Notch1/Jagged1(CD339)在肾移植受者外周血淋巴细胞及移植肾组织中的表达
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摘要
目的和意义:
肾脏移植是临床上治疗终末期肾脏疾病的有效手段,但移植排斥反应仍是目前移植领域难以克服的重要问题,在同种异体肾移植中,急性排斥反应(Acuterejection,AR)是术后最常见的并发症,它通常发生于术后3个月内,最常见于术后1～3周,术后几年亦有发生。AR如果能得到早期诊断并正确治疗的话,90%以上患者的排斥反应可被逆转。AR是慢性排斥反应(Chronic rejection,CR)发生的高危因素,也是导致移植物长期存活失败和移植肾失去功能的最重要危险因素之一。急性排斥反应和急性肾小管坏死(Acute tubular necrosis,ATN)都是。肾移植术后常见并发症,两者术后都是以少尿、无尿为主要特点,术后早期较难鉴别。因此,如何早期诊断急性排斥反应非常重要。移植肾穿刺活检病理进行分型诊断是了解移植肾状态的经典指标,但其有创性及反复动态监测的困难性限制了它的临床应用。血肌酐升高作为排斥指标既不够敏感,又缺乏特异性。因此,探索非侵入性急性排斥反应诊断方法一直是肾移植临床研究的热点之一。
Notch信号通路是一个古老的信号传导途径,Notch信号是一组跨膜受体,Notch信号通路由Notch受体、Notch配体和CSL-DNA结合蛋白、其它的效应物、Notch的调节分子等组成。Notch受体最早发现于果蝇,因该基因的部分功能缺失会在果蝇翅膀边缘造成一些缺刻(Notchs)而命名。哺乳动物中共发现4类Notch基因,编码4种Notch受体,分别为Notch1～4。Notch的配体在哺乳动物中Notch配体共有5种,分别为Delta-like1、3、4和Jagged1、2,Notch的配体与其受体一样均为跨膜蛋白。Jagged1(新近被命名为CD339)是存在于哺乳动物细胞膜上Notch受体的主要配体之一,为单次跨膜糖蛋白,表达于骨髓、胎儿肝基质细胞和胸腺上皮细胞等多种组织,参与调控许多组织的生长发育。
最近的许多研究提示,Notch信号在外周免疫系统的分化和调节中发挥作用。移植肾排斥反应发生时,移植物作为同种异体抗原刺激机体免疫系统,引起Notch受体表达上调。Notch信号通路又通过一系列途径促进淋巴细胞分化、增殖。活化的淋巴细胞分泌细胞因子,进一步促进淋巴细胞增殖并产生免疫效应。研究表明,Notch信号可以通过调节淋巴细胞表面CD25的表达以及增强NF-kappaB的活性,促进IFNγ的分泌等途径来促进淋巴细胞的增殖。Notch信号通路活化在细胞因子或CD分子分泌表达之前,因此与其他的检测指标相比,Notch1受体表达水平的检测可能更有助于急性排斥反应的早期诊断。
本课题拟采用流式细胞术检测Notch1/Jagged1在肾移植术后受者外周血淋巴细胞中的表达,并应用免疫组化SP法检测各组移植肾组织中的Notch1/Jagged1蛋白表达,探讨检测外周血淋巴细胞中Notch1/Jagged1的有效性及实用性,并就Notch1/Jagged1在急性排斥反应的诊断及鉴别诊断中是否有意义以及对急性排斥的治疗是否具有指导价值等方面展开一些探讨和研究。
方法:
1、选取2007年1月～2008年12月接受肾移植手术的患者,以术后早期(1个月内)发生急性排斥反应(AR)的患者15例为急性排斥组,同期术后急性肾小管坏死(ATN)6例为肾小管坏死组,同期术后顺利恢复、肾功能稳定的15例为肾功能稳定组。采用流式细胞术检测所有患者术前和手术后1d、3d、7d、14d以及急性排斥组和肾小管坏死组发病(发生排斥或肾小管坏死)时及相应治疗(抗排斥或血液透析等)后1d、3d、7d、14d的Notch1/Jagged1的淋巴细胞阳性表达率,对比各组手术前后、发病前后以及AR患者抗排斥治疗前后不同时间点间的Notch1/Jagged1表达差异。
2、选取2007年1月～2008年12月我中心收治的晚期急性排斥反应(＞1年)患者8例为急性排斥组,诊断为慢性移植肾肾病(CAN)的15例患者为慢性移植肾病组,同期我院就诊行定期常规复查的肾移植术后肾功能正常稳定的10例患者作为肾功能正常组。采用流式细胞术检测患者外周血淋巴细胞的Notch1/Jagged1表达,对比三组外周血的Notch1/Jagged1表达差异,再按不同的免疫抑制方案和慢性移植肾病者是否行血液透析分别进行分组对比,了解Notch1/Jagged1表达与免疫抑制方案及血液透析的关系。
3、2007年1月～2008年12月采集的肾组织标本分为以下三组:(1)、急性排斥组:15例,均为肾移植术后发生急性排斥反应时行移植肾穿刺获取的患者移植肾组织;(2)、慢性移植肾肾病(CAN)组:15例,均为肾移植术后发生慢性移植肾肾病患者的移植肾组织;(3)、正常对照组:10例,为正常肾组织,其中6例为外伤后行肾切除者,4例为肾癌根治术癌旁5cm以外正常组织石蜡标本。所有肾脏组织标本采用免疫组化SP法检测组织中Notch1/Jagged1蛋白表达,对比各组Notch1/Jagged1的表达差异,并将急性排斥组和慢性移植肾病组按病理结果进行分级,对比不同病理分级间的Notch1/Jagged1表达差异,了解Notch1/Jagged1表达与病理分级之间的关系。
4、统计学方法:采用SPSS13.0软件包建立数据库,计量资料采用均数±标准差((?)±s)表示,以P＜0.05为差异具有统计学意义。术后早期各组多时间点间的比较采用重复测量的方差分析,每个分组在不同时间点上的两两比较采用Bonferron法,同一时间点上不同分组之间的两两比较采用LSD法。术后晚期及移植肾组织两组之间的比较采用独立样本t检验,急性排斥组抗排斥治疗前后的比较采用配对样本t检验,多组之间的比较采用单因素方差分析One-wayANOVA),组间两两比较采用LSD法。Notch1与Jagged1表达的相关性用Pearson相关分析,Notch1/Jagged1表达与病理分级的相关性用spearman相关分析。
结果:
1、肾功能稳定组、急性排斥组、肾小管坏死组之间肾移植手术前的Notch1表达无显著性差异(F=0.277,P=0.760),术后1d、3d时各组的Notch1表达差异亦无统计学意义(F=0.123,P=0.885和F=1.726,P=0.104),而手术后第7、14d时各组间的Notch1表达差异显著(F=3.354,P=0.027和F=2.672,P=0.039),其中急性排斥组的Notch1表达高于肾功能稳定组和肾小管坏死组(均P<0.05),而相同时间点间肾功能稳定组和肾小管坏死组间无显著性差异(均P>0.05)。肾功能稳定组手术后1d的Notch1阳性淋巴细胞率较术前升高(P=0.000),手术后3d时恢复至手术前水平(P=0.703),之后维持平稳,至术后14d时略低于术前水平,但差异无统计学意义(P=0.485);急性排斥组手术后1d时的Notch1阳性淋巴细胞率亦较术前升高(P=0.000),手术后3d时有所下降,术后7d、14d又显著升高并且再次高于术前Notch1水平(均P=0.000);手术后1d时的Notch1阳性淋巴细胞率亦较术前升高(P=0.000),3d、7d、14d时均略高于手术前水平,但差异无统计学意义(均P>0.05)。
术前各组之间的Jagged1表达无显著性差异(F=0.298,P=0.744),手术后1d、3d时各组间的Jagged1表达差异也并不显著(F=0.032,P=0.968和F=0.656,P=0.526),在术后7d、14d时各组间的Jagged1表达有显著性差异(F=1.959,P=0.045和F=3.120,P=0.027),其中急性排斥组高于肾功能稳定组和肾小管坏死组(均P<0.05),而相同时间点间肾小管坏死组和肾功能稳定组间Jagged1表达均无显著性差异(均P>0.05)。肾功能稳定组手术后1d时的Jagged1阳性淋巴细胞率较术前升高(P=0.000),手术后3d时与术前水平无统计学差异(P=0.102),之后开始平稳下降,术后14d时略低于手术前水平,但差异无统计学意义(P=0.225);急性排斥组手术后1d时的Jagged1阳性淋巴细胞率亦较术前升高(P=0.000),手术后3d时有所下降,但术后第7、14d时又开始升高,并且再次显著高于术前水平(均P=0.000);肾小管坏死组手术后1d时的Jagged1阳性淋巴细胞率亦较术前升高(P=0.000),术后3d时仍高于术前水平(P=0.071),术后7d、14d时与术前水平无显著性差异(P＝0.057和P＝0.534)。
