摘要
慢性肾功能衰竭(chronic renal failure,CRF)严重时又称尿毒症,不是一种独立的疾病,是各种病因引起肾脏损害并进行性恶化至终末期、肾功能接近于正常的十分之一左右时出现的一系列综合症状。其发病机制目前尚未明了。除外目前解释CRF的肾小球高灌注、高压力、高滤过,肾小球囊内高压,肾小管高代谢及肾脏代偿性肥大等理论,肾小球和肾间质的变化：肾小球、肾小管间质细胞及肾脏浸润细胞产生的大量细胞因子,也参与了肾功能恶化的过程。近年来,人类白细胞抗原(human leukocyte antigen, HLA)以及一些免疫分子受体也成为研究CRF的热点。同种异体肾移植是目前治疗慢性肾衰竭尿毒症期的最有效手段,而移植后的急性排斥反应(acute rejection, AR)是影响移植肾长期生存的最重要因素之一,虽然近年关于AR的基础和临床研究取得较大进展,但其发生的具体机制仍不十分清楚。
NK细胞是固有免疫系统中骨髓来源的大颗粒淋巴细胞,无需抗原预先致敏,就能直接杀伤某些病理细胞或异体细胞。NK细胞表面的杀伤细胞免疫球蛋白样受体(killer cell immunoglobulin-like receptor, KIR)能特异性识别主要组织相容性复合体(major histocompatibility complex, MHC) I类分子,传导抑制或激活性信号,调节NK细胞的活性。
KIR属于表达于NK细胞和部分T细胞表面的一系列免疫球蛋白样超家族分子,它由14个KIR功能基因和2个沉默基因X和Z组成。KIR的基本结构由胞外区、跨膜区和胞内区组成。根据KIR胞内区的长短可将其可分为L(long)型和S(short)型。其中L型胞内区主要传导抑制性信号,为抑制型KIR (inhibitory KIR);S型胞内区传导激活信号,为活化型KIR (activated KIR)。生理条件下NK细胞表面抑制型受体的作用占主导地位,主要向细胞内传递抑制性信号,使NK细胞处于非活化状态,其生理意义在于阻止NK细胞对自身细胞发挥杀伤作用。
HLA-Cw属于经典的HLA-I类基因,其广泛分布于有核细胞表面,不仅呈递内源性多肽给CD8+T细胞诱发特异性细胞杀伤效应,还可作为KIR的配体参与免疫反应。HLA-Cw在机体抗感染免疫、肿瘤免疫、移植免疫中均发挥重要作用。
由于在造血干细胞移植中供受者之间KIR受体-HLA配体匹配程度对预后有显著影响,启发研究者开始探索在肾移植领域KIR受体与其HLA配体的相互作用。移植后适当的KIR表达有助于降低发生免疫排斥反应的风险,但同时也会降低患者的免疫力,增加感染的几率。如何合理地控制KIR表达量以达到理想的平衡点将是KIR研究的一个重要方向。随着对KIR的不断深入研究,KIR与其HLA配体之间的复杂关系将会被进一步阐明,使得通过主动干预调节KIR与其HLA配体间的相互作用进行免疫治疗成为可能,对改善移植患者的预后有重要意义。
本课题以尿毒症患者、健康人群以及接受肾移植手术的供受者对为研究对象,采用序列特异性引物聚合酶链反应(polymerase chain reaction sequence specific primer, PCR-SSP)法检测尿毒症患者KIR、HLA-Cw基因并分析其多态性,与健康人群进行比较,结合KIR与HLA-Cw之间识别方式,探讨两者的关系以及与尿毒症发病机制的相关性。最后应用回顾性分析的方法,通过分析肾移植受者KIR基因、基因型与其相应的供者HLA-Cw配体的匹配情况,探讨二者与肾移植急性排斥反应的相关性,作为移植结果的预测因素为临床制定治疗方案提供参考。
第一章尿毒症患者杀伤细胞免疫球蛋白样受体KIR基因多态性分析
目的检测尿毒症患者和正常健康人群杀伤细胞免疫球蛋白样受体KIR基因,分析其基因频率的分布特点,探讨KIR基因与尿毒症发病机制的相关性。
方法对55例尿毒症患者(病例组)和60例无血缘关系随机健康个体(对照组)抽取静脉血。采用PCR-SSP法检测两组KIR基因,统计每个个体的KIR基因类型。应用SPSS13.0统计分析软件分析数据,KIR基因表现型频率(phenotype frequencies,PF)=观察到的KIR基因阳性个数(n)／被研究人数,基因频率(gene frequencies,GF)应用公式GF=1-(1-PF)1/2计算。两组基因表现型频率、基因型频率的比较采用四格表χ2检验。年龄、个体KIR基因数目均数的比较采用独立样本t检验。以P<0.05为差异具有统计学意义。
结果病例组与对照组两组14个KIR基因表现型频率均无显著统计学差异(P>0.05)。KIR2DL4、3DL2、3DL3在两组表达率均为100%。两组中KIR2DL1、2DL3、3DL1、2DS4表达均较高,KIR2DL2、2DL5、2DS1、2DS2、2DS3、2DS5、3DS1的表达则相对较低。个体KIR基因数目均数比较显示两组均以抑制性KIR基因表达为主。两组共检出33种KIR基因型,其中病例组21种,对照组25种。抑制性KIR基因型AA类型病例组为21例,对照组为26例；非抑制性KIR基因型AB类型病例组为30例,对照组为31例；BB类型病例组为4例,对照组为3例。抑制性KIR基因型AA类型2DL1+,2DL2-,2DL3+,2DL4+,2DL5-,3DL1+,3DL2+,3DL3+,2D S1-,2DS2-,2DS3-,2DS4+,2DS5-,3DS1-类型在两组表达率均最高,分别为32.7%(18／55)、36.7%(22／60),但二者相比无显著统计学差异(P＞0.05)。
