| 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 ObjectiveTo 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.MethodVenous 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.ResultsThere 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).ConclusionsPCR-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 patientsObjectiveTo 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.MethodVenous 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. ResultsThere 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).ConclusionsHLA-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 transplantationObjectiveTo 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.MethodVenous 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 (Gâ…¡: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.ResultsDistribution of donor HLA-Cw and recipient KIR between G I and Gâ…¡: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 Gâ…¡(10.5%vs 44.1%). The phenotype frequencies for recipient KIR2DL2/2DS2, KIR2DL3 in G I were lower than that in Gâ…¡(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 Gâ…¡(55.9%,97.1%).ConclusionsTissue 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). |
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