2、急性排斥组和肾小管坏死组术前、术后发病时(发生排斥或肾小管坏死)时及相应治疗(抗排斥或血液透析等)后1d、3d、7d、14d比较显示:发病时及治疗后1d、3d时,急性排斥组的Notch1表达均显著高于肾小管坏死组(t＝49.521,P＝0.000;t＝37.284,P＝0.000和t＝7.295,P＝0.000),治疗后7d时,急性排斥组的Notch1表达仍高于肾小管坏死组(t＝2.249,P＝0.037),至治疗后14d时,两组差异无统计学意义(t＝1.741,P＝0.098)。急性排斥组出现排斥反应时Notch1阳性淋巴细胞率较术前明显升高(P＝0.000),经抗排斥治疗后逐渐下降,抗排斥治疗后3d、7d、14d时Notch1表达均明显低于抗排斥前(发生排斥反应时)Notch1水平(均P＝0.000),但抗排斥后7d时的Notch1表达仍高于术前(P＝0.041),至抗排斥后14d时与术前水平无统计学差异(P＝0.072);肾小管坏死组发生肾小管坏死时,Notch1阳性淋巴细胞率亦较术前升高(P＝0.002);经血液透析等治疗后,Notch1表达下降,至治疗后3d时已接近术前水平(P＝0.067),之后维持平稳。发病时及治疗后1d、3d时,急性排斥组的Jagged1表达均显著高于肾小管坏死组(t＝60.598,P＝0.000;t＝44.716,P＝0.000和t＝8.455,P＝0.000),治疗后7d、14d时,两组间的Jagged1表达无显著性差异(t＝0.022,P＝0.884和t＝0.097,P＝0.613)。急性排斥组出现排斥反应时Jagged1阳性淋巴细胞率较术前明显升高(P＝0.000),经抗排斥治疗后逐渐下降,抗排斥治疗后3d、7d、14d时的Jagged1表达均显著低于发生排斥反应时的Jagged1表达(均P＜0.05),至抗排斥治疗后7d时与术前水平无统计学差异(P＝0.512),抗排斥治疗后14d时略低于术前水平,但差异亦无统计学意义(P＝0.615);肾小管坏死组发生肾小管坏死时,Jagged1阳性淋巴细胞率较术前升高(P＝0.005),经血液透析等治疗后,Jagged1表达下降,至治疗后3d时接近术前水平(P＝0.054)。
3、肾移植术后晚期,急性排斥组发病时与肾功能正常组和慢性移植肾病三组间外周血淋巴细胞Notch1表达有显著性差异(F＝43.992,P＝0.000),急性排斥组的Notch1表达显著高于慢性移植。肾病组和肾功能正常组(均P＝0.000),而肾功能正常组和慢性移植肾病组的外周血Notch1阳性淋巴细胞率无显著性差异(P＝0.734)。急性排斥组抗排斥治疗前后Notch1表达差异显著(t＝5.511,P＝0.001),抗排斥治疗后Notch1表达明显下降。
三组间外周血淋巴细胞Jagged1表达差异显著(F＝61.652,P＝0.000),急性排斥组的Jagged1表达显著高于慢性移植肾病组和肾功能正常组(均P＝0.000),而肾功能正常组和慢性移植肾病组的外周血Jagged1阳性淋巴细胞率无显著性差异(P＝0.555)。急性排斥组抗排斥治疗前后Jagged1表达差异显著(t＝3.983,P＝0.005),抗排斥治疗后Jagged1表达明显下降。
急性排斥组Tac方案和CsA方案在排斥时和抗排斥冲击治疗后的Notch1表达以及慢性移植肾病组Tac方案和CsA方案的Notch1表达无显著性差异(分别t＝0.567,P＝0.591、t＝0.210,P＝0.841、t＝0.161,P＝0.875);不同免疫抑制方案间的Jagged1表达亦无显著性差异(分别t＝0.119,P＝0.907、t＝0.926,P＝0.095、t＝0.249,P＝0.807)。
慢性移植肾病组受者外周血淋巴细胞Notch1/Jagged1表达与是否行血液透析无明显关系(t＝0.459,P＝0.654和t＝1.638,P＝0.125)。
4、正常肾组织中Notch1表达极低(MOD＝0.012±0.003),仅在肾小管胞浆偶有较弱表达;急性排斥组肾组织的Notch1表达(MOD＝0.131±0.012)明显高于正常对照组(P＝0.000),其表达主要定位于肾小管上皮细胞的胞浆和胞膜,间质细胞有较弱表达;慢性移植肾肾病组肾组织Notch1表达(MOD＝0.013±0.003)也极弱,其Notch1表达与正常对照组无显著性差异(P＝0.756),但明显低于急性排斥组(P＝0.000)。正常肾组织中Jagged1几乎为阴性表达(MOD＝0.009±0.003);急性排斥组肾组织的Jagged1表达(MOD＝0.130±0.010)较正常对照组明显升高(P＝0.000),其表达亦主要定位于肾小管上皮细胞的胞浆和胞膜,间质细胞有较弱表达:慢性移植肾肾病组肾组织Jagged1表达(MOD＝0.012±0.002)也极微弱,其Jagged1表达与正常对照组无显著性差异(P＝0.335),但明显低于急性排斥组(P＝0.000)。
急性排斥组病理Ⅰ级的Notch1表达值为0.126±0.013,Ⅱ级Notch1表达值为O.136±0.011,Ⅱ级的Notch1表达略高于Ⅰ级,但其差异尚无统计学意义(t＝1.541,P＝0.147);而病理Ⅱ级的Jagged1表达(MOD＝0.136±0.005)明显高于Ⅰ级(MOD＝0.123±0.008)(t＝3.745,P＝0.002)。慢性移植肾病组各病理分级间肾组织的Notch1表达无显著性差异(F＝0.811,P＝0.467);各病理分级间肾组织的Jagged1表达亦无显著性差异(F＝0.811,P＝0.467)。
5、相关分析显示,肾移植术后早期外周血淋巴细胞的Notch1与Jagged1表达呈显著的正相关(r＝0.994,P＝0.000);移植肾组织中的Notch1与Jagged1表达亦呈显著的正相关(r＝0.983,P＝0.000)。
结论:
1、肾移植术后早期急性排斥反应发生时外周血淋巴细胞表面Notch1/Jagged1表达水平显著增高,检测外周血淋巴细胞的Notch1/Jagged1表达水平有助于急性排斥反应的诊断。
2、急性排斥反应经抗排斥治疗后,Notch1/Jagged1表达水平显著下降,因此,测定外周血淋巴细胞的Notch1/Jagged1表达水平有助于急性排斥反应治疗效果的动态监测。
3、肾移植手术后1d时各组受者外周血Notch1/Jagged1表达水平均有升高,早期ATN患者外周血淋巴细胞的Notch1/Jagged1表达与肾功能稳定患者无显著性差异,但明显低于急性排斥反应患者,对鉴别ATN和AR有一定帮助。
4、检测肾移植晚期受者外周血淋巴细胞的Notch1/Jagged1表达不能用于CAN的诊断,但仍有助于肾移植晚期受者急、慢性排斥反应的鉴别以及监测抗排斥治疗的效果。
5、CAN患者外周血淋巴细胞表面Notch1/Jagged1表达水平与免疫抑制方案及是否行血液透析无关。
6、急性排斥反应肾组织Notch1/Jagged1蛋白表达明显高于正常肾组织和慢性移植肾肾病肾组织,提示移植肾组织Notch1/Jagged1蛋白表达可作为监测移植肾形态学变化、功能状态以及预后判断的重要指标。
Objective and Significance