结论PCR-SSP能有效地进行KIR基因分型。尿毒症患者KIR基因多态性与正常健康人无明显差异。通常情况下,个体以抑制性KIR基因表达为主。仅从KIR基因角度分析,未发现其与尿毒症发病机制的相关性。
第二章尿毒症患者人类白细胞抗原HLA-Cw基因多态性分析
目的检测尿毒症患者和正常健康人群HLA-Cw基因,分析其基因频率分布特点,结合KIR与HLA-Cw识别方式,初步探讨两者的关系及它们在尿毒症’发病机制中可能的作用。
方法对55例尿毒症患者(病例组)和60例无血缘关系随机健康个体(对照组)抽取静脉血。采用PCR-SSP法检测两组HLA-Cw基因,统计每个个体的HLA-Cw基因类型。采用SPSS13.0统计分析软件进行分析,按照HLA-Cw分子α重链上第80位氨基酸的不同将HLA-Cw分为两类即HLA-C1(HLA-CAsn80)类型和HLA-C2(HLA-CIys80)类型,两种类型在病例组和对照组组间以及各组内的比较采用独立样本t检验。基因表现型频率、KIR2D-HLA-Cw受体配体对阳性率的比较采用四格表χ2检验。以P＜0.05为差异具有统计学意义。
结果病例组与对照组两组之间12个HLA-Cw基因表现型频率均无显著统计学差异(P>0.05)。HLA-Cw*03、07、01、08四个基因频率在两组均较高,HLA-Cw*02、04、05、06、12、14、15、16基因频率相对较低,两组均未检测到HLA-Cw*13、17、18。病例组和对照组组内HLA-C1与HLA-C2之间的比较均有显著统计学差异(P=0.023,P=0.020), HLA-C1类型基因频率均高于HLA-C2类型。抑制性和活化性KIR2D-HLA-Cw受体配体对阳性率在病例组和对照组之间的比较无显著统计学差异(P＞0.05)。
结论HLA-Cw基因具有较为丰富的多态性,尿毒症患者和正常健康人HLA-Cw基因多态性无明显差异。作为KIR配体将HLA-Cw分为两类,两组均以HLA-C1类型(即包含HLA-Cw*03、07、01、08四个具有较高基因频率的基因)为主。通常情况下,个体中抑制性KIR2D-HLA-Cw受体配体对阳性率较高。KIR2D-HLA-Cw受体配体对在尿毒症发病机制中的作用尚不确定。
第三章受者KIR／供者HLA-Cw匹配与肾移植术后急性排斥反应的关系
目的探讨供受者对中受者KIR／供者HLA-Cw匹配所介导的信号传导通路在肾移植受者术后出现急性排斥反应(acute rejection, AR)中可能的作用。
方法对53对肾移植供受者抽取静脉血,采用PCR-SSP法检测受者KIR基因和供者HLA-Cw基因。受者按照手术后肾功能状态分为急性排斥组(n=19)和肾功能稳定组(n=34),急性排斥反应的诊断根据临床表现、实验室检查、移植肾彩色多普勒超声检查、移植肾穿刺活检等综合判断。探讨供者HLA-Cw不同类型、受者KIR不同基因型以及受者KIR／供者HLA-Cw三种不同匹配类型与肾移植急性排斥反应发生的相关性。采用SPSS13.0统计软件进行统计学分析,受者性别、原发病因、HLA错配位点、术前抗体、移植次数的分布、供者HLA-Cw基因不同类型分布、受者KIR不同基因型分布以及受者KIR／供者HLA三种不同匹配类型分布的比较采用R×C列联表χ2检验,受者KIR基因表现型频率、急性排斥反应发生率的比较采用四格表χ2检验,年龄、冷热缺血时间的比较采用独立样本t检验。以P<0.05为差异具有统计学意义。
结果供者HLA-Cw基因不同类型、受者KIR不同基因型在急性排斥组和肾功能稳定组的分布：供者HLA-Cw基因不同类型两组间分布无显著性差异(P>0.05)。受者KIR不同基因型两组间分布有显著统计学差异(P=0.014),受者KIR基因型AA类型在急性排斥组低于肾功稳定组(10.5% vs 44.1%)。受者KIR基因表现型频率：KIR2DL2/2DS2、KIR2DL3表现型频率在急性排斥组低于‘肾功稳定组(26.3% vs 55.9%,P=0.038；73.7% vs 97.1%,P=0.034)。供者HLA-Cw基因不同类型、受者KIR不同基因型急性排斥反应发生率的比较：供者HLA-C1/C1类型低于非HLA-C1/C1类型(31.6% vs 46.7%,P>0.05),受者KIR基因型AA类型低于非AA类型(11.8% vs 47.2%,P=0.012)。受者KIR／供者HLA-Cw三种匹配类型在急性排斥组与肾功稳定组的分布：KIR2DL2/HLA-C1和KIR2DL3/HLA-C1的三种匹配类型在两组间分布具有显著统计学差异(P=0.021,P=0.011),肾功能稳定组KIR2DL2/HLA-C1和KIR2DL3/HLA-C1信号匹配相合(match)率较高(55.9%,97.1%)。
结论按照供者HLA-Cw基因型为HLA-C1／C1类型,受者KIR基因型为AA类型,受者KIR基因型表达抑制型受体KIR2DL2、KIR2DL3,受者KIR2DL2、KIR2DL3与供者相应HLA-C1匹配相合的原则进行供受者配型选择可以降低肾移植术后急性排斥反应的发生率。
