淋巴细胞为研究对象，对百令胶囊-（CORBRIN CAPSULES，人工培养冬虫夏草菌丝粉）对异基因混合淋巴细胞反应的影响及对淋巴细胞凋亡、细胞因子及可溶性粘附分子的影响进行实验研究。
1 材料与方法
1.1 药品 人工培养冬虫夏草———百令胶囊菌丝粉提取液及纯化品由杭州中美华东制药有限公司药物研究所提供。百令胶囊菌丝粉经醇提、水提，蒸馏水稀释为10mg/ml、100mg/ml高压灭菌-20℃低温冰箱冻存备用。
1.2 试剂 RPMI-1640（SIGMA）培养液，含10%灭活小牛血清（四季青生物工程技术研究所，杭州），L-谷酰胺（2mmol/L），青、链霉素各100U/ml，pH调至7.0。IL-1β、IL-2、IL-6、IL-10（ELISA）试剂盒（COULTER公司，购自法国），sICAM-1（ELISA）试剂盒（CATALOG公司，购自比利时）
1.3 人外周血单个核细胞（PBMC）分离与异基因淋巴细胞双向混合培养（MLC） 三组同型健康人外周血分别肝素抗凝，淋巴细胞分离液密度梯度离心分离PBMC，PBS充分洗涤，计数，RPMI1640配成淋巴细胞悬液，加入药液，多块24孔培养板双向混合培养，37℃（5%CO 2 ）72h。细胞浓度为5×10/ml。