Renal transplantation is an effective clinic method to cure end-stage renal deseases.In renal transplantation field,acute rejection(AR) is the most frequent post-operation complication.AR often took place in the first 3 months,especially 1-3 weeks after operation.AR can lead to chronic rejection(CR) and is one of the most dangerous factors to result in allograft function losing.If AR gains early diagnosis and correct treatment,90%of ARs can be reversed.Acute tubular necrosis(ATN) is also a kind of common complication after renal transplantation.Main symptom of ATN and AR are both oliguria and anuria.ATN and AR are difficultly to be identified in early stage after operation.So it is important to early diagnose AR. Blood CR is not sensitive and absence lack of specificity.Renal needle biopsy is a traumatic examination.It is a hot point of clinic research to study non-traumatic diagnosis methods to AR.
Notch signal pathway is a group of transmembrane receptors.It is made up of Notch receptor,Notch ligand,CSL-DNA fixation protein,other effector and Notch regulatory molecules.Four kinds of Notch gene were found mammalian which encode four Notch receptors(Notch1～4).There are 5 kinds of Notch ligands (Delta-like1,3,4 and Jagged1,2) in mammalian.Notch ligands are transmembrane receptors too.Jagged1(CD339) is one of the main Notch ligands.
Recently,some research discovered that Notch signal can effect on differentiation and accommodation of periphery immune system.When renal allograft rejection occurred,allograft stimulates immune system and then Notch receptor expression increases.Notch signal pathway promotes lymphocytes differentiation and proliferation.Activated lymphocytes excretes cytokine which promotes lymphocytes proliferation and brings out immunologic effect.Notch signal can enhance the activity of NF-kappa B through modifying CD25 expression on the surface of lymphocytes,and can also promote lymphocytes proliferation through promoting IFNγ,secretion.Activation of Notch signal pathway is ahead of the secreting of cytokine or CDs.So examination of Notch1 receptor expression is perhaps more helpful on early diagnosis of acute rejection.
Materials and Methods
1 15 patients suffering early acute rejection after renal transplantation(＜1 month) in 2007.1～2008.12 are arranged as acute rejection(AR) group.6 patients with post-operation acute tubular necrosis are arranged as ATN group.15 patients with normal post-operation renal function are arranged as control group.Flow cytometry is used to detect Notch1/Jagged1 expression on lymphocytes before operation,in one week after operation and in one week after AR and ATN occurrence.Notch1/Jagged1 expressions before operation,after operation and after morbidity are compared.
2 8 patients suffering late acute rejection(＞1 year) in 2007.1～2008.12 are arranged as late acute rejection(LAR) group.15 patients with chronic allograft nephropathy(CAN) are arranged as CAN group.10 patients with stable and normal post-operation renal function are arranged as control group.Flow cytometry is used to detect Notch1/Jagged1 expression on lymphocytes.Notch1/Jagged1 expressions of every group are compared.
3 Nephridial tissue samples collected in 2007.1～2008.12 are divided into 3 groups as follow:(1) AR group,15 samples gained through kidney puncturation when acute rejection occurred.(2) CAN group,15 samples gained from CAN patients.(3) Control group,10 samples are normal kidney tissue.The expression of Notch1 and Jagged1 were detected by immunohistochemistrical SP staining method.Notch1/ Jagged1 expressions of every group and among different pathological grades are compared.
