【作者】 张帆；

【导师】 卢一平；

【作者基本信息】 四川大学 ， 外科学（泌尿）， 2023， 博士

【摘要】 目的:随着针对移植受者ABO血型抗体预处理技术的发展,ABO血型不相合(ABO blood group incompatibility,ABOi)活体肾移植已逐渐成为一项临床常规手术,其受者和移植物的存活率均与ABO血型相合(ABO blood group compatibility,ABOc)活体肾移植相当。ABO血型抗体介导的急性或超急性排斥反应是由受者体内ABO血型抗体与移植物内的相应血型抗原结合进而激活补体系统引起。研究证实,ABOi肾移植预后和受者初始血型抗体效价之间存在一定程度的相关性,但其预后与供者/供肾ABO血型抗原表达之间的相关性尚缺乏研究。此外,尽管通过严格的术前预处理将ABOi受者的血型抗体效价降低至极低的“安全”水平,但是术后仍有部分受者会发生由血型抗体介导的急性或超急性排斥反应,其发生原因尚未阐明。我们推测,这可能与移植肾ABO血型抗原表达的个体差异相关。人的ABO血型由ABH血型抗原决定,按ABH血型抗原是否可溶于水而分为分泌型血型抗原和固定于组织细胞表面的固定型血型抗原。在本研究中,我们总结了本移植中心ABOi活体肾移植的预处理方案及安全性、有效性,探讨了活体供者A/B血型抗原表达差异(分泌型和/或固定型A/B血型抗原)、供肾A/B血型抗原表达强度(A/B血型抗原强弱表达)与ABOi肾移植的预后的关系,并探索了影响供肾ABO血型抗原表达强度的关键因素。材料和方法:2014年9月1日至2020年8月31日期间,我院共实施了100例ABOi活体肾移植。我们在广泛参考国内外相关文献的基础上,制定并逐步改进了本移植中心基于供者初始抗ABO血型抗体效价的个体化预处理方案:(1)对于初始抗供者ABO血型抗体效价≤1:8的ABOi肾移植受者,在移植前仅接受口服免疫抑制剂进行预处理;(2)对于初始抗供者ABO血型抗体效价=1:16的ABOi肾移植受者,除接受口服免疫抑制剂外还接受抗体去除治疗(血浆置换和/或血浆双重滤过);(3)对于初始抗供者ABO血型抗体效价≥1:32的ABOi肾移植受者,在(2)的基础上加用利妥昔单抗进行预处理。我们通过倾向性评分匹配了本移植中心同期施行的200例ABO血型相合(ABOc)活体肾移植作为对照进行比较研究。通过竞争风险模型分析比较ABOi和ABOc活体肾移植的预后,包括感染、急性排斥反应、移植物丢失和受者死亡的累积发生率。我们纳入了2019年9月1日至2020年12月30日完成的32例ABOi活体肾移植,探究体内是否拥有分泌型A/B血型抗原与ABOi肾移植预后的关系。我们采用Sanger测序技术确定分泌型A/B血型抗原的决定基因FUT2的基因型,通过Wiener凝集试验检测唾液样本确定FUT2基因的血清学表型,通过凝胶卡技术动态监测ABOi肾移植受者围手术期血型抗体效价水平,探索ABOi活体肾移植的预后与分泌型A/B血型抗原的相关性。我们纳入了2019年1月1日至2021年12月30日完成的24例死亡捐献案例,获取了7个部位的组织,包括肾动脉、肾静脉、肾组织、肝动脉、肝静脉、门静脉和肝组织,研究了不同部位间固定型A/B血型抗原表达的差异。采用蛋白质免疫印迹法分析比较了血型抗原糖蛋白A/B的表达强度。我们前瞻性收集了2021年1月1日至2021年12月30日的28例ABOi肾移植病例,于供肾冷保存期间使用一次性穿刺活检枪获取供肾组织,通过免疫组织化学染色检测供肾固定型A/B血型抗原的表达水平,并通过图像分析软件半定量抗原表达强度,以探究供肾固定型A/B血型抗原的表达强度与ABOi肾移植预后的相关性。我们还对不同A/B血型抗原表达强度的供肾穿刺活检标本进行了转录组测序、差异基因筛选、韦恩分析、富集分析等研究,以探寻与供肾组织中固定型A/B血型抗原表达强度相关的信号通路和关键基因,并在移植肾穿刺活检组织样本中验证基因表达水平与A/B血型抗原表达强度的相关性。我们通过PCR扩增和Sanger测序技术检测了HK2细胞的ABO血型基因型。我们还通过构建B3GALT5过表达的HK2稳转细胞模型,使用实时定量PCR和蛋白质免疫印迹法检测了B3GALT5的表达水平。我们使用免疫荧光技术比较了B3GALT5过表达的HK2稳转细胞与对照组HK2细胞A/B血型抗原表达的差异,探索B3GALT5过表达对A/B血型抗原表达强度的影响。结果:中位随访25.8个月(范围:3.5-75.6个月)后,ABOi和ABOc组之间的移植物存活率(P=0.79)和受者存活率(P=0.39)均无显著性差异。在本院个体化预处理方案的稳定期,ABOi和ABOc组间受者急性排斥反应的累积发生率相近(10.0%vs 10.5%,P=0.346)。在ABOi肾移植组内,与接受利妥昔单抗预处理的受者(71例)相比,未接受利妥昔单抗预处理的受者(29例)急性排斥反应的累积发生率略高(8.4%vs 17.4%),但差异无统计学意义(P=0.362)。多变量竞争风险模型分析显示,无利妥昔单抗的预处理方案显著降低感染风险(HR:0.305,95%CI:0.120-0.781,P=0.013)。值得注意的是,受者抗A/B血型抗体效价术后的反弹在无利妥昔单抗预处理组的ABOi受者中更为常见(P=0.013),抗体效价反弹的受者发生急性排斥反应的风险是未反弹受者的2.72倍(HR:2.72,95%CI:1.01-7.31,P=0.048)。尽管相关的术前预处理已经将受者血型抗体的效价降至极低的“安全”水平,但是ABOi肾移植受者在移植后第一年内发生抗体介导的急性排斥反应的概率仍较ABOc活体肾移植组的受者显著升高(P=0.008),我们推测其可能与供肾A/B血型抗原相关并进行进一步研究。在探索分泌型A/B血型抗原与ABOi肾移植预后相关性的队列(32例受者及对应的32例活体供者)研究中,23例(71.9%)受者和27例(84.4%)供者具有FUT2分泌型基因型,9例(28.1%)受者和5例(15.6%)供者为非分泌型基因型。具有分泌型基因型个体的血清学表型可表现为强分泌型或弱分泌型,而非分泌型基因型个体则表现为弱分泌型或非分泌型表型。ABOi活体肾移植术后的抗A/B血型抗体水平变化的速率与供肾的分泌型状态相关。接受分泌型A/B血型抗原供肾的受者在接受ABOi肾移植术后30天内移植肾功能显著更好(P=0.003),且在术后一年内急性排斥反应的发生率更低(P=0.004)。我们使用蛋白质免疫印迹法检测了24例死亡捐献供者7个不同组织部位的固定型A/B血型抗原的表达水平,结果发现A/B血型抗原的表达在肾组织和门静脉中最高,在肾动脉中最低。在不同供者的同一组织学部位,或同一供者的不同组织学部位之间观察到固定型血型糖蛋白抗原的表达水平具有显著差异。单变量分析显示,固定型A/B血型抗原的表达受到供者年龄和性别的影响,其在男性和年轻人中的表达水平更高。供肾固定型血型抗原A存在三种表达水平(强、中和弱表达),血型抗原B则仅存在两种表达水平(强和弱表达)。接受固定型血型抗原强表达移植物的受者,其Ig M抗体效价反弹的比例显著升高(P=0.017)。与接受固定型血型抗原弱表达移植物的受者相比,接受固定型血型抗原强表达移植物的受者术后第1个月内的移植肾功能显著更差(P=0.047)。未观察到分泌型血型抗原(即分泌型状态)与固定型血型抗原表达强度之间存在显著的相关性。通过转录组测序,我们确定了三组与供肾组织固定型血型抗原表达强度相关的差异性表达基因集,分别包括983个、1021个和897个差异表达基因。通过韦恩分析,我们获得一组包括349个交集基因的共同差异表达基因集。经GO/KEGG富集分析发现,共同差异表达基因集与2条KEGG通路相关,分别是糖脂合成通路和神经活性配体受体相互作用通路。在糖脂合成通路中富集到三个共同差异表达基因,分别是B3GALT5、B3GNT3和FUT9。对供肾术中穿刺活检标本的免疫组化染色显示,B3GALT5、B3GNT3和FUT9过表达的供肾组织中血型抗原的表达强度较低。我们成功构建了B3GALT5过表达的HK-2稳转细胞模型,并通过Sanger测序发现HK-2细胞的血型基因型为A101/A101。我们通过过表达B3GALT5发现,HK-2细胞表面的A抗原显著降低。由于我们的研究发现,移植肾固定型血型抗原的表达水平越高,ABOi活体肾移植的预后越差,据此我们推测,可以通过过表达B3GALT5来减少供肾表面血型抗原的表达,从而改善ABOi活体肾移植的预后。结论:ABOi活体肾移植的个体化预处理方案可以实现与ABOc活体肾移植相当的移植物和受者存活率。