| Objective: (1)To develop an oligonucleotide array for SNP typing cytokines such as IL-4,IL-6,IL-10,TNF-a and TGF-β 1 and their receptors. (2)To establish a relationship among cytokines gene polymorphisms, the levels of cytokines (IL-10,TNF-a and TGF- β1) expression and the outcome of allograft function.Methods: (1)Accoding to documents, SNP database of NCBI and SNP 500Cancer database of NCI,choose out SNP locus and sequence of cytokines (IL-4,IL-6,IL-10,TNF-a and TGF- β1) and matched cytokine receptors with clinical value. 58 synthesized oligonucleotide probes were immobilized on a glass support,then the primers and Cy5-dCTP were used in poly-PCR, thus the products were labeled with Cy5. The labeled PCR products were hybridized with the probes in the array, and the signals were scanned by Scanner and then analyzed by Image software.30 DNA samples of kidney transplantation recipients were tested by this array for 3 times.Others DNA samples were also typed by the array. (2)Genomic DNA samples from the peripheral blood lymphocytes of 144 kidney transplant recipients and 103 donors were tested by an oligoneucleotide array. The distribution of 21 single nucleotide polymorphisms in cytokines and cytokine receptors gene were compared between two groups according to the presence or absence of acute kidney rejection.(3) The serum levels of TNF-α IL-10 and TGF- β1 in 65 healthy Han's people and 102 kidney allograft recipients were determined both at pre- and early post-transplantation (day 1 and day 4) by the means of enzyme-linked immunosorbent assay with avidin-biotin-HRP (horse radish peroxidase) complex (ELISA-ABC).Results: (1) 30 DNA samples of kidney transplantation recipients had been tested by cytokine oligonucleotide array for 3 times.The rate of repeatability was 100%. DNA samples from 114 recipients and 103 donors had been genotyped by cytokine array successfully. (2)In recipients,the gene polymorphisms distribution in rejection group and non-rejection group were significantly different,as follows:TNF-a(-3O8A/A,A/G,G/G), IL-10(-597A/A,C/C,A/C,-824T/T,C/C,C/T, -1087A/A,A/G), TGF- P 1 (+869C/C,C/T,T/T) and the recipient alleles TNF-a(-308A/G), IL-10(-597A/C,-824T/C,-1087A/G), TGF- P 1 (+869C/T). (3) Indonors,the genotypes and alleles distribution in rejection group and non-rejectiongroup was insignificantly different,as follows: TNF-a(-238,-308),TNF-aR(168,587),IL-4(-590),IL-4R(1902,Ile50Val),IL-6(-597,-573,-174),IL-6R(-183,D358),IL-10(-597,-824,-1087),IL-10R(536,1112),TGF- P l(+869) and TGF- P1R(1167). (4) A greater proportion of the recipients with themselves' TNF-ahigh/their corresponding donors' TNF- a high producer genotype had acuterejection as compared with all other genotypes.Conversely, significantly fewerpatients with themselves'TNF- a low/ their corresponding donors' TNF- a lowproducer genotype had acute rejection as compared with all other genotypes.Significantly fewer recipients with IL-10 high/ intermediate producer genotype(also donors) had acute rejection as compared with all other genotypes.Conversely, a greater proportion of patients with IL-10 low producer genotype(also donors) had acute rejection as compared with all other genotypes. TGF- P 1was as the same.(5) 8 of 48 patients with acute rejection lost their transplantkidneys. Among them the frequency of TNF- a high producer and TGF- P 1 lowproducer genotype were 75% and 37.5% respectively comparing 37.5% and 5%respectively in recovery group patients (P<0.05).(6) Analysing the distributionof cytokines and cytokine receptors gene polymorphisms pre- and post-operationin 12 acute rejection recipients,we found that there was not any change during theoccurrence of acute rejection or one month later comparing pre-operation.Thesame as it was when compared the distribution of cytokines and cytokinereceptors gene polymorphisms in 8 resected renal specimens with that in theirdonors.(7) The plasma level of TNF-a in genotype A/A and A/G(56.63±12.01pg/mi) was significantly higher than that in genotype G/G(31.58 ±8.76pg/ml,P<0.01). The plasma level of IL-10 in genotype GCC/GCC(45.13±11.94pg/ml) was significantly higher than that in genotype GCC/ACC orGCC/ATA(24.57±7.46pg/ml,P<0.01).The latter was significantly higher thanthat in genotype ACC/ACC,ACC/ATA or ATA/ATA(13.61 ±4.82pg/ml,P<0.01).Similarly, the plasma level of TGF- P 1 in genotype T/T(1015.87 ± 107.61 ng/ml)was significantly higher than that in genotype T/C(454.43±76.59ng/ml,P<0.01).The latter was significantly higher than that in genotype C/C(134.I6 ±65.97ng/ml, PO.01). (8) Detecting the cytokine level in 69 recipients with stable graft function(SGF) and 33 recipients with rejections(REs),we found that:by day 1 and 4, the level of TNF- a in group with SGF was significantly higher than that in group with REs.Pre-operation,the level of IL-10 in group with SGF was significantly lower than that in group with REs. By day 1, the group with SGF demonstrated a significant increase of IL-10,while the group with REs showed only a mild increase of IL-10. Pre-operation,the level of TGF- P 1 in group with SGF was significantly higher than that in group with REs. By day 1 and 4, the group with SGF demonstrated a significant increase of TGF- |3 1,while the group with REs showed only a mild increase of TGF- 0 1. (9) A greater proportion of the recipients with TNF-a high producer genotype/CsA low dosage (17/27, 62.96% ) had acute rejection compared with recipients with the other genotype/CsA dosage (22/99, 22.22%; PO.01). A greater proportion of recipients with IL-10 low producer genotype/CsA low dosage (35/96, 36.46%) had acute rejection compared with recipients with the other genotype/CsA dosage (4/30, 13.33%,P=0.017). Similarly, a greater proportion of recipients with TGF- 0 1 low producer genotype/CsA low dosage( 33/89,37.08% )had acute rejection compared with recipients with the other genotype/CsA dosage (6/37, 16.22%,P=0.021) .Conclusion: (1) Oligonucleotide array technique is an ideal molecular method for SNP genotyping of cytokines and cytokine receptors,wih high specificity, high sensitivity and good repeatability.(2)TNF-a(Thl type cytokine) was associated with acute allograft rejection.IL-10(Th2 type cytokine) and TGF-P l(Th3 type cytokine) were found in correlation with allograft protection. While in recipients and donors,the genotypes and alleles distribution in rejection group and non-rejection group was insignificantly different,as follows:TNF-aR(l68-< 587)n IL-4(-590, -33)x IL-4R(1902, IleSOVaK -3223)> IL-6(-597, -573, -174), IL-6R(-183, D358), IL-10R(536, 1112) and TGF- P 1R(1167). (3) The cytokines gene polymorphisms of donors may affect the outcome of allograft in some given situation. (4) TNF-a and TGF- P 1 gene polymorphisms had significant influence on the severity of acute rejection in renal transplantations. (5) The distribution of cytokines and cytokine receptors gene polymorphisms in recipients and donors had not any change pre- and post-operation. (6) TNF-a, TGF-3 1 or IL-10 genotypes are determinants of their corresponding serum levels in healthy adult Han's people. (7) The plasma levels of TNF-ouTGF-3 1 or IL-10 were associated...
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