Results
1 Preoperative Notch1 expression on lymphocytes among AR group,ATN group and control group didn't show difference(F＝0.277,P＝0.760).Notch1 expression among 3 groups didn't show difference on post-operative 1d,3d either(F＝0.123, P＝0.885 and F＝1.726,P＝0.104).However,Notch1 expression among 3 groups showed significant difference on post-operative 7d,14d(F＝3.354,P＝0.027 and F＝2.672,P＝0.039).Notch1 expression of AR group was higher than that of control group and ATN group(all P＜0.05).Notch1 expression of control group and that of ATN group didn't show difference on every day(all P＞0.05).Notch1 expression on post-operative 1d was higher than preoperative ones on every group(P＝0.000). Notch1 expression of control group decreased and reached preoperative level on post-operative 3d(P＝0.703).Notch1 expression of AR group decreased on post-operative 3d too,but increased on post-operative 7d,14d(P＝0.000 vs preoperative level).Notch1 expression of ATN group decreased slowly within post-operative 1 week.Notch1 expression of ATN group decreased and reached preoperative level from post-operative 3d(P＞0.05).
Preoperative Jagged1 expression among AR group,ATN group and control group didn't show difference(F＝0.298,P＝0.744).Jagged1 expression among 3 groups didn't show difference on post-operative 1d,7d either(F＝0.032,P＝0.968 and F＝0.656,P＝0.526).Jagged1 expression of AR group was higher than that of control group and ATN group on post-operative 7d,14d(all P＜0.05).Jagged1 expression of control group and that of ATN group didn't show difference on every day(all P＞0.05). Jagged1 expression on post-operative 1d was higher than preoperative ones in all group(all P＝0.000).Jagged1 expression of control group decreased from post-operative 3d and reached preoperative level(P＝0.102).Jagged1 expression of AR group decreased on post-operative 3d too,but increased on post-operative 7d,14d and higher than preoperative level(P＝0.000).Notch1 expression of ATN group decreased on post-operative 3d too(P＝0.071),and reached preoperative level on post-operative 7d,14d(P＝0.057 and P＝0.534).
2 Notch1 expression of AR group was significantly higher than that of ATN group on 1d after morbidity and on post-treatment 1d,3d 7d(t＝49.521,P＝0.000; t＝37.284,P＝0.000;t＝7.295,P＝0.000;t＝2.249,P＝0.037).They didn't show difference on post-treatment 14d(t＝1.741,P＝0.098).When acute rejection occurred, Notch1 expression of AR group was higher than preoperative one(P＝0.000).After anti-rejection,Notch1 expression decreased continually and reached preoperative level on 7d(P＝0.072).Notch1 expression of ATN group was higher than preoperative one(P＝0.002).After therapy.Notch1 expression decreased(P＝0.067 on 3d).
Jagged1 expression of AR group was significantly higher than that of ATN group on morbidity day and post-operative 1d,3d(t＝60.598,P＝0.000;t＝44.716, P＝0.000 and t＝8.455,P＝0.000).They didn't show difference on post-operative 7d, 14d(t＝0.022,P＝0.884;t＝0.097,P＝0.613).Jagged1 expression of AR group was higher than preoperative on post-operative 1d(P＝0.000).After anti-rejection,Jagged1 expression decreased continually and reached preoperative level on 7d(P＝0.512). Jagged1 expression of ATN group was higher than preoperative on post-operative 1d(P＝0.005).After therapy.Jagged1 expression decreased(P＝0.054 on 3d).
3 In late post-operative time,Notch1 expression on lymphocytes among AR group,CAN group and control group show significant difference(F＝43.992, P＝0.000).Notch1 expression of AR group was significant higher than that of CAN group and control group(both P＝0.000).Notch1 expression of CAN group show no difference with that of control group(P＝0.734).Notch1 expression of AR group decreaSed clearly after anti-rejection(t＝5.511,P＝0.001).
Jagged1 expression among AR group,CAN group and control group show significant difference too(F＝61.652,P＝0.000).Jagged1 expression of AR group was significant higher than that of CAN group and control group(both P＝0.000).Jagged1 expression of CAN group show no difference with that of control group(P＝0.555). Jagged1 expression of AR group decreased clearly after anti-rejection(t＝3.983, P＝0.005).
Notch1/Jagged1 expression of CAN group wasn't in relation to hemodialysis (t＝0.459,P＝0.654;t＝1.638,P＝0.125).There weren't difference between CsA and Tac immunological suppression programs(all P＞0.05).
4 Notch1/Jagged1 mainly expressed in kytoplasm and membrame of renal tubular epithelial cell.Notch1/Jagged1 expression of normal renal tissues was very low(MOD＝0.012±0.003 and MOD＝0.009±0.003).Notch1/Jagged1 expression of AR renal tissues were higher than that of normal renal tissues(P＝0.000).Notch1/Jagged1 expression of CAN renal tissues were very weak too(MOD＝0.013±0.003 and MOD＝0.012±0.002).It was clearly lower than that of AR group(both P＝0.000),but showed no difference with that of normal renal tissues(P＝0.756 and P＝0.335).
Notch1 expression of pathological gradeⅠof AR renal tissue(MOD＝0.126±0.013) showed no difference with that of pathological gradeⅡ(MOD＝0.136±0.011) (t＝1.541,P＝0.147).But Jagged1 expression of pathological gradeⅡ(MOD＝0.136± 0.005) was higher than that of pathological gradeⅠ(MOD＝0.123±0.008)(t＝3.745, P＝0.002).Notch1/Jagged1 expression of CAN renal tissues showed no difference among different pathological grades(F＝0.811,P＝0.467 and F＝0.811,P＝0.467).
5 Notch1 and Jagged1 expression on peripheral blood lymphocytes showed significant positive correlation(r＝0.994,P＝0.000).Notch1 and Jagged1 expression on allograft tissue showed significant positive correlation too(r＝0.983,P＝0.000).
Conclusions
1 When post-renal-transplantation acute rejection occurred,Notch1/Jagged1 expression on lymphocytes increased significantly.Examination of Notch1/Jagged1 expression on lymphocytes is helpful to diagnosis of acute rejection.
2 When acute rejection was reversed,Notch1/Jagged1 expression decreased significantly.So detection of Notch1/Jagged1 expression on lymphocytes can help understand the effect of anti-rejection.
3 On the 1d after renal transplantation,Notch1/Jagged1 expression increased in all groups.Notch1/Jagged1 expression of ATN group didn't show difference with control group,but significantly lower than that of AR group,which was helpful to differentiate ATN and AR.
4 Examination of Notch1/Jagged1 expression in late stage after renal transplantation can't diagnose CAN,but still was helpful to identify acute rejection and chronic rejection,and was helpful to understand anti-rejection effect.
5 Notch1/Jagged1 expression of CAN patients isn't in relation to hemodialysis.
6 Notch1/Jagged1 expression in acute rejection renal tissue was higher than that in CAN renal tissue,which shows that examination of Notch1/Jagged1 expression in allograft can help understand pathological change and prognosis of allograft.