接受利妥昔单抗预处理的ABOi活体肾移植受者术后感染并发症的发生风险更高;对初始血型抗体效价较低的受者进行无利妥昔单抗的预处理方案可同样有效地预防急性排斥反应,且会降低受者的感染风险。相比ABOc肾移植受者,ABOi肾移植受者在移植后第一年内发生抗体介导的急性排斥反应的概率较高,并可能与供肾分泌型血型抗原或/和固定型血型抗原相关。FUT2基因发生突变的个体体内不存在或仅存在少量分泌型血型抗原。分泌型A/B血型抗原影响ABOi活体肾移植的预后,当供者为分泌型时ABOi活体肾移植术后的移植肾功能较好,且急性排斥反应发生的概率较低。不同个体的相同组织部位之间,或同一个体的不同组织部位之间,固定型A/B血型抗原的表达水平存在显著的差异。在人类肾脏中,固定型A血型抗原分为强、中和弱三种表达强度;固定型B血型抗原分为强和弱两种表达强度。供肾固定型A/B血型抗原的表达强度影响ABOi活体肾移植的预后,供肾A/B血型抗原的表达水平越高,ABOi活体肾移植术后发生抗体反弹的概率越大,术后1个月内的移植肾功能越差。糖脂合成通路与供肾固定型A/B血型抗原的表达强度密切相关;供肾血型抗原的表达水平越高,B3GALT5、B3GNT3及FUT9的表达水平均越低;过表达B3GALT5可以减少HK2细胞血型抗原的表达强度。体外实验的结果为我们后续的动物实验模型验证提供了依据。更多还原

【Abstract】 Objective:With proper pretreatment technology aiming at the reduction of recipient blood group antibodies,ABO blood group incompatibility(ABOi)living donor kidney transplantation has now become a routine operation,and the survival rate of both recipients and grafts is comparable to that of ABO blood group compatible(ABOc)living donor kidney transplantation.Hyperacute or acute rejection mediated by ABO blood group antibodies is caused by the combination of ABO blood group antibodies in the recipient and the corresponding blood group antigens in the graft to activate the complement system.Studies have confirmed that there is a correlation between the prognosis of ABOi kidney transplantation and the recipient’s initial blood group antibody titer,but the correlation between the prognosis and the expression of ABO blood group antigen in the donor/donor kidney is still lacking.In addition,although the blood group antibody titer of ABOi recipients has been reduced to an extremely "safe" level through strict preoperative pretreatment,some recipients still experience acute or hyperacute rejection mediated by blood group antibodies after surgery,the cause of which has not yet been elucidated.We speculate that this may be related to individual differences in the expression of ABO blood group antigens in transplanted kidneys.The human ABO blood type is determined by the ABH blood group antigen.According to whether the ABH blood group antigen is soluble in water,it can be divided into secreted blood group antigens and fixed blood group antigens fixed on the surface of tissue cells.In this study,we summarized the pretreatment regimen,efficacy and safety of ABOi living donor kidney transplantation in our transplant center,and explored the differences in the expression of A/B blood group antigens(secretory and/or fixed A/B blood group antigen),correlation between donor kidney A/B blood group antigen expression intensity(A/B blood group antigen strong or weak expression)and the prognosis of ABOi kidney transplantation,and explored the key factors affecting the donor kidney ABO blood group antigen expression intensity.Materials and methods:From September 1,2014 to August 31,2020,our hospital performed a total of100 cases of ABOi living donor kidney transplantation.On the basis of extensive reference to relevant studies,we have formulated and gradually improved the individualized pretreatment plan based on the initial anti-ABO blood group antibody titer of the donor in our transplant center:(1)For the initial anti-ABO blood group antibody titer of the donor ABOi kidney transplant recipients ≤1:8,only receive oral immunosuppressant pretreatment before transplantation;(2)For ABOi kidney transplant recipients whose initial anti-donor ABO blood group antibody titer = 1:16,oral immunosuppressants and antibody removal therapy(plasma exchange and/or plasma double filtration)is conducted;(3)for ABOi kidney transplant recipients whose initial anti-donor ABO blood group antibody titer ≥ 1:32,rituximab was added on the basis of(2).We matched 200 cases of ABO blood