【作者】 杜国盛；

【导师】 石炳毅；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2009， 博士

【摘要】 目的:探讨白介素23（IL-23）、白介素17（IL-17）在小鼠皮肤移植后不同免疫状态下外周循环血浆中及移植物组织病理的变化;了解IL-23、IL-17与调节T细胞（Treg cell）在不同免疫状态下的相互作用;探讨IL-23/IL-17通路在小鼠皮肤移植后的免疫调节作用。为临床移植后排斥反应的预防、诊断和治疗转归及免疫耐受提供理论指导。方法:小鼠皮肤移植动物模型的建立（n=10）:第1组、同种同基因移植组（BALB/c→BALB/c）、第2组、同种异基因移植组（C57BL/6→BALB/c）,第3组、供体脾细胞输注组（C57BL/6→BALB/c）。观察三组不同时间移植皮瓣外观变化及镜下三组移植皮肤HE切片的病理改变;采用ELISA方法检测小鼠血浆IL-17、IL-23的浓度;RT-PCR反应检测移植皮肤及脾脏的IL-23mRNA、IL-17mRNA的表达;流式细胞仪检测脾脏Treg细胞百分比。结果:三组小鼠移植后大体外观及HE染色镜下所见:同基因移植组小鼠移植皮术后皮片生长完好,无排斥反应发生;异基因小鼠移植皮出现典型的急性排斥反应;术前输注脾细胞异基因小鼠移植组,排斥反应明显减轻。三组小鼠皮肤移植后,IL-17、IL-23及IL-23mRNA、IL-17mRNA术后第1天内均表达升高,差异无统计学意义（P＞0.05）;随着移植时间的延长,2、3组IL-17、IL-23及IL-23mRNA、IL-17mRNA的表达明显升高,与第1组相比差异有统计学意义（P＜0.01）;2与3组术后3、5、7天的IL-23、IL-23mRNA的表达与1组相比,差异有统计学意义（P＜0.05）;2、3组相比无统计学意义（P＞0.05）;三组术后第3、5、7天的IL-17、IL-17mRNA表达结果显示:1、3组与第2组之间相比差异均有统计学意义（P＜0.01）。1、3组之间无统计学差异（P＞0.05）。流式细胞术检测显示:三组CD4⁺FoxP3⁺Treg细胞术后第1天占CD4⁺T细胞的比例分别为（0.9±0.6）%、（0.7±0.5）%、（0.8±0.4）%,差异无统计学意义（P＞0.05）。在皮肤移植术后第7天,第3组CD4⁺FoxP³⁺Treg占CD⁴⁺T细胞的比例高于2组,其分别为（5.1±2.8）%和（4.54±2.5）%,差别有统计学意义（P=0.00）:Th17/CD4⁺FoxP3⁺Treg比值在第2组中的表达高于第3组差别有统计学意义（P=0.00）。结论:（1）、移植后早期发生急性排斥反应时,IL-23,IL-17及IL-23mRNA、IL-17mRNA处于高表达水平;无排斥反应条件下IL-23、IL-17及IL-23mRNA、IL-17mRNA早期升高,随着炎症减轻其数值降低,IL-23,IL-17可作为预测和纠正排斥反应发生的观察指标。（2）、在急性排斥反应模型中IL-23与IL-17呈正相关性表达,IL-23作为IL-17的上游因子,对Th17细胞的成熟及其IL-17分泌有调控作用。抑制IL-23的分泌可降低IL-17的产生,可能降低排斥反应的发生。（3）、受体输注供体脾细胞,可明显延长移植物的存活,其机制可能与IL-17的分泌降低CD4⁺FoxP3⁺Treg增多有关。（4）、受体输注供体脾细胞可刺激Treg细胞生成,Thl7/CD4⁺FoxP3⁺Treg比值可能影响移植后的免疫状态。更多还原

【Abstract】 Objective:To probe the changes of interleukin23(IL-23) and interleukinl 7(IL-17) in the peripheral circulation and the pathological tissues and organ after the skin transplantation in mice;to disscuss the interaction during IL-23,IL-17 and regulatory T cells under the situation of different immune state;to understand the effect of immulogical regulation of IL-23/IL-17 axis after the skin transplantation in mice and to provide the guidance of the theory for the prevention,diagnosis,treatment and immune tolerance of rejection after transplantation in clinic.Method:The construction of animal model of skin transplantation in mice(n=10): groupl,isotransplantation(BALB/c→BALB/c);group2,allotransplantation (C57BL/6→BALB/c);group3,the donor spleenocytes were transfused to the recipient before transplantation(C57BL/6→BALB/c).To observe the three groups appearance changes of the transplanted skin and the pathological sections difference with Hematoxylin-eosine(HE) at different timing;to detect the concentration of IL-17,IL-23 in blood serum with ELISA method,IL-23mRNA,IL-17mRNA with R-T-PCR and Tregs in spleen with Flow cytometry.Result:With the observation and sections of H.E:the transplanted skin is well in the isotransplantation group without acute rejection,the typical acute rejection was occurred in the allotransplantation group and the rejection was significantly reduce in the group of preoperative infusion of allogeneic spleen cells in mice.There is no statistical significance in three groups among IL-17,IL-23,IL-23mRNA and IL-17mRNA at the first day after skin transplantation in mice(P＞0.05);to compare group2,group 3 with the groupl,there is statistical significance with the expressions of IL-23,IL-23mRNA at the 3,5,7 day(P＜0.01) and at the same time there is no statistical significance between 2rd and 3th group(P＞0.05);with the expressions of IL-17,IL-17mRNA,there is also significant difference between group2 and group1 or 3,and no statistical significance between 1 and 3 group at 3,5,7day after skin transplantation(P＜0.01).The percentage of CD4~+FoxP3+Treg account for CD4+T of the three groups is (0.9±0.6)%,(0.7±0.5)%,(0.8±0.4)%respectively at 1 day after transplantation and no statistical significance of three groups(P＞0.05).At the 7 day,the percentage of 3 group is higer than that of 2 group,(5.1±2.8)%,(4.54±2.5)%respectively with statistically significant(p=0.00);the relative value of Th17/CD4~+FoxP3+Treg in the 2 group is higer than that in the 3 group(p=0.00).Conclusion:1.Early post-transplant acute rejection occurs,expression of IL-23,IL-17 and IL-23mRNA,IL-17mRNA is in high-level;under conditions of no rejection and immune tolerance,IL-23,IL-17 and IL-23mRNA,IL-17mRNA rise in early stage,reducing its value as reduced inflammation,IL-23,IL-17 could serve as a preditor of acute rejection and prognostic indicator.2.In the acute rejection model,IL-23 and IL-17 expression was positively correlated.As the upper reaches of the IL-17 factor,IL-23 regulates Th17 cell growth and its secretion of IL-17.Inhibition of IL-23 may reduce IL-17 production,which might decrease the incidence of rejection.3.Infusion of donor spleen cells,can induce immune tolerance in skin allograft recipient mice.The mechanism may be related to the reduction of IL-17 and the increase of CD4+FoxP3+Tregs.4.Infusion of donor spleen cells may stimulate the generation of CD4+FoxP3+ Tregs in skin allograft recipient mice.Th17/CD4+FoxP3+Treg ratio may affect the immune system after transplantation.更多还原