肾脏移植是临床上治疗终末期肾脏疾病的有效手段,但移植排斥反应仍是目前移植领域难以克服的重要问题,在同种异体肾移植中,急性排斥反应(Acuterejection,AR)是术后最常见的并发症,它通常发生于术后3个月内,最常见于术后1～3周,术后几年亦有发生。AR如果能得到早期诊断并正确治疗的话,90%以上患者的排斥反应可被逆转。AR是慢性排斥反应(Chronic rejection,CR)发生的高危因素,也是导致移植物长期存活失败和移植肾失去功能的最重要危险因素之一。急性排斥反应和急性肾小管坏死(Acute tubular necrosis,ATN)都是。肾移植术后常见并发症,两者术后都是以少尿、无尿为主要特点,术后早期较难鉴别。因此,如何早期诊断急性排斥反应非常重要。移植肾穿刺活检病理进行分型诊断是了解移植肾状态的经典指标,但其有创性及反复动态监测的困难性限制了它的临床应用。血肌酐升高作为排斥指标既不够敏感,又缺乏特异性。因此,探索非侵入性急性排斥反应诊断方法一直是肾移植临床研究的热点之一。
Notch信号通路是一个古老的信号传导途径,Notch信号是一组跨膜受体,Notch信号通路由Notch受体、Notch配体和CSL-DNA结合蛋白、其它的效应物、Notch的调节分子等组成。Notch受体最早发现于果蝇,因该基因的部分功能缺失会在果蝇翅膀边缘造成一些缺刻(Notchs)而命名。哺乳动物中共发现4类Notch基因,编码4种Notch受体,分别为Notch1～4。Notch的配体在哺乳动物中Notch配体共有5种,分别为Delta-like1、3、4和Jagged1、2,Notch的配体与其受体一样均为跨膜蛋白。Jagged1(新近被命名为CD339)是存在于哺乳动物细胞膜上Notch受体的主要配体之一,为单次跨膜糖蛋白,表达于骨髓、胎儿肝基质细胞和胸腺上皮细胞等多种组织,参与调控许多组织的生长发育。
最近的许多研究提示,Notch信号在外周免疫系统的分化和调节中发挥作用。移植肾排斥反应发生时,移植物作为同种异体抗原刺激机体免疫系统,引起Notch受体表达上调。Notch信号通路又通过一系列途径促进淋巴细胞分化、增殖。活化的淋巴细胞分泌细胞因子,进一步促进淋巴细胞增殖并产生免疫效应。研究表明,Notch信号可以通过调节淋巴细胞表面CD25的表达以及增强NF-kappaB的活性,促进IFNγ的分泌等途径来促进淋巴细胞的增殖。Notch信号通路活化在细胞因子或CD分子分泌表达之前,因此与其他的检测指标相比,Notch1受体表达水平的检测可能更有助于急性排斥反应的早期诊断。
本课题拟采用流式细胞术检测Notch1/Jagged1在肾移植术后受者外周血淋巴细胞中的表达,并应用免疫组化SP法检测各组移植肾组织中的Notch1/Jagged1蛋白表达,探讨检测外周血淋巴细胞中Notch1/Jagged1的有效性及实用性,并就Notch1/Jagged1在急性排斥反应的诊断及鉴别诊断中是否有意义以及对急性排斥的治疗是否具有指导价值等方面展开一些探讨和研究。
方法:
1、选取2007年1月～2008年12月接受肾移植手术的患者,以术后早期(1个月内)发生急性排斥反应(AR)的患者15例为急性排斥组,同期术后急性肾小管坏死(ATN)6例为肾小管坏死组,同期术后顺利恢复、肾功能稳定的15例为肾功能稳定组。采用流式细胞术检测所有患者术前和手术后1d、3d、7d、14d以及急性排斥组和肾小管坏死组发病(发生排斥或肾小管坏死)时及相应治疗(抗排斥或血液透析等)后1d、3d、7d、14d的Notch1/Jagged1的淋巴细胞阳性表达率,对比各组手术前后、发病前后以及AR患者抗排斥治疗前后不同时间点间的Notch1/Jagged1表达差异。
2、选取2007年1月～2008年12月我中心收治的晚期急性排斥反应(＞1年)患者8例为急性排斥组,诊断为慢性移植肾肾病(CAN)的15例患者为慢性移植肾病组,同期我院就诊行定期常规复查的肾移植术后肾功能正常稳定的10例患者作为肾功能正常组。采用流式细胞术检测患者外周血淋巴细胞的Notch1/Jagged1表达,对比三组外周血的Notch1/Jagged1表达差异,再按不同的免疫抑制方案和慢性移植肾病者是否行血液透析分别进行分组对比,了解Notch1/Jagged1表达与免疫抑制方案及血液透析的关系。
3、2007年1月～2008年12月采集的肾组织标本分为以下三组:(1)、急性排斥组:15例,均为肾移植术后发生急性排斥反应时行移植肾穿刺获取的患者移植肾组织;(2)、慢性移植肾肾病(CAN)组:15例,均为肾移植术后发生慢性移植肾肾病患者的移植肾组织;(3)、正常对照组:10例,为正常肾组织,其中6例为外伤后行肾切除者,4例为肾癌根治术癌旁5cm以外正常组织石蜡标本。所有肾脏组织标本采用免疫组化SP法检测组织中Notch1/Jagged1蛋白表达,对比各组Notch1/Jagged1的表达差异,并将急性排斥组和慢性移植肾病组按病理结果进行分级,对比不同病理分级间的Notch1/Jagged1表达差异,了解Notch1/Jagged1表达与病理分级之间的关系。
4、统计学方法:采用SPSS13.0软件包建立数据库,计量资料采用均数±标准差((?)±s)表示,以P＜0.05为差异具有统计学意义。术后早期各组多时间点间的比较采用重复测量的方差分析,每个分组在不同时间点上的两两比较采用Bonferron法,同一时间点上不同分组之间的两两比较采用LSD法。术后晚期及移植肾组织两组之间的比较采用独立样本t检验,急性排斥组抗排斥治疗前后的比较采用配对样本t检验,多组之间的比较采用单因素方差分析One-wayANOVA),组间两两比较采用LSD法。Notch1与Jagged1表达的相关性用Pearson相关分析,Notch1/Jagged1表达与病理分级的相关性用spearman相关分析。
结果:
1、肾功能稳定组、急性排斥组、肾小管坏死组之间肾移植手术前的Notch1表达无显著性差异(F=0.277,P=0.760),术后1d、3d时各组的Notch1表达差异亦无统计学意义(F=0.123,P=0.885和F=1.726,P=0.104),而手术后第7、14d时各组间的Notch1表达差异显著(F=3.354,P=0.027和F=2.672,P=0.039),其中急性排斥组的Notch1表达高于肾功能稳定组和肾小管坏死组(均P<0.05),而相同时间点间肾功能稳定组和肾小管坏死组间无显著性差异(均P>0.05)。肾功能稳定组手术后1d的Notch1阳性淋巴细胞率较术前升高(P=0.000),手术后3d时恢复至手术前水平(P=0.703),之后维持平稳,至术后14d时略低于术前水平,但差异无统计学意义(P=0.485);急性排斥组手术后1d时的Notch1阳性淋巴细胞率亦较术前升高(P=0.000),手术后3d时有所下降,术后7d、14d又显著升高并且再次高于术前Notch1水平(均P=0.000);手术后1d时的Notch1阳性淋巴细胞率亦较术前升高(P=0.000),3d、7d、14d时均略高于手术前水平,但差异无统计学意义(均P>0.05)。
术前各组之间的Jagged1表达无显著性差异(F=0.298,P=0.744),手术后1d、3d时各组间的Jagged1表达差异也并不显著(F=0.032,P=0.968和F=0.656,P=0.526),在术后7d、14d时各组间的Jagged1表达有显著性差异(F=1.959,P=0.045和F=3.120,P=0.027),其中急性排斥组高于肾功能稳定组和肾小管坏死组(均P<0.05),而相同时间点间肾小管坏死组和肾功能稳定组间Jagged1表达均无显著性差异(均P>0.05)。肾功能稳定组手术后1d时的Jagged1阳性淋巴细胞率较术前升高(P=0.000),手术后3d时与术前水平无统计学差异(P=0.102),之后开始平稳下降,术后14d时略低于手术前水平,但差异无统计学意义(P=0.225);急性排斥组手术后1d时的Jagged1阳性淋巴细胞率亦较术前升高(P=0.000),手术后3d时有所下降,但术后第7、14d时又开始升高,并且再次显著高于术前水平(均P=0.000);肾小管坏死组手术后1d时的Jagged1阳性淋巴细胞率亦较术前升高(P=0.000),术后3d时仍高于术前水平(P=0.071),术后7d、14d时与术前水平无显著性差异(P＝0.057和P＝0.534)。
