预注射供体凋亡细胞对移植胰岛存活及外周血T淋巴细胞功能的影响

The Influences of Pre-injection of Donor Apoptotic Cells on Survival of Islet Grafts and Function of T Lymphocytes

目的研究预先注射供体来源的凋亡细胞对移植胰岛存活时间及外周血T淋巴细胞功能的影响。方法分别利用直线加速器照射及热休克(56℃水浴箱震荡摇1 h)的方法获取供体凋亡细胞和坏死细胞。利用链脲佐菌素(STZ)腹腔注射的方法将接受胰岛移植的大鼠42只诱导为糖尿病模型大鼠后,随机分为4组,分别在胰岛移植前1周注射生理盐水(9只)、正常细胞(12只)、凋亡细胞(12只)及坏死细胞(9只);在注射处理后第7天进行肾包囊下胰岛移植。通过血糖变化评估胰岛存活时间,并在胰岛移植前(注射后1周)、胰岛移植后1周、2周以及发生排斥反应后取3只大鼠采取外周血,利用四甲基偶氮唑盐(MTT)法检测外周血T淋巴细胞的增殖功能;利用多功能流式点阵仪检测外周血T淋巴细胞亚群细胞因子干扰素(IFN)-γ、白介素(IL)-10的水平及酶联免疫方法测定转化生长因子(TGF)-β1水平。结果凋亡细胞预处理能明显延长胰岛移植物存活时间,并抑制外周血T淋巴细胞对刀豆蛋白A刺激的增殖能力;同时在移植后1周、2周抑制了外周血IFN-γ的分泌,而增加了IL-10、TGF-β1的分泌(均P<0.05)。结论凋亡细胞可能通过抑制外周血T淋巴细胞的增殖活化能力,改变不同T细胞亚群细胞因子的分泌调节受体的免疫反应,从而抑制排斥反应,延长移植物存活。

Objective To study the influence of pre-injection of donor apoptotic cells in the survival of islet grafts and the function of T lymphocytes in the peripheral blood. Methods The donor apoptotic cells and necrotic cells were ob-tained respectively by X-irradiation from electron linear accelerator and a heat-shock procedure （water bath box 56℃, 1 h）. The diabetic rats for islet transplantation （n=42） were induced by a single intraperitoneal injection of streptozotocin （STZ）, then were randomly divided into four groups：rats were injected by physiological saline group （n=9）, normal cells group （n=12）, apoptotic donor cell group （n=12） and necrotic donor cell group （n=9）. On the seventh day, each group received islet transplantation under the renal capsule. The blood glucose level was detected to reflect the survival of the islets. The periph-eral blood samples of three rats in each group were obtained at different observation times. The proliferative activity of T lym-phocytes was determined by MTT method. The levels of cytokines interferon （IFN）-γ, interleukin （IL）-10 in peripheral blood were measured by Luminex 100 Integrated System, and transforming growth factor （TGF）-β1 by ELISA respectively at 0 d, 1 week, 2 weeks and after rejection. Results The survival time of islets was significantly prolonged by the pre-intervention of apoptotic cells, and the proliferative activity of T lymphocytes stimulated by ConA was inhibited. Meanwhile, the extent of the increased level of IFN-γwas inhibited significantly at 1 week and 2 weeks after transplantation （P〈0.05）, the levels of IL-10 and TGF-β1 were significantly increased before transplantation, 1 week and 2 weeks after transplantation （P〈0.05）. Conclusion Our results demonstrated that the pre-treatment of donor apoptotic cells can regulate the recipient’s immune reactive state by inhibiting the proliferative activity of T lymphocytes and changing the levels of cytokines from different sub-sets of T lymphocytes, and finally resulted in the prolonging of the survival of islet grafts.