复合扩增荧光标记STR-PCR定量检测技术在异基因造血干细胞移植术中的应用
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摘要
目的利用微卫星片段(Short tandem repeat ,STR)遗传多态性的特点,结合聚合酶链式反应(Polymerase chain reaction,PCR)和毛细管电泳技术,采用复合扩增荧光标记短串联重复序列STR-PCR结合毛细管电泳检测15个STR基因座和1个性别位点的方法,建立异基因造血干细胞移植术(Allogeneic stem cell transplantation,Allo-HSCT)后嵌合体组成的定量检测方法。分别检测骨髓(Bone marrow transplantation,BMT)联合外周血造血干细胞术(Peripheral blood stem cell transplantation, PBSCT)或单纯PBSCT后30天造血细胞嵌合体组成情况以及非血缘脐血移植(Unrelated Cord blood transplantation, UCBT)后30天内造血细胞嵌合体的动态变化,判断移植物植入情况,同时在移植术后长期跟踪检测造血细胞嵌合体组成以评价疾病状态、预测疾病转归等,并将此方法和其他临床检测手段相比较,观察其实际临床应用价值。
方法建立识别个体的荧光标记复合PCR扩增STR位点结合毛细管电泳检测嵌合体方法,使用Promage公司的PowerPlex16个体识别试剂盒扩增荧光标记多重STR,特异性STR位点选择包括:D3S1358、TH01、D21S11、D18S51、PentaE、D5S818、D13S317、D7S820、D16S539、CSF1PO、PentaD、vWA、D8S1179、TPOX、FGA 15个基因座和1个性别基因Amelogenin。扩增产物经ABI公司的3100型遗传分析仪进行毛细管电泳,GenScan?等分析软件分析数据,检测异基因造血干细胞移植术后移植受者造血细胞嵌合体组成情况,判断移植物植入情况,同时,在定性检测的基础上,依据毛细管电泳后DNA信息峰曲线下面积的大小,定量计算供者细胞的嵌合率。用此技术检测2006年至2007年间在安徽省立医院血液科接受Allo-HSCT术的44例血液系统恶性肿瘤患者移植后不同时间点造血细胞嵌合体组成情况。35例Allo-HSCT受者(其中33例BMT联合PBSCT、2例PBSCT)检测移植后30天造血细胞嵌合体组成以判断移植物植入情况,9例UCBT患者连续检测移植后30天内(移植后7、14、21、30天)造血细胞嵌合体组成变化,并同时和相应的外周血象比较,以观察移植物的植入规律并提前预测移植效果。移植后定期跟踪检测移植受者造血细胞嵌合体变化以预测移植物永久植入情况和疾病的转归。
结果全部移植受者在移植后不同时间点经复合扩增荧光标记STR-PCR定量技术检测造血细胞嵌合体组成中均检测到供者成分。35例BMT/PBSCT受者全部获得造血重建,34例移植受者术后30天经STR-PCR定量技术检测造血细胞嵌合体组成呈完全供者型(Full donor chimera,FDC),1例63岁ALL患者行减低强度PBSCT,移植后30天嵌合体呈混合嵌合(Mixed chimera,MC),移植后90天嵌合体呈FDC。9例UCBT受者在移植后不同时间均检测到供者成分,发现获得造血重建的7例移植受者,在移植后7天造血未恢复外周血中嵌合体均呈FDC,而2例未获得造血重建受者移植后7天嵌合体呈MC,移植后30天外周血和骨髓中均未检测到供者基因成分。跟踪随访中,7例骨髓复发患者均提前出现嵌合体中供者成分减低的现象,2例髓外复发患者其嵌合体仍呈FDC。
结论1,复合扩增荧光标记STR一PCR结合毛细管电泳技术能准确有效地判断出Allo-HSCT术后早期移植物植入状况,并能连续动态监测移植后移植物永久植入情况,及时预测疾病的转归,为临床早期干预治疗提供可靠的实验依据;
2,UCBT后30天内连续检测嵌合体组成情况,在外周血象尚未恢复时(移植后7天)即可提前预测移植物被植入,与沿用的移植成功指标ANC具有很好的关联性,并可揭示脐血移植植入动力学的规律,该技术可作为早期评估UCBT是否成功最灵敏特异的指标。3,该方法灵敏度高、特异性强、标本需要量少、适应范围广,具有很好的临床应用价值。
Objective Using microsatellites genetic polymorphism , polymerase chain reaction and capillary electrophoresis to establish quantitative detection of chimerism by multiple PCR amplification of short tandem repeats markers and capillary electrophoresis with fluorescence detection for 16 STR locus after Allogeneic hematopoietic stem cell transplantation(Allo-HSCT). The chimerism examination were made for 30 dyas after Bone marrow transplantation(BMT) combined with peripheral blood stem cells transplantation(PBSCT) or singel PBSCT while the chimerism was continuous detected within 30 days after Unrelated Cord blood transplantation(UCBT) to judge transplants implantation. At the same time, the evaluation of diseases and prediction of diseases outcome were made by chimerism examination long-term after Allo-HSCT and observe its practical clinical application value by comparing with other detection means.