2、急性排斥组和肾小管坏死组术前、术后发病时(发生排斥或肾小管坏死)时及相应治疗(抗排斥或血液透析等)后1d、3d、7d、14d比较显示:发病时及治疗后1d、3d时,急性排斥组的Notch1表达均显著高于肾小管坏死组(t＝49.521,P＝0.000;t＝37.284,P＝0.000和t＝7.295,P＝0.000),治疗后7d时,急性排斥组的Notch1表达仍高于肾小管坏死组(t＝2.249,P＝0.037),至治疗后14d时,两组差异无统计学意义(t＝1.741,P＝0.098)。急性排斥组出现排斥反应时Notch1阳性淋巴细胞率较术前明显升高(P＝0.000),经抗排斥治疗后逐渐下降,抗排斥治疗后3d、7d、14d时Notch1表达均明显低于抗排斥前(发生排斥反应时)Notch1水平(均P＝0.000),但抗排斥后7d时的Notch1表达仍高于术前(P＝0.041),至抗排斥后14d时与术前水平无统计学差异(P＝0.072);肾小管坏死组发生肾小管坏死时,Notch1阳性淋巴细胞率亦较术前升高(P＝0.002);经血液透析等治疗后,Notch1表达下降,至治疗后3d时已接近术前水平(P＝0.067),之后维持平稳。发病时及治疗后1d、3d时,急性排斥组的Jagged1表达均显著高于肾小管坏死组(t＝60.598,P＝0.000;t＝44.716,P＝0.000和t＝8.455,P＝0.000),治疗后7d、14d时,两组间的Jagged1表达无显著性差异(t＝0.022,P＝0.884和t＝0.097,P＝0.613)。急性排斥组出现排斥反应时Jagged1阳性淋巴细胞率较术前明显升高(P＝0.000),经抗排斥治疗后逐渐下降,抗排斥治疗后3d、7d、14d时的Jagged1表达均显著低于发生排斥反应时的Jagged1表达(均P＜0.05),至抗排斥治疗后7d时与术前水平无统计学差异(P＝0.512),抗排斥治疗后14d时略低于术前水平,但差异亦无统计学意义(P＝0.615);肾小管坏死组发生肾小管坏死时,Jagged1阳性淋巴细胞率较术前升高(P＝0.005),经血液透析等治疗后,Jagged1表达下降,至治疗后3d时接近术前水平(P＝0.054)。
3、肾移植术后晚期,急性排斥组发病时与肾功能正常组和慢性移植肾病三组间外周血淋巴细胞Notch1表达有显著性差异(F＝43.992,P＝0.000),急性排斥组的Notch1表达显著高于慢性移植。肾病组和肾功能正常组(均P＝0.000),而肾功能正常组和慢性移植肾病组的外周血Notch1阳性淋巴细胞率无显著性差异(P＝0.734)。急性排斥组抗排斥治疗前后Notch1表达差异显著(t＝5.511,P＝0.001),抗排斥治疗后Notch1表达明显下降。
三组间外周血淋巴细胞Jagged1表达差异显著(F＝61.652,P＝0.000),急性排斥组的Jagged1表达显著高于慢性移植肾病组和肾功能正常组(均P＝0.000),而肾功能正常组和慢性移植肾病组的外周血Jagged1阳性淋巴细胞率无显著性差异(P＝0.555)。急性排斥组抗排斥治疗前后Jagged1表达差异显著(t＝3.983,P＝0.005),抗排斥治疗后Jagged1表达明显下降。
急性排斥组Tac方案和CsA方案在排斥时和抗排斥冲击治疗后的Notch1表达以及慢性移植肾病组Tac方案和CsA方案的Notch1表达无显著性差异(分别t＝0.567,P＝0.591、t＝0.210,P＝0.841、t＝0.161,P＝0.875);不同免疫抑制方案间的Jagged1表达亦无显著性差异(分别t＝0.119,P＝0.907、t＝0.926,P＝0.095、t＝0.249,P＝0.807)。
慢性移植肾病组受者外周血淋巴细胞Notch1/Jagged1表达与是否行血液透析无明显关系(t＝0.459,P＝0.654和t＝1.638,P＝0.125)。
4、正常肾组织中Notch1表达极低(MOD＝0.012±0.003),仅在肾小管胞浆偶有较弱表达;急性排斥组肾组织的Notch1表达(MOD＝0.131±0.012)明显高于正常对照组(P＝0.000),其表达主要定位于肾小管上皮细胞的胞浆和胞膜,间质细胞有较弱表达;慢性移植肾肾病组肾组织Notch1表达(MOD＝0.013±0.003)也极弱,其Notch1表达与正常对照组无显著性差异(P＝0.756),但明显低于急性排斥组(P＝0.000)。正常肾组织中Jagged1几乎为阴性表达(MOD＝0.009±0.003);急性排斥组肾组织的Jagged1表达(MOD＝0.130±0.010)较正常对照组明显升高(P＝0.000),其表达亦主要定位于肾小管上皮细胞的胞浆和胞膜,间质细胞有较弱表达:慢性移植肾肾病组肾组织Jagged1表达(MOD＝0.012±0.002)也极微弱,其Jagged1表达与正常对照组无显著性差异(P＝0.335),但明显低于急性排斥组(P＝0.000)。
急性排斥组病理Ⅰ级的Notch1表达值为0.126±0.013,Ⅱ级Notch1表达值为O.136±0.011,Ⅱ级的Notch1表达略高于Ⅰ级,但其差异尚无统计学意义(t＝1.541,P＝0.147);而病理Ⅱ级的Jagged1表达(MOD＝0.136±0.005)明显高于Ⅰ级(MOD＝0.123±0.008)(t＝3.745,P＝0.002)。慢性移植肾病组各病理分级间肾组织的Notch1表达无显著性差异(F＝0.811,P＝0.467);各病理分级间肾组织的Jagged1表达亦无显著性差异(F＝0.811,P＝0.467)。
5、相关分析显示,肾移植术后早期外周血淋巴细胞的Notch1与Jagged1表达呈显著的正相关(r＝0.994,P＝0.000);移植肾组织中的Notch1与Jagged1表达亦呈显著的正相关(r＝0.983,P＝0.000)。
结论:
1、肾移植术后早期急性排斥反应发生时外周血淋巴细胞表面Notch1/Jagged1表达水平显著增高,检测外周血淋巴细胞的Notch1/Jagged1表达水平有助于急性排斥反应的诊断。
2、急性排斥反应经抗排斥治疗后,Notch1/Jagged1表达水平显著下降,因此,测定外周血淋巴细胞的Notch1/Jagged1表达水平有助于急性排斥反应治疗效果的动态监测。
3、肾移植手术后1d时各组受者外周血Notch1/Jagged1表达水平均有升高,早期ATN患者外周血淋巴细胞的Notch1/Jagged1表达与肾功能稳定患者无显著性差异,但明显低于急性排斥反应患者,对鉴别ATN和AR有一定帮助。
4、检测肾移植晚期受者外周血淋巴细胞的Notch1/Jagged1表达不能用于CAN的诊断,但仍有助于肾移植晚期受者急、慢性排斥反应的鉴别以及监测抗排斥治疗的效果。
5、CAN患者外周血淋巴细胞表面Notch1/Jagged1表达水平与免疫抑制方案及是否行血液透析无关。
6、急性排斥反应肾组织Notch1/Jagged1蛋白表达明显高于正常肾组织和慢性移植肾肾病肾组织,提示移植肾组织Notch1/Jagged1蛋白表达可作为监测移植肾形态学变化、功能状态以及预后判断的重要指标。
Objective and Significance
