| Objectives:To establish real-time PCR(RT-PCR)method for detecting mRNA expression of killer immunoglobulin-like receptor(KIR)2DS1 gene on the surface of natural killer(NK)cells,and to evaluate its performance.Then,the established method was used to explore the expression pattern of KIR2DS1 gene in allo-HSCT and the correlation of KIR2DS1 expression with aGvHD and relapse.Methods:The specific primers and probe of KIR2DS1 gene were designed for Taqman-MGB fluorescence quantitative PCR detection system.Then,the performance parameters of the detecting system such as coincidence rate,repeatability,sensitivity,the scope of application of the instrument and the reproducibility of technicians were evaluated and validated.Then,taking 72 cases of ALL and AML as research objects,the self-established RT-PCR method was used to dynamically detect the expression level of KIR2DS1 mRNA in donor-recipient pairs.Combined donor-patient KIR genotyping results,to analyze the source of KIR2DS1 expression in leukemia patients after HSCT,and to investigate the expression pattern of KIR2DS1 gene after allo-HSCT.At the same time,combined with clinical data,study the correlation of KIR2DS1 expression with aGvHD and relapse.Results:1.Establishment of a method for detection of mRNA expression of KIR2DS1 gene on the surface of NK cell(1)The PCR-SSO and PCR-SSP methods were used to screen the specific KIR2DS1 gene base sequence,and probes and fluorescence were designed.The universal PCR reaction system was used to establish a fluorescent quantitative PCR reaction system.(2)Taking KIR SSO Genotyping Test as the gold standard,the result of 35 samples showed the accuracy of self-built assay for positive or negative detection of KIR2DS1 was 100%.(3)Three samples with high,median and low value of Ct were used to repeatability test.The intra-assay and inter-assay coefficient of variation were ranged from 0.09%to 0.46%and 0.71%to 1.13%respectively.(4)The sensitivity of this method can be up to 102 copies/?L at least,and the coefficient of variation of the three samples that equalled 102 copies/?L having been tested for five times was 5.37%,2.71%and 5.51%,respectively.(5)The regression analysis of samples detected by ABI-7500 and LC-480 fluorescence-quantitative PCR instrument was Y=0.9736X+0.1183(R2=0.9619).(6)The reproducibility of ten KIR2DS1-positive samples operated by two technicians showed that the biases were all less than 5%.2.Clinical value of detection method for NK cell surface receptor KIR2DS1 mRNA expression level(1)Through KIR-SSO testing,it was found that the KIR genotype of 72 patients all transformed to the donor's after the first month of allo-HSCT.And there was no statistically significant difference in the expression level of KIR2DS1 mRNA between the donor's specific KIR2DS1 gene group and the donor's non-specific KIR2DS1 gene group in each month after transplantation(P>0.05).(2)The Median expression of KIR2DS1 in donor's on transplant's day was 1898.79×104/ABLcopies.KIR2DS1 recognition group(HLA-C2 positive group)expression level was higher than KIR2DL1 don't recognize group(HLA-C2 negative group),were 2238.89×104/ABL copies and 1683.81×104/ABL copies respectively,compared two groups was no statistically difference(P=0.431).(3)At the 1M,2M,3M,4-6M,7-9M,10-12M after allo-HSCT,the median expression level of KIR2DS1 was 2711.92 2865.93,2663.60,2043.93,1956.50,2160.41×104/ABL copies in AML group and 2263.17,2382.20,2607.68,1825.76,2011.68,2183.70×104/ABL copies in ALL group.Compared with the expression level of the donor on the day of transplantation,the mRNA expression of KIR2DS1 in ALL patients was statistically significant at the 1st,2nd and 3rd month after transplantation(P=0.028,P=0.034,P=0.034).The expression level of KIR2DS1 mRAN in AML patients was statistically significant at the first and second month after transplantation(P=0.019,P=0.001).The expression pattern of KIR2DS1 in the AML and ALL groups is generally consistent,and it was at a high expression level for 1-3 months in the early stage of transplantation.After reaching its highest median transcription level,KIR2DS1 began to decline,and finally remained slightly higher than that of the donor's level.(4)Within the first 3 months after transplantation,the expression level of KIR2DS1 mRNA in the aGvHD group was lower than that in the non-aGvHD group,compared two groups was statistically difference at 2 months after transplantation(P=0.014).(5)At the 1-3M,4-6M,7-9M after allo-HSCT,the expression level of KIR2DS1 mRNA in immune tolerance group was higher than that in relapse group,but there was no significant difference between the two groups.Conclusion:The Taqman-MGB fluorescence quantitative PCR method was established to detect the mRNA expression level of KIR2DS1 gene,and was initially used to study the expression of KIR2DS1gene in allo-HSCT.It was found that the expression level of KIR2DS1 was dynamically changed after allo-HSCT and the low expression of KIR2DS1 mRNA was associated with the occurrence of ?-? aGvHD.It set up the experimental basis for the clinical application of KIR2DS1 gene quantitative detection as a diagnostic indicator of GvHD. |
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