转染IκB激酶2的显性负性突变体的受者树突状细胞诱导产生的CD4＋CD25-T细胞延长肾移植大鼠生存时间的研究

The study of prolongation of rat renal allograft survival by CD4 ＋ CD25-T cells induced by recipient dendritic cells transfected with dominant negative form of IκB kinases 2

目的 探讨转染Adv-IκB激酶2的显性负性突变体（IKK2dn）基因并负载供者抗原的受者树突状细胞（DC）诱导产生的CD4＋ CD25-T细胞回输是否延长同种异体肾移植大鼠的存活时间,并探讨其机制.方法 获取和培养受者Lewis大鼠骨髓源性DC,转染IKK2dn基因并负载BN大鼠可溶性抗原,与BN大鼠的T细胞初次混合淋巴细胞反应（MLR）,72 h后采用免疫磁珠分选法筛选CD4＋ CD25-T细胞.肾移植受者为Lewis大鼠,分为初始T细胞组、Adv0-CD4＋T细胞组、CD4＋CD25-T细胞组、排斥组,术前7d分别输注1×107个初始CD4＋T细胞、Adv-0-DC诱导产生的CD4＋ CD25-T细胞、Adv-IKK2dn-DC诱导产生的CD4＋ CD25-T细胞和等量生理盐水,供者均为BN大鼠.另设第三方供者组,术前处理同治疗组,供者为Wistar大鼠.移植术后观察各组大鼠存活时间和发生排斥反应;测定受者T淋巴细胞增殖能力;检测血清白细胞介素（IL）-2、IL-10、γ-干扰素（IFN-γ）以及转化生长因子-β（TGF-β）表达水平;监测血肌酐（Cr）水平;病理学检查评定排斥级别.结果 CD4＋ CD25-T细胞组肾移植大鼠生存时间为（28.50 ±2.36）d,较其他各组显著延长（P＜0.01）;与其他各组比较,CD4＋ CD25-T细胞组表达高水平IL-10和TGF-β（P＜0.05）;与排斥组、Adv0-CD4＋T细胞组、第三方供者组比较,CD4＋ CD25-T细胞组低表达IL-2和IFN-γ（P＜0.05）;CD4＋ CD25-T细胞组T淋巴细胞的增殖能力明显低于其他4组（P＜0.05）;病理学检查CD4＋CD25-T细胞组发生排斥反应的病理级别较低.结论 转染IKK2dn基因并负载供者抗原的受者DC诱导产生的CD4＋ CD25-T细胞回输可以延长同种异体肾移植大鼠生存时间,并具有针对供者的特异性,其机制可能与诱导的IL-10、TGF-β的高分泌有关.

Objective To observe whether CD4 ＋ CD25-T cells induced by recipient-derived immature dendritic cells （imDC） transfected with dominant negative form of IκB kinases 2 （IKK2dn） gene and loaded with donor antigen can prolong the survival time of kidney transplantation in rats and investigate its probably.Methods DC were cultured from recipient rats （Lewis） bone marrow,transfected with AdvIKK2dn and loaded with donor antigen,then cultured with Lewis rats lymphocytes in primary mixed lymphocyte reaction （MLR）.The CD4 ＋ CD25-T cells were separated by immune magnetic beads method after 72 hours.Male Brown Norway rats and Lewis rats were used as donors and recipients respectively,Four groups were set up（naive T cells group,Adv0-CD4 ＋ T cells group,CD4 ＋ CD25-T cells group and rejection group）,receiving 1 × 107 naive CD4 ＋ T-cell,Adv-0-DC-generated CD4 ＋ CD25-T-cell,Adv-IKK2dnDC-generated CD4 ＋ CD25 T-cell and equal volume of normal saline,respectively 7 days before transplantation.In the third party donor group,wistar rats as donors were treated the same as CD4 ＋ CD25 T cells group before transplantation.After transplantation,the survival time of recipients were observed; the T lymphocyte proliferation in recipients was measured; the levels of serum interleukin （IL）-2,IL-10,interferon-γ（IFN-γ）,transforming growth factor-β （TGF-β） were detected; serum creatinine levels were monitored ; pathological changes were examined to identify the grade of rejection,Results Compared with other groups,CD4 ＋ CD25-T cells group markedly prolonged the survival time of renal allografts [（28.50 ± 2.36） d,P ＜0.01],and had higher expression of IL-10 and TGF-β （P ＜0.05）.Compared with Adv0CD4 ＋ T cells group,rejection group,the third party donor group,expression levels of IL-2 and IFN-γwere lower（P ＜0.05）.In contrast to other groups,CD4 ＋ CD25-T cells group elicited markedly lower proliferative responses （P ＜ 0.05） and lower grade as estimated by pathological examination.Conclusion These findings suggested that CD4 ＋ CD25 T cells induced by recipient-derived immature DC transfected by IKK2dn and loaded with donor antigen could prolong renal allograft survival in a donor-specific manner,and it maybe by expressing high levels of IL-10 and TGF-β.