Renal transplantation is an effective clinic method to cure end-stage renal deseases.In renal transplantation field,acute rejection(AR) is the most frequent post-operation complication.AR often took place in the first 3 months,especially 1-3 weeks after operation.AR can lead to chronic rejection(CR) and is one of the most dangerous factors to result in allograft function losing.If AR gains early diagnosis and correct treatment,90%of ARs can be reversed.Acute tubular necrosis(ATN) is also a kind of common complication after renal transplantation.Main symptom of ATN and AR are both oliguria and anuria.ATN and AR are difficultly to be identified in early stage after operation.So it is important to early diagnose AR. Blood CR is not sensitive and absence lack of specificity.Renal needle biopsy is a traumatic examination.It is a hot point of clinic research to study non-traumatic diagnosis methods to AR.
Notch signal pathway is a group of transmembrane receptors.It is made up of Notch receptor,Notch ligand,CSL-DNA fixation protein,other effector and Notch regulatory molecules.Four kinds of Notch gene were found mammalian which encode four Notch receptors(Notch1～4).There are 5 kinds of Notch ligands (Delta-like1,3,4 and Jagged1,2) in mammalian.Notch ligands are transmembrane receptors too.Jagged1(CD339) is one of the main Notch ligands.
Recently,some research discovered that Notch signal can effect on differentiation and accommodation of periphery immune system.When renal allograft rejection occurred,allograft stimulates immune system and then Notch receptor expression increases.Notch signal pathway promotes lymphocytes differentiation and proliferation.Activated lymphocytes excretes cytokine which promotes lymphocytes proliferation and brings out immunologic effect.Notch signal can enhance the activity of NF-kappa B through modifying CD25 expression on the surface of lymphocytes,and can also promote lymphocytes proliferation through promoting IFNγ,secretion.Activation of Notch signal pathway is ahead of the secreting of cytokine or CDs.So examination of Notch1 receptor expression is perhaps more helpful on early diagnosis of acute rejection.
Materials and Methods
1 15 patients suffering early acute rejection after renal transplantation(＜1 month) in 2007.1～2008.12 are arranged as acute rejection(AR) group.6 patients with post-operation acute tubular necrosis are arranged as ATN group.15 patients with normal post-operation renal function are arranged as control group.Flow cytometry is used to detect Notch1/Jagged1 expression on lymphocytes before operation,in one week after operation and in one week after AR and ATN occurrence.Notch1/Jagged1 expressions before operation,after operation and after morbidity are compared.
2 8 patients suffering late acute rejection(＞1 year) in 2007.1～2008.12 are arranged as late acute rejection(LAR) group.15 patients with chronic allograft nephropathy(CAN) are arranged as CAN group.10 patients with stable and normal post-operation renal function are arranged as control group.Flow cytometry is used to detect Notch1/Jagged1 expression on lymphocytes.Notch1/Jagged1 expressions of every group are compared.
3 Nephridial tissue samples collected in 2007.1～2008.12 are divided into 3 groups as follow:(1) AR group,15 samples gained through kidney puncturation when acute rejection occurred.(2) CAN group,15 samples gained from CAN patients.(3) Control group,10 samples are normal kidney tissue.The expression of Notch1 and Jagged1 were detected by immunohistochemistrical SP staining method.Notch1/ Jagged1 expressions of every group and among different pathological grades are compared.