group matching(ABOc)living donor kidney transplantation performed during the same period.A competing risks analysis was performed to compare ABOi and ABOc living donor kidney transplant outcomes,including the cumulative incidence of infection,acute rejection,graft loss,and recipient death.We also included 32 ABOi living donor kidney transplants completed from September 1,2019 to December 30,2020,to explore the relationship between the presence of secreted A/B blood group antigens and the prognosis of ABOi kidney transplantation.We used Sanger sequencing technology to determine the genotype of FUT2,the determinant gene of secreted A/B blood group antigen,detected the serological phenotype of FUT2 gene by Wiener agglutination test,and dynamically monitored the circumference of ABOi kidney transplant recipients by gel card technology.Blood group antibody titer level during operation,and explore the correlation between prognosis of ABOi living donor kidney transplantation and secreted A/B blood group antigen.We included 24 death donation cases from January 1,2019 to December 30,2021,and obtained tissues from 7 parts,including renal artery,renal vein,renal tissue,hepatic artery,hepatic vein,portal vein and liver tissue.The differences in the expression of fixed blood group antigens among different sites were explored.The expression intensity of blood group antigen glycoprotein A and B was compared by Western blot analysis.We prospectively collected 28 cases of ABOi kidney transplantation from January 1,2021 to December 30,2021.During cold preservation of the donor kidney,we used a disposable biopsy gun to obtain the donor kidney tissue,and detected the donor kidney by immunohistochemical staining.The expression level of fixed A/B blood group antigen was analyzed,and the antigen expression intensity was displayed semi-quantitatively by image analysis software,so as to explore the correlation between the expression intensity of donor kidney fixed A/B blood group antigen and the prognosis of ABOi kidney transplantation.In order to explore factors which may correlate with the intensity of A/B blood group antigen,we also conducted transcriptome sequencing,differential gene screening,Wayne analysis,and enrichment analysis on donor kidney biopsy specimens with different A/B blood group antigen expression intensities.Signal pathways and differential genes related to antigen expression intensity,and the correlation between gene expression level and A/B blood group antigen expression intensity was verified in biopsy tissue samples of transplanted kidney.We detected the ABO genotype of HK2 cells by PCR amplification and Sanger sequencing.After constructing a B3GALT5-overexpressed HK2 stable cell model,we detected the expression level of B3GALT5 by real-time quantitative PCR and Western blotting.We used immunofluorescence to compare the expression of A/B blood group antigens between B3GALT5 overexpressed HK2 stable cells and control HK2 cells,and explored the effect of B3GALT5 overexpression on the expression intensity of A/B blood group antigens.Results:After a median follow-up of 25.8 months(range: 3.5-75.6 months),there were no significant differences in graft survival(P=0.79)or recipient survival(P=0.39)between the ABOi and ABOc groups.During the stable period of the individualized conditioning regimen in our hospital,the cumulative incidence of acute rejection was similar between the ABOi and ABOc groups(10.0% vs 10.5%,P=0.346).In the ABOi kidney transplant group,compared with recipients receiving rituximab pretreatment(71 cases),the cumulative incidence of acute rejection was slightly lower(17.4% vs8.4%)in