异基因造血干细胞移植中非血缘供者KIR2DL1、KIR3DL1免疫重建规律的研究

Immune reconstruct regularity profile of KIR2DL1 and KIR3DL1 in unrelated-donor allogeneic hematopoietic stem cell transplantation

目的在非血缘供者为杀伤细胞免疫球蛋白样受体（ⅪR）．AA基因型的异基因造血干细胞移植（allo．HSCT）中研究KIR2DLl、KIR3DLl的免疫重建规律。方法采用序列特异性引物PCR、流式细胞术和实时荧光定量PCR法，对75对供受者进行KIR基因分型，确定供者均为KIR．AA基因型，并动态检测移植当天及移植后不同时间澎植后0．5个月～〈1．5个月（1个月）、移植后1．5个月～〈2．5个月（2个月）、移植后2．5个月～〈3．5个月（3个月）、移植后3．5个月～〈6．5个月（3-6个月）、移植后6．5个月～〈9．5个月（6-9个月）、移植后9．5个月〈12．5个月（9～12个月）]KIR2DLl、KIR3DLl膜蛋白和mRNA的表达水平。结果KIR2DL膜蛋白中位表达水平：非血缘供者移植当天为21．60％，受者在1、2、3、3-6个月时表达水平分别为7．40％、12．00％、16．92％、17．64％；KIR2DLl的mRNA中位表达水平：非血缘供者移植当天为265．14／10。ABL拷贝数，受者移植后1、2、3、3-6、6-9、9-12个月时表达水平分别为332．17、438．31、723．25、414．17、187．60及234．67／10。ABL拷贝数。KIR2DLl的mRNA和膜蛋白表达水平在移植后各个时间点均逐渐升高，且在3个月后达到最高，但mRNA表达水平升高早于膜蛋白。（2）KIR3DLl膜蛋白中位表达水平：非血缘供者移植当天为18．56％，受者在移植后1、2、3、3-6个月时表达水平分别为23．83％、22．57％、23．02％、21．60％；KIR3DLlmRNA中位表达水平：非血缘供者移植当天为572．29／10。ABL拷贝数，受者移植后1、2、3、3-6、6-9、9-12个月时表达水平分别为1233．74、1140．42、876．73、1057．07、739．02、514．43／10。ABL拷贝数。KIR3DLl的mRNA和膜蛋白在移植后1个月的表达水平均已超过了非血缘供者，并在移植后各个时间点均稳定高表达，且mRNA与膜蛋白表达水平同步升高。结论KIR2DLl和KIR3DLl基因移植后的免疫重建规律性不同，为在移植后选择最佳时间点定量检测KIR2DLl、KIR3DLl表达水平提供了实验依据。

Objective To investigate the immune reconstruct regularity profile of K1R2DL1 and KIR3DL1 in unrelated-donor allogeneic hematopoietic stem cell transplantation （allo-HSCT） with KIR- AA genotype. Method 75 donor-recipient pairs were performed by KIR genotying using PCR-SSP, and all donors were identified with KIR-AA genotype. Dynamic detections （including unrelated-donor on the day of transplantation and the recipient each month post allo-HSCT） of the expression of KIR2DL1/3DL1 on NK cell and mRNA level were performed in 291 cases using flow cytometry （FCM） and real-time fluorescent quantitation PCR （RT-qPCR）. Result （1）The median expression of KIR2DLI in unrelated- donor on transplant＇ s day was 21.60%, the median expression of KIR2DL 1 in recipient 1 M, 2M, 3M and 3- 6M after transplantation were 7.40%, 12.00%, 16.92%, 17.64% respectively. The median expression of KIR2DL1 in unrelated-donor on transplant＇ s day was 265.14 copies/10 000abl copies, the medianexpression of KIR2DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 332.17, 438.31,723.25,414.17, 180.76 and 234.67 copies/10 000abl copies respectively. The median expression of KIR2DL1 on NK cells and mRNA level gradually increased at all time points after transplantation, and reached the highest expression at 3 months after transplantation. But mRNA expression levels increased earlier than NK cell membrane proteins. @The median expression of KIR3DL1 in unrelated-donors on transplant＇ s day was 18.56%, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M after transplantation were 23.83%, 22.57%, 23.02%, 21.60% respectively. The median expression of KIR3DL1 in unrelated-donor on transplant＇ s day was 572.29 copies/10 000abl copies, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 1 233.74, 1 140.42, 876.73, 1 057.07, 739.02 and 514.43 copies/10 000abl copies respectively. The median expression of KIR3DL1 on NK cells and mRNA level were higher than donors at 1 month after transplantation, and stable expression at all time points after transplantation, so mRNA and NK cell membrane proteins expression increased at the same time. Conclusion The immune reconstruct regularity of KIR2DL1 and KIR3DL1 gene were different, which provided an experimental basis for selecting the best time to detect the expressions of KIR2DL1 and 3DL1 after transplantation.