Results
1 Preoperative Notch1 expression on lymphocytes among AR group,ATN group and control group didn't show difference(F＝0.277,P＝0.760).Notch1 expression among 3 groups didn't show difference on post-operative 1d,3d either(F＝0.123, P＝0.885 and F＝1.726,P＝0.104).However,Notch1 expression among 3 groups showed significant difference on post-operative 7d,14d(F＝3.354,P＝0.027 and F＝2.672,P＝0.039).Notch1 expression of AR group was higher than that of control group and ATN group(all P＜0.05).Notch1 expression of control group and that of ATN group didn't show difference on every day(all P＞0.05).Notch1 expression on post-operative 1d was higher than preoperative ones on every group(P＝0.000). Notch1 expression of control group decreased and reached preoperative level on post-operative 3d(P＝0.703).Notch1 expression of AR group decreased on post-operative 3d too,but increased on post-operative 7d,14d(P＝0.000 vs preoperative level).Notch1 expression of ATN group decreased slowly within post-operative 1 week.Notch1 expression of ATN group decreased and reached preoperative level from post-operative 3d(P＞0.05).
Preoperative Jagged1 expression among AR group,ATN group and control group didn't show difference(F＝0.298,P＝0.744).Jagged1 expression among 3 groups didn't show difference on post-operative 1d,7d either(F＝0.032,P＝0.968 and F＝0.656,P＝0.526).Jagged1 expression of AR group was higher than that of control group and ATN group on post-operative 7d,14d(all P＜0.05).Jagged1 expression of control group and that of ATN group didn't show difference on every day(all P＞0.05). Jagged1 expression on post-operative 1d was higher than preoperative ones in all group(all P＝0.000).Jagged1 expression of control group decreased from post-operative 3d and reached preoperative level(P＝0.102).Jagged1 expression of AR group decreased on post-operative 3d too,but increased on post-operative 7d,14d and higher than preoperative level(P＝0.000).Notch1 expression of ATN group decreased on post-operative 3d too(P＝0.071),and reached preoperative level on post-operative 7d,14d(P＝0.057 and P＝0.534).
2 Notch1 expression of AR group was significantly higher than that of ATN group on 1d after morbidity and on post-treatment 1d,3d 7d(t＝49.521,P＝0.000; t＝37.284,P＝0.000;t＝7.295,P＝0.000;t＝2.249,P＝0.037).They didn't show difference on post-treatment 14d(t＝1.741,P＝0.098).When acute rejection occurred, Notch1 expression of AR group was higher than preoperative one(P＝0.000).After anti-rejection,Notch1 expression decreased continually and reached preoperative level on 7d(P＝0.072).Notch1 expression of ATN group was higher than preoperative one(P＝0.002).After therapy.Notch1 expression decreased(P＝0.067 on 3d).
Jagged1 expression of AR group was significantly higher than that of ATN group on morbidity day and post-operative 1d,3d(t＝60.598,P＝0.000;t＝44.716, P＝0.000 and t＝8.455,P＝0.000).They didn't show difference on post-operative 7d, 14d(t＝0.022,P＝0.884;t＝0.097,P＝0.613).Jagged1 expression of AR group was higher than preoperative on post-operative 1d(P＝0.000).After anti-rejection,Jagged1 expression decreased continually and reached preoperative level on 7d(P＝0.512). Jagged1 expression of ATN group was higher than preoperative on post-operative 1d(P＝0.005).After therapy.Jagged1 expression decreased(P＝0.054 on 3d).
3 In late post-operative time,Notch1 expression on lymphocytes among AR group,CAN group and control group show significant difference(F＝43.992, P＝0.000).Notch1 expression of AR group was significant higher than that of CAN group and control group(both P＝0.000).Notch1 expression of CAN group show no difference with that of control group(P＝0.734).Notch1 expression of AR group decreaSed clearly after anti-rejection(t＝5.511,P＝0.001).
Jagged1 expression among AR group,CAN group and control group show significant difference too(F＝61.652,P＝0.000).Jagged1 expression of AR group was significant higher than that of CAN group and control group(both P＝0.000).Jagged1 expression of CAN group show no difference with that of control group(P＝0.555). Jagged1 expression of AR group decreased clearly after anti-rejection(t＝3.983, P＝0.005).
Notch1/Jagged1 expression of CAN group wasn't in relation to hemodialysis (t＝0.459,P＝0.654;t＝1.638,P＝0.125).There weren't difference between CsA and Tac immunological suppression programs(all P＞0.05).
4 Notch1/Jagged1 mainly expressed in kytoplasm and membrame of renal tubular epithelial cell.Notch1/Jagged1 expression of normal renal tissues was very low(MOD＝0.012±0.003 and MOD＝0.009±0.003).Notch1/Jagged1 expression of AR renal tissues were higher than that of normal renal tissues(P＝0.000).Notch1/Jagged1 expression of CAN renal tissues were very weak too(MOD＝0.013±0.003 and MOD＝0.012±0.002).It was clearly lower than that of AR group(both P＝0.000),but showed no difference with that of normal renal tissues(P＝0.756 and P＝0.335).
Notch1 expression of pathological gradeⅠof AR renal tissue(MOD＝0.126±0.013) showed no difference with that of pathological gradeⅡ(MOD＝0.136±0.011) (t＝1.541,P＝0.147).But Jagged1 expression of pathological gradeⅡ(MOD＝0.136± 0.005) was higher than that of pathological gradeⅠ(MOD＝0.123±0.008)(t＝3.745, P＝0.002).Notch1/Jagged1 expression of CAN renal tissues showed no difference among different pathological grades(F＝0.811,P＝0.467 and F＝0.811,P＝0.467).
5 Notch1 and Jagged1 expression on peripheral blood lymphocytes showed significant positive correlation(r＝0.994,P＝0.000).Notch1 and Jagged1 expression on allograft tissue showed significant positive correlation too(r＝0.983,P＝0.000).
Conclusions
1 When post-renal-transplantation acute rejection occurred,Notch1/Jagged1 expression on lymphocytes increased significantly.Examination of Notch1/Jagged1 expression on lymphocytes is helpful to diagnosis of acute rejection.
2 When acute rejection was reversed,Notch1/Jagged1 expression decreased significantly.So detection of Notch1/Jagged1 expression on lymphocytes can help understand the effect of anti-rejection.
3 On the 1d after renal transplantation,Notch1/Jagged1 expression increased in all groups.Notch1/Jagged1 expression of ATN group didn't show difference with control group,but significantly lower than that of AR group,which was helpful to differentiate ATN and AR.
4 Examination of Notch1/Jagged1 expression in late stage after renal transplantation can't diagnose CAN,but still was helpful to identify acute rejection and chronic rejection,and was helpful to understand anti-rejection effect.
5 Notch1/Jagged1 expression of CAN patients isn't in relation to hemodialysis.
6 Notch1/Jagged1 expression in acute rejection renal tissue was higher than that in CAN renal tissue,which shows that examination of Notch1/Jagged1 expression in allograft can help understand pathological change and prognosis of allograft.
引文
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