recipients who did not receive rituximab pretreatment(n=29),but the difference was not statistically significant(P=0.362).Multivariate competing risk analysis showed that the conditioning regimen without rituximab significantly reduced the risk of infection(HR: 0.305,95% CI: 0.120-0.781,P=0.013).It is found that postoperative rebound of recipients’ anti-A/B blood group antibody titers was more common in the non-rituximab pretreatment recipient group(P=0.013).The risk of acute rejection was 2.72 times that of non-rebound recipients(HR: 2.72,95% CI:1.01-7.31,P=0.048).Although the relevant preoperative pretreatment has reduced the titer of recipient blood group antibodies to an extremely "safe" level,the probability of antibody-mediated acute rejection in ABOi kidney transplant recipients in the first year after transplantation is still relatively increased than that of ABOc living donor kidney transplant recipients(P = 0.008).We speculate that it may be related to donor kidney A/B blood group antigens and further researches was performed.In a cohort study(32 recipients and corresponding 32 living donors)exploring the correlation between secretory A/B blood group antigens and ABOi kidney transplantation prognosis,23 recipients(71.9%)and 27 donors(84.4%)had the FUT2 secreting genotype,and 9 recipients(28.1%)and 5 donors(15.6%)had the nonsecreting genotype.The serological phenotype of an individual with a secretory genotype can be either a strong secretor or a weak secretor,whereas an individual with a non-secretor genotype can be a weak secretor or a non-secretor.The rate of change in anti-A/B blood group antibody levels after ABOi living donor kidney transplantation correlates with the secretory status of the donor.Recipients who received secretory A/B blood group antigen donor kidneys had significantly better graft function within 30 days after ABOi kidney transplantation(P=0.003),and had a lower incidence of acute rejection within one year after surgery(P =0.004).We detected the expression levels of fixed A/B blood group antigens in 7different tissue sites of 24 deceased donors by Western blot,and found that the expression levels of A and B blood group antigens were highest in renal tissue and portal vein,and lowest in renal arteries.Significant differences in the expression levels of the fixed glycophorin antigen were observed in the same histological site from different donors,or between different histological sites in the same donor.Univariate analysis showed that the expression level of fixed A/B blood group antigens was affected by donor age and sex,with higher expression levels in males and young adults.Donor fixed blood group antigen A had three expression levels(strong,moderate,and weak expression),while blood group antigen B had only two expression levels(strong and weak expression).The proportion of Ig M antibody titer rebound was significantly increased in recipients who received grafts with strong expression of fixed blood group antigens(P=0.017).Compared with recipients who received grafts with weak expression of fixed blood group antigens,recipients who received grafts with strong expression of fixed blood group antigens had significantly worse graft function in the first month after operation(P=0.047).No significant correlation was observed between secreted blood group antigens(i.e.,secreted status)and fixed blood group antigen expression intensity.Through transcriptome sequencing,we identified three sets of differentially expressed genes related to the expression intensity of fixed blood group antigens in donor kidney tissues,including 983,1021,and 897 differentially expressed