Chronic renal failure (CRF) or uremia in severe condition is not an independent disease. A variety of reasons cause kidney damage and progressive deterioration. A series of complex symptom will appear when renal function decreased to about 10% of normal. Etiological mechanisms of CRF are yet unclear. Currently, hypothesis about CRF mainly on glomerular perfusion, high-pressure, high filtration; glomerular capsule pressure, high metabolism of renal tubular and compensatory hypertrophy, glomerular and interstitial changes:renal glomerular cells, tubular cells and infiltrating cells produce a large number of cytokines which involve in the process of renal function deterioration. In recent years, human leukocyte antigen (HLA) and some immune receptor molecules become hot spot. Kidney transplantation is an effective clinic method to cure end-stage renal deases. However, rejection response after transplantation is still an important factor affecting the allograft long-term survival. Although basic and clinical researches about acute rejection have made great progress recently, the specific mechanism remains elusive.
NK cells are bone marrow-derived large granular lymphocyte in innate immune systems. They can directly kill certain pathological cells or allogeneic cells without prior antigen sensitization. Killer cell immunoglobulin-like receptors(KIRs) as NK cell surface receptors can recognize specific MHC class I molecules, transmit inhibitory or activated signal to regulate NK cell activity.
KIRs which belong to immunoglobulin superfamily are expressed mainly on the surface of NK cells and some T cells. KIR family consists of 14 KIR genes and two pseudogenes(X, Z). Their basic structures are composed of extracellular region, transmembrane region and intracellular region. According to the length of intracellular region, KIR can be divided into long type (L-type) and short typ (S-type). L-type as inhibitory KIRs mainly transmit inhibitory signal, While S-type as activated KIRs mainly transmit activated signal. Under physiological condition, inhibitory receptors on NK cell surface are dominant, so NK cells are in non-activated state. Its physiological significance is to prevent NK cell from killing autologous source of cells.
HLA-Cw belongs to the classical HLA-I genes, they are widely distributed on the surface of nucleated cells. HLA-Cw genes not only present endogenous peptides to CD8+T cells which induce specific cell-killing effects, but also involve in immune response as KIR ligand. Research shows, HLA-Cw plays an important role in anti-infection immunity, tumor immunity and transplantation immunity.
