白细胞介素10修饰树突状细胞在异基因肝肾联合移植大鼠体内迁移及免疫保护

背景:供体抗原递呈细胞迁移至受体,可能诱导受体T细胞对供体产生免疫耐受,导致移植物最终被接受.利用白细胞介素10(interleukin-10,IL-10)对供体树突状细胞进行修饰,使其保持在所需的分化状态,对提高肝肾联合移植的肾脏保护作用是一种有效策略.目的:观察经IL-10修饰的树突状细胞在肝肾联合移植大鼠体内的迁移情况,探讨其免疫保护的诱导作用.设计、时间及地点:随机对照动物实验,于2004-06/2006-02在中山大学医学院完成.材料:清洁级成年DA雄性供鼠、Lewis雌性受鼠各60只.受鼠随机分为3组:IL-10修饰细胞组、单纯细胞组、模犁对照组,20只/组.方法:采用肝、肾联合法切取供体鼠的肝、肾,各组受体鼠均施行原位肿移植和原位左肾移植,建立肝肾联合移植免疫排斥模犁.无菌状态下分离供体鼠的股骨和胫骨,用DMEM培养基冲出骨髓,去除红细胞后贴壁法分离培养树突状细胞,加入终浓度20 μg/L的IL-10修饰72 h,经阴茎背静脉输入IL-10修饰细胞组大鼠体内,2×10(7)个细胞/只;单纯细胞组同法输入等量树突状细胞;模型对照组不给予任何干预措施.主要观察指标:肝肾联合移植后大鼠的生命体征、尿量及肝肾功能等指标的变化,病理组织学检测结果,原位杂交检测供体树突状细胞在受体鼠体内的分布.结果:单纯细胞组、模型对照组在移植后5～6 d尿量均减至0 mL,均呈重度急性排斥反应.IL-10修饰细胞组移植后2周内尿量维持在6～12 mL,肝肾移植的急性排斥反应受到明显抑制,平均存活时间为(20.0±2.6)d,明显长于单纯细胞组、模型对照组,Log Rank法检验P<0.05.受体肠系膜淋巴结等组织内有较多Y染色体标记的树突状细胞迁入.结论:IL-10修饰的树突状细胞可以对联合移植的肝肾起到免疫保护作用;供体的成熟前期树突状细胞迁移到受体组织内,能够抑制肝肾联合移植急性排斥反应,并延长肝、肾移植物和受体大鼠的存活时间.

In vivo migration and immunoprotection of interleukin-10-modified dendritic cells in rats after heterogenic simultaneous liver-kidney transplantation

BACKGROUND: Donor antigen presenting cells immigrated into the recipient can induce the immunological tolerance of recipient T cells to donor, leading to a final acceptance to grafts, lnterleukin-10 (IL-10) modification maintains dendritic cells at a desirable differentiating state, which is an effective method to promote the protection to kidney in the simultaneous liver-kidney transplantation.OBJECTIVE: To observe the immigration of IL-10-modified dendritic cells in rats subjected to simultaneous liver-kidney transplantation and to investigate the mechanism of action.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed in the Medical College of Sun Yat-sen University between June 2004 and February 2006.MATERIALS: Male DA donor rats (n=60) and Lewis recipient rats (n=60), both were adult and of clean grade, were included.Sixty Lewis rats were randomly and evenly divided into 3 groups: IL-10-modified cell group, simple cell group, and model control group.METHODS: Donor rat liver and kidney were harvested by simultaneous liver-kidney transplantation. Recipient rats in each group were subjected to orthotopic liver and left kidney transplantation to establish models of immunological rejection. Under sterile condition, donor rat femur and tibia were harvested. Dnlbecco's modified eagle's medium (DMEM) was used to wash out the bone marrow. After removal of red cells, dendritic cells were isolated and cultured by adherent method. After modified with 20 μg/L IL-10 for 72 hours, dendritic cells were intravenously transfused into rat bodies in the IL-10-modified group, 2×10(7) cells/rat, In the simple cell group, rats were treated with donor dendritic cells without modification with IL-10. Rats in the model control group received no any interventions.MAIN OUTCOME MEASURES: 1]Dynamic changes of vital sign, urine volume, liver and renal function in recipient tissues;2] Pathohistological detection results;3]Distribution of donor dendritic cells in the recipient rats by in situ hybridization.RESULTS: In the simple cell and model control groups, urinary volume was reduced to 0 mL 5-6 days after transplantation. In addition, both groups presented with severe acute rejection. In the IL-10-modified cell group, urinary volume maintained at 6-12mL within 2 weeks after transplantation. The acute rejection of liver and kidney transplantation was obviously inhibited, surviving for(20.0±2.6) days on average, which was significantly longer than that in the simple cell group and model control group. A probability value of less than 0.05 in the Log Rank test was considered statistically significant. There were many Y chromosome-labeled dendritic cells immigrated into the mesenteric lymph node in the recipient rats.CONCLUSION: IL-10-modified dendritic cells play an immunoprotective effect on the liver and kidney transplanted simultaneously. Donor immature dendritic cells immigrated into recipient tissue could reduce acute rejections and prolong the survival time of liver and kidney grafts and recipients.