genes,respectively.Through Wayne analysis,we obtained a set of common differentially expressed genes including 349 intersecting genes.The GO/KEGG enrichment analysis found that the common differentially expressed gene set was related to two KEGG pathways,namely the glycolipid synthesis pathway and the neuroactive ligand-receptor interaction pathway.Three commonly differentially expressed genes were enriched in the glycolipid synthesis pathway,namely B3GALT5,B3GNT3 and FUT9.Immunohistochemical staining of needle biopsy specimens during donor kidney surgery showed that the expression intensity of blood group antigens in donor kidney tissues with overexpression of B3GALT5,B3GNT3 and FUT9 was lower.We successfully constructed a stable HK-2 cell model with overexpression of B3GALT5,and found that the blood type genotype of HK-2 cells was A101/A101 by Sanger sequencing.We found that the A antigen on the surface of HK-2 cells was significantly reduced by overexpressing B3GALT5.Since our study found that the higher the expression level of fixed blood group antigens in transplanted kidneys,the poorer the prognosis of ABOi living donor kidney transplantation,we speculated that overexpression of B3GALT5 can reduce the expression of donor kidney surface blood group antigens,thereby improving the prognosis of ABOi living donor kidney transplantation.Conclusion:Individualized conditioning regimens for ABOi living donor kidney transplantation can achieve graft and recipient survival rates comparable to those of ABOc living donor kidney transplantation.ABOi living donor kidney transplant recipients who received rituximab preconditioning had a higher risk of postoperative infectious complications.Recipients with lower initial blood group antibody titers could be treated with a preconditioning regimen without rituximab Effectively prevent acute rejection and reduce the recipient’s risk of infection.Compared with ABOc kidney transplant recipients,ABOi kidney transplant recipients have a higher probability of antibody-mediated acute rejection within the first year after transplantation,which may be related to donor kidney secreted blood group antigens or/and fixed blood group antigens.Individuals with mutations of FUT2 gene have no or only small amounts of secreted blood group antigens.Secretory A/B blood group antigens affect the prognosis of ABOi living donor kidney transplantation.When the donor graft can secrete A/B blood group antigens,the graft function is better and the probability of acute rejection is lower.Significant differences exist in the expression levels of fixed A/B blood group antigens between the same tissue sites in different individuals,or between different tissue sites in the same individual.In the human kidney,fixed A blood group antigens are divided into strong,medium and weak three expression intensities;fixed B blood group antigens are divided into strong and weak expression intensities.The expression intensity of fixed A/B blood group antigen in donor kidney affects the prognosis of ABOi living donor kidney transplantation.The higher the expression level of donor kidney A/B blood group antigen,the greater the probability of antibody rebound after ABOi living donor kidney transplantation and the worse the function of the transplanted kidney within one month.Glycolipid synthesis pathway is closely related to the expression intensity of fixed A/B blood group antigen in donor kidney;the higher the expression level of blood group antigen in donor kidney,the lower the expression level of B3GALT5,B3GNT3 or FUT9.Overexpression of B3GALT5 can reduce the intensity of blood group antigen expression in HK2 cells.更多还原