Because KIR/HLA ligand matching has a significant influence on the prognosis of hematopoietic stem cell transplantation, researchers begin to explore KIR and its HLA ligand interaction in kidney transplantation. After transplantation, appropriate KIR expression will be helpful to reduce risk of immune rejection, but it also compromises the immune system, causes infection at the same time. How can achieve the ideal balance by controlling the expression of KIR will be an important direction in transplantation research. With continuous in-depth study, KIR and its HLA ligand complex relationship will be clarified in the near future. It's possible to intervene interaction of KIR and its HLA ligand actively by immune therapy which has important significance in improving the prognosis of transplant patients.
Uremic patients, healthy individuals and donor/recipient pairs of kidney transplantation as our study object. Using polymerase chain reaction sequence specific primer (PCR-SSP) to detect KIR gene, HLA-Cw gene in uremic patients. Gene distribution was compared with healthy control. Relationship between KIR and HLA-Cw was analyzed preliminary. Futhermore, the effect of recipient KIR/donor HLA ligand matching on acute rejection (AR) after kidney transplantation was explored. It may be used as a prediction of graft outcome to provide reference for the clinical treatment.
Chapter 1 Polymorphism of killer cell immunoglobulin-like receptor (KIR) gene in uremic patients Objective
To detect the killer cell immunoglobulin-like receptor (KIR) gene frequencies in uremic patients and normal healthy individuals, analyze KIR gene distribution. Aim to explore their relationship with the pathogenesis of uremia.
Method
Venous blood was collected of 55 uremic patients (case group) and 60 random unrelated healthy individuals (control group). KIR genes were typed by PCR-SSP. 14 KIR genes were analyzed in each individual. Statistical analysis was performed by SPSS13.0 for windows. Direct counting method was used to calculate phenotype frequencies (PF). Gene frequencies (GF) were calculated by formula:GF=1-(1-PF) 1/2. Gene phenotype frequencies and genotype frequencies between two groups were analyzed by Chi-square test. T-test of independent sampler was used for comparison of age and average number of indibidual KIR genes. A value of P<0.05 was considered statistical different.
Results
There were no significant differences of 14 KIR gene phenotype frequencies between uremic patients and healthy controls (P>0.05). KIR2DL4,3DL2,3DL3 were expressed in all individuals as framework gene. KIR2DL1,2DL3,3DL1,2DS4 had a higher expression in both groups, while KIR2DL2,2DL5,2DS1,2DS2,2DS3,2DS5, 3DSlwere relatively lower. The average number of indibidual KIR genes showed that inhibitory KIR genes were more than activated KIR genes in both groups.33 genotype constituted by 14 functional genes were detected in all cases,21 genotype in case group and 25 genotype in control group. Inhibitory KIR genotype (L-KIR, AA) was found 21 cases in uremic patients and 26 cases in healthy controls. The non-inhibitory KIR genotype contains two types:AB and BB. AB was found 30 cases in uremic patients and 31 cases in healthy controls; BB was found 4 cases in uremic patients and 3 cases in healthy controls. The genotype of 2DL1+,2DL2-,2DL3+,2DL4+,2DL5-,3DL1+,3DL2+, 3DL3+,2DS1-,2DS2-,2DS3-,2DS4+,2DS5-,3DS1-which belong to inhibitory KIR genotype was most prevalent genotype in uremic patients (32.7%) and healthy controls(36.7%), but there was no significant difference between two groups (P>0.05).
Conclusions
PCR-SSP is an effective method for KIR genotyping. KIR gene polymorphisms have no difference between uremic patients and normal healthy controls. Under normal condation, inhibitory KIR gene is dominant in every individual. Just from the perspective of KIR gene, there is no relevance between KIR and pathogenesis of uremia.
Chapter 2 Polymorphism of human leukocyte antigen Cw(HLA-Cw) gene in uremic patients
Objective
To detect the HLA-Cw gene frequencies in uremic patients and normal healthy individuals, analyze HLA-Cw gene distribution. Aim to investigate the contact between KIR and HLA-Cw, their relationship with the pathogenesis of uremia.
