抗体介导的小鼠移植肾急性排斥反应模型的构建与改良

Construction and modification of an antibody-mediated acute rejection model of renal graft in mice

目的构建并改良同种异体小鼠肾移植急性抗体介导排斥反应(ABMR)模型。方法选用雄性6~8周龄、主要组织相容性复合体(MHC)全错配的C57BL/6和BALB/c小鼠各31只(南京医科大学医药实验动物中心),分别作为供受体。采用简单随机分组分为以下4组,ABMR组(10只):行C57BL/6H2b→BALB/cH2d小鼠全层皮肤移植术预致敏后5 d,采用肾动脉带瓣Patch的端侧吻合法行同种异体肾移植术(C57BL/6H2b→BALB/cH2d);细胞介导排斥反应(TCMR)组(5只):采用肾动脉带瓣Patch的端侧吻合法行C57BL/6H2b→BALB/cH2d肾移植术;同基因对照(SYN)组(5只):采用肾动脉带瓣Patch的端侧吻合法行BALB/cH2d→BALB/cH2d肾移植术;皮肤移植(ST)组(5只):仅行C57BL/6H2b→BALB/cH2d小鼠全层皮肤移植术。记录受体存活时间;使用小动物B型多普勒超声系统测量小鼠移植肾血流速度及血流分布。在肾移植术后3、5、7 d行移植肾病理染色以及Banff诊断,并检测小鼠移植肾组织CD4+T细胞、补体片段C3d沉积、外周血内IgG类供体特异性抗体(DSA)、血清肌酐(Cr)及血尿素氮(BUN)水平。组间均数比较使用t检验、非参数检验,并采用Kaplan-Meier法进行生存分析,以P<0.05为差异有统计学意义。结果SYN组与TCMR组小鼠术后5 d均存活,而ABMR组小鼠术后5 d存活率为66.7%,生存状况远低于SYN组及TCMR组(χ^(2)=17.02,P<0.05)。在肾移植术后5 d,ABMR组血清Cr高于TCMR组、SYN组和ST组[(233.70±22.13)μmol/L比(68.06±11.10)、(20.08±2.42)、(20.92±2.27)μmol/L(F=325.5,P<0.01)];此外,ABMR组血清BUN也高于TCMR组、SYN组和ST组[(120.20±13.46)μmol/L比(24.12±3.68)、(20.48±5.39)、(17.00±2.55)μmol/L,F=216.4,P<0.01]。彩色多普勒超声显示,SYN组小鼠移植肾术后7 d移植肾血流分布及肾动脉收缩期最大流速与正常BALB/c小鼠差异无统计学意义[(373.40±6.44)mm/s比(387.90±6.90)mm/s,t=2.674,P>0.05]。与SYN组比较,ABMR组病理结果显示术后第3、5、7天时病理损伤逐渐加重,以术后第5天时最为典型。在肾移植术后第5天时,TCMR组移植肾CD4+T细胞浸润数量高于ABMR组(20310±5079比131083±8994,t=18.580,P<0.01),且TCMR组外周血内IgG类DSA低于ABMR组(426.9±37.4比900.2±30.0,F=828.3,P<0.01),根据Banff标准符合ABMR诊断。结论通过改进皮肤移植预致敏和血管吻合方法,成功改良并鉴定同种异体小鼠肾移植ABMR模型,并使用C57BL/6H2b、BALB/cH2d小鼠作为供受者,便于后续基因敲除动物实验的开展。

Objective To construct and improve an acute antibody-mediated rejection(ABMR)model in allogeneic mouse kidney transplantation.Methods Totally,31 each of male C57BL/6 and BALB/c mice(6-8) weeks old,with complete mismatch of major histocompatibility complex(MHC),obtained from Nanjing Medical University Medical Experimental Animal Center,were used as donors and recipients,respectively.The mice were randomly divided into 4 groups:ABMR group(10 mice):undergoing full-thickness skin transplantation from C57BL/6H2b to BALB/cH2d as a pre-sensitization procedure 5 days prior to allogeneic kidney transplantation using the end-to-side anastomosis method with renal artery patch;T-cell mediated rejection(TCMR)group(5 mice):undergoing C57BL/6H2b to BALB/cH2d kidney transplantation;syngeneic control(SYN)group(5 mice):undergoing BALB/cH2d to BALB/cH2d kidney transplantation;skin transplantation(ST)group(5 mice):undergoing only C57BL/6H2b to BALB/cH2d full-thickness skin transplantation.Recipient survival time was recorded.Renal blood flow velocity and distribution in transplanted kidneys were measured using a small animal Doppler ultrasound system.Renal pathology staining and Banff diagnosis were performed on days 3,5,and 7 after transplantation.CD4+T cells,complement fragment C3d deposition,donor-specific antibodies(DSA),peripheral blood IgG,serum creatinine(Cr),and blood urea nitrogen(BUN)levels were detected.Comparisons of means between groups were performed using T-tests,non-parametric tests,and survival analyses using the Kaplan-Meier method.P<0.05 was considered statistically significant.Results On the 5th day post-operation,all mice in the SYN and TCMR groups survived,while the survival rate in the ABMR group was 66.7%,significantly lower than that in the SYN and TCMR groups(χ^(2)=17.02,P<0.05).On day 5 post-transplantation,serum Cr in the ABMR group was higher than in the TCMR,SYN,and ST groups[(233.70±22.13)μmol/L vs.(68.06±11.10),(20.08±2.42),(20.92±2.27)μmol/L,respectively,F=325.5,P<0.01].Additionally,serum BUN in the ABMR group was also higher than in the TCMR,SYN,and ST groups[(120.20±13.46)μmol/L vs.(24.12±3.68),(20.48±5.39),(17.00±2.55)μmol/L,respectively,F=216.4,P<0.01)].Color Doppler ultrasound showed no significant difference in renal blood flow distribution and peak systolic velocity in the SYN group compared to normal BALB/c mice at 7th day after transplantation[(373.40±6.44)mm/s vs.(387.90±6.90)mm/s,t=2.674,P>0.05].Compared to the SYN group,pathological results in the ABMR group showed progressively worsening damage on days 3,5,and 7 post-operation,with the most typical damage observed on day 5.At day 5 post-transplantation,the number of CD4+T-cell infiltration in transplanted kidneys was greater in the TCMR group than in the ABMR group(20310±5079 vs.131083±8994,t=18.580,P<0.01),and the peripheral intracellular blood IgG class DSA was lower in the TCMR group than in the ABMR group(426.9±37.4 vs.900.2±30.0,F=828.3,P<0.01),consistent with the Banff criteria for ABMR diagnosis.Conclusion By improving the skin transplantation sensitization and vascular anastomosis methods,the allogeneic mouse kidney transplantation ABMR model was successfully modified and identified,using C57BL/6H2b and BALB/cH2d mice as donors and recipients respectively,facilitating subsequent gene knockout animal experiments.