大鼠胸腺联合后肾原基环磷酰胺预处理移植研究

Preliminary study on tolerance induction by combined of rat thymus and metanephros anlages transplantation preconditioned with cyclophosphamide protocol

目的将大鼠胚胎胸腺联合后肾原基同种和异种移植至环磷酰胺(CP)预处理去胸腺受体鼠,探讨能否诱导供体特异性免疫耐受.方法从受孕第16天(E16)的Lewis大鼠胚胎中提取成对胸腺和后肾原基,分别植入以CP预处理(BN鼠80 mg/kg、C57BL/6鼠200 mg/kg,腹腔注射,q6d×4)的去胸腺实验(Ⅰ)组或对照(Ⅱ、Ⅲ、Ⅳ)组受体腹腔,术后CP用量减半并联合环孢素[CsA,BN鼠8 mg/(kg·d)、C57BL/6鼠CsA12 mg/(kg·d)]皮下注射,每日一次×12 d,给药至第13天停药.移植后行胸腺、后肾移植物的形态学及功能检查,检测受体鼠血清白细胞介素(IL)-2和干扰素-γ(IFN-γ)含量、单向混合淋巴细胞反应(MLR)及皮肤移植试验.率的比较采用x2检验,两独立样本比较采用t检验,其他参数作方差分析.结果异种移植后14 d,C57BL/6鼠未见Lewis鼠胸腺和后肾原基生长.同种移植后21 d,部分实验(Ⅰ)组BN鼠胸腺及后肾发育尚好,显示一定的泌尿功能.实验(Ⅰ)组BN鼠Banff排斥反应分级最轻(x2=6.316,P<0.05),血清IL-2和IFN-γ含量分别为(388.92±55.98)ng/L(F=9.794,P<0.01)和(51.37±7.43)ng/L(F=4.120,P<0.05),与对照(Ⅱ、Ⅲ、Ⅳ)组差异有统计学意义.对供体Lewis和无关Wistar鼠移植皮片平均存活时间分别为(8.7±0.8)d和(5.8±1.2)d,两者差异有统计学意义(t=4.867,P<0.01);与对照(Ⅱ、Ⅲ、Ⅳ)组差异有统计学意义(F=17.421,P<0.01);对MLR脾细胞刺激指数(SI)分别为1.7±0.2和3.7±0.4,两者差异有统计学意义(t=-11.688,P<0.01);与对照(Ⅱ、Ⅲ、Ⅳ)组差异有统计学意义(F=12.787,P<0.01);各(Ⅰ、Ⅱ、Ⅲ、Ⅳ)组对第三品系Wistar鼠皮片存活时间(F=0.756,P>0.05)和SI差异无统计学意义(F=0.920,P>0.05).移植后5~7周,移植后肾和胸腺排斥反应加重,逐渐形成结缔组织瘢痕.结论使用CP预处理方案,Lewis鼠E16胸腺和后肾原基,在异种C57BL/6鼠腹腔内未能生长发育;在同种BN鼠腹腔内可继续发育,形成器官,尽管未诱导出免疫耐受,但可产生供体特异性免疫低反应.

Objective To investigate whether allo-or xenotransplantation tolerance can be induced by transplanted thymus combined with metanephros primordia,from rats to cyclophosphamide(CP)pretreated recipients.Methods Two pairs of thymus and metanephros primordia were extracted from the Lewis rats on embryonic day16(E16)and implanted into experiment(Ⅰ)group or control(Ⅱ,Ⅲ,Ⅳ)group renal capsules and omenta of thymectomized recipients,which had been pretreated with CP(80 mg/kg in BN rats and 200 mg/kg in C57BL/6 mice,ip,q6d×4).Post-transplantation,subcutaneous injection(sc,qd×12 d)of cyclosporine[8 mg/(kg·d)in BN rat and 12 mg/(kg·d)in C57BL/6 mouse]were simultaneously administrated till the 13th day.Histopathological examination of thymus and metanephros grafts and functional of developed metanephroi were performed post-transplantation.Rat seruminterleukin(IL)-2 and interferon-γ(IFN-γ)levels,one-way mixed lymphocyte reaction(MLR)and skin transplantation tests were measured.Results On the 14th day post-xenotransplantation,the thymus and metanephros primordia of Lewis rats were not found in the C57BL/6 mouse.On the 21st day after allotransplantation,the thymus and metanephros of some experimental(Ⅰ)group developed well,displaying partial urinary function.In experimental(Ⅰ)group,Banff rejection score(χ^2=6.316,P<0.05)was the lightest,and serum levels of IL-2 and IFN-γwere(388.92±55.98)ng/L(F=9.794,P<0.01)and(951.37±7.43)ng/L(F=4.120,P<0.05)respectively and the difference was statistically significant compared to control(Ⅱ,Ⅲ,Ⅳ)group.The mean skin graft survival time of donor Lewis and unrelated Wistar rat were(8.7±0.8)d and(5.8±1.2)d,respectively,and the difference between them(t=4.867,P<0.01)and to the control(Ⅱ,Ⅲ,Ⅳ)group were statistically significant(F=17.421,P<0.01).The stimulation index(SI)of MLR splenocytes were 1.7±0.2 and 3.7±0.4,respectively(t=-11.688,P<0.01).And the difference to control(Ⅱ,Ⅲ,Ⅳ)group(F=12.787,P<0.01)were also statistically significant;the skin graft survival time of each(Ⅰ,Ⅱ,Ⅲ,Ⅳ)group on the third strain Wistar(F=0.756,P>0.05)and SI had no significant difference(F=0.920,P>0.05).5-7 weeks post-transplantation,acute rejection of metanephros and thymus deteriorated and connective tissue scars gradually formed.Conclusion Using the CP pretreatment protocol,E16 Lewis thymus and metanephros anlagens xenografted into C57BL/6 mice failed to grow and function,whereas fail to induce allogeneic immune tolerance but can grow and develop in the peritoneal cavity of BN rat,produce donor-specific immune hyporesponsiveness.