Method
Venous blood was collected of 55 uremic patients (case group) and 60 random unrelated healthy individuals (control group). HLA-Cw genes were typed by PCR-SSP. Statistical analysis was performed by SPSS13.0 for windows. HLA-Cw gene was analyzed in each individual. According to the difference of HLA-Cw at Position 80 of the a-1 helix, HLA-Cw was divided into two types:HLA-C1 which characterized by an asparagine residue at position 80 of the a-1 helix and HLA-C2 which characterized by a lysine residue at position 80 of the a-1 helix. T-test of independent sampler was used for comparison of HLA-C1, HLA-C2 in both groups and between groups. Gene frequencies and positive rate of KIR2D-HLA-Cw were analyzed by Chi-square test. A value of P< 0.05 was considered statistical different.
Results
There were no significant differences of HLA-Cw gene frequencies between uremic patients and healthy controls (P>0.05). HLA-Cw*03,07,01,08 had a higher expression in both groups. HLA-Cw*02,04,05,06,12,14,15,16 were relatively lower, HLA-Cw *13,17,18 were not detected in all individuals. Gene frequencies of HLA-C1 were much higher than that of HLA-C2 in case group and control group (P=0.023, P=0.020). There were no significant differences for positive rate of KIR2D-HLA-Cw between two groups (P>0.05).
Conclusions
HLA-Cw genes have richer polymorphisms. HLA-Cw gene polymorphisms have no difference between uremic patients and normal healthy controls. As KIR ligand HLA-Cw is divided into two types (HLA-C1 and HLA-C2). HLA-C1 which contains HLA-Cw*03, 07,01,08 is predominant compared with HLA-C2 in both groups. In general, each individual's inhibitory KIR2D-HLA-Cw receptor-ligand pairs are dominant, but the role of KIR2D-HLA-Cw pair in pathogenesis of uremia is not clear.
Chapter 3 Relationship between matching of recipient KIR/donor HLA-Cw and acute rejection after kidney transplantation
Objective
To explore the effect of recipient KIR/donor HLA-Cw ligand matching which mediates inhibitory or activated signal pathways on acute rejection (AR) after kidney transplantation.
Method
Venous blood was collected of 53 donor/recipient pairs of kidney transplantation. HLA-Cw and KIR genotype were typed by PCR-SSP. All recipients were divided into AR group (G I:n=19) and stable renal function group (GⅡ:n=34). Diagnosis of acute rejection depended on clinical symptoms, lab test, Color Doppler sonography and renal biopsy. The impact of donor HLA-Cw, recipient KIR and distinct recipient KIR/donor HLA-Cw matching on acute rejection after kidney transplantation were studied. Statistical analysis was performed by SPSS 13.0 for windows. Distribution of receptor's gender, primary disease type, HLA mismatch site, PRA, transplant views, different types of donor/receptor's gene type composition, receptor's KIR phenotype frequencies, the incidence of acute rejection and three different types of recipient KIR/donor HLA-Cw matching were analyzed by Chi-square test. T-test of independent sampler was used for comparison of age, cold and warm ischemia time. A value of P< 0.05 was considered statistical different.
Results
Distribution of donor HLA-Cw and recipient KIR between G I and GⅡ:There were no statistically significant differences of donor HLA-C1/2 between two groups (P>0.05). The distribution of recipient's different KIR genotypes were significant different between two groups (P=0.014), number of KIR genotype (AA) in G I was lower than that in GⅡ(10.5%vs 44.1%). The phenotype frequencies for recipient KIR2DL2/2DS2, KIR2DL3 in G I were lower than that in GⅡ(26.3%vs 55.9%, P=0.038; 73.7%vs 97.1%,P=0.034). Incidence of AR for donor HLA-C1/2 and recipient KIR genotype:donor HLA-C1/1 was lower than that in non-C1/1(31.6%vs 46.7%, P>0.05), recipient KIR genotype (AA) was lower than that in non-AA(11.8% vs 47.2%,P=0.012). Distribution of three different types recipient KIR/donor HLA-Cw matching:KIR2DL2/HLA-C1 and KIR2DL3/HLA-C1 were significant different between two groups(P=0.021,P=0.011). A higher number of matches for KIR2DL2/HLA-C1 and KIR2DL3/HLA-C1 were observed in GⅡ(55.9%,97.1%).
Conclusions
Tissue matching may reduce the incidence of acute rejection according to the following principles:donor's HLA-Cw genotype is HLA-C1/C1, recipient's KIR genotype is AA, recipient's KIR genotype expression of inhibitory receptor KIR2DL2, KIR2DL3, the more matches of distinct recipient KIR/donor HLA-Cw matching (such as KIR2DL2/HLA-C1 and KIR2DL3/HLA-C1).
引文
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