Method To build the STR typing system by fluorescence labeled multiplex-PCR and capillary electrophoresis technique for personal identification. Amplified fluorescence labeled multiplex-STR with PowerPlex16 personal identification kits from Promage corporation. Specific str locus contains fifteen gene locus such as D3S1358、TH01、D21S11、D18S51、PentaE、D5S818、D13S317、D7S820、D16S539、CSF1PO、PentaD、vWA、D8S1179、TPOX、FGA and one sexual gene locus Amelogenin. The amplified products were electrophoreticed by 3100 genetic analyzer from ABI corporation, and data analysis by GenScan analysis software. At the same time, under the basement of qualitative detection, quantitative detection of chimerism were investigated according to the Proportion of the Peak areas corresponding to DNA signal after electrophoresis. The method was used to detected chimerism at different time after Allo-HSCT for 44 hematologic malignancies patients who received Allo-HSCT in Anhui provincial hospital between 2006-2007. In the all 44 hematologic malignancies patients, chimerism was detected at 30 days to judge grafts implantation after Allo-HSCT for 33 BMT combined with PBSCT and 2 singel PBSCT. While in the 9 UCBT, chimerism was detected four times within 30 days after Allo-HSCT, and compared with peripheral hemogram to observe the rule of grafts implantation or predict the transplantation effect in advance. The grafts implantation permanently and diseases outcome were predicted by the continuous tracking of chimerism detection after Allo-HSCT.
Result The chimerism can be detected in all the transplantation recipients. 35 Allo-HSCT obtained hematopoietic reconstitution successfully. 34 PBSCT/BMT transplantation recipients formed FDC at 30 days after Allo-HSCT, 1 63 year old patient received decreased intensity PBSCT and formed MC at 30 days and FDC at 90 days after Allo-HSCT. The chimerism can be detected in all the UCBT recipients, 7 patients who obtained hematopoietic reconstitution successfully have FDC in the peripheral blood hemopoietic cells.other 2 patients formed MC at 7 days after Allo-HSCT and whose hematopoietic reconstitution were failed.it is failed to find donor at the chimerism at 30 days after Allo-HSCT, In the tracking follow-up, 7 bone marrow relapse have the sign of donor decreased in the chimerism while 2 out of bone marrow relapse were formed FDC all the time.
conclusion 1.The STR typing system by fluorescence labeled multiplex-PCR and capillary electrophoresis technique can dudge graft accurately at early time after Allo-HSCT. The method can can predict graft implantation permanently and disease prognosis by Chimerism track after Allo-HSCT, provid reliable experimental basis for clinical treatment. 2. Detect the Chimerism continuously within 30 days after UCBT can provide the information of graft implantation, Graft can be detected as early as 7 day after UCBT while peripheral hemogram could not recover and have a good coherence with implantation index ANC, the method can reveal UCBT engraftment kinetics and evaluate UCBT engraftment sensitivity and specificity 3.The method has high sensitivity and specificity and have a good coherence with other detection method , the sampling quantity was low and clinical application value was good.
方法建立识别个体的荧光标记复合PCR扩增STR位点结合毛细管电泳检测嵌合体方法,使用Promage公司的PowerPlex16个体识别试剂盒扩增荧光标记多重STR,特异性STR位点选择包括:D3S1358、TH01、D21S11、D18S51、PentaE、D5S818、D13S317、D7S820、D16S539、CSF1PO、PentaD、vWA、D8S1179、TPOX、FGA 15个基因座和1个性别基因Amelogenin。扩增产物经ABI公司的3100型遗传分析仪进行毛细管电泳,GenScan?等分析软件分析数据,检测异基因造血干细胞移植术后移植受者造血细胞嵌合体组成情况,判断移植物植入情况,同时,在定性检测的基础上,依据毛细管电泳后DNA信息峰曲线下面积的大小,定量计算供者细胞的嵌合率。用此技术检测2006年至2007年间在安徽省立医院血液科接受Allo-HSCT术的44例血液系统恶性肿瘤患者移植后不同时间点造血细胞嵌合体组成情况。35例Allo-HSCT受者(其中33例BMT联合PBSCT、2例PBSCT)检测移植后30天造血细胞嵌合体组成以判断移植物植入情况,9例UCBT患者连续检测移植后30天内(移植后7、14、21、30天)造血细胞嵌合体组成变化,并同时和相应的外周血象比较,以观察移植物的植入规律并提前预测移植效果。移植后定期跟踪检测移植受者造血细胞嵌合体变化以预测移植物永久植入情况和疾病的转归。
结果全部移植受者在移植后不同时间点经复合扩增荧光标记STR-PCR定量技术检测造血细胞嵌合体组成中均检测到供者成分。35例BMT/PBSCT受者全部获得造血重建,34例移植受者术后30天经STR-PCR定量技术检测造血细胞嵌合体组成呈完全供者型(Full donor chimera,FDC),1例63岁ALL患者行减低强度PBSCT,移植后30天嵌合体呈混合嵌合(Mixed chimera,MC),移植后90天嵌合体呈FDC。9例UCBT受者在移植后不同时间均检测到供者成分,发现获得造血重建的7例移植受者,在移植后7天造血未恢复外周血中嵌合体均呈FDC,而2例未获得造血重建受者移植后7天嵌合体呈MC,移植后30天外周血和骨髓中均未检测到供者基因成分。跟踪随访中,7例骨髓复发患者均提前出现嵌合体中供者成分减低的现象,2例髓外复发患者其嵌合体仍呈FDC。
结论1,复合扩增荧光标记STR一PCR结合毛细管电泳技术能准确有效地判断出Allo-HSCT术后早期移植物植入状况,并能连续动态监测移植后移植物永久植入情况,及时预测疾病的转归,为临床早期干预治疗提供可靠的实验依据;
2,UCBT后30天内连续检测嵌合体组成情况,在外周血象尚未恢复时(移植后7天)即可提前预测移植物被植入,与沿用的移植成功指标ANC具有很好的关联性,并可揭示脐血移植植入动力学的规律,该技术可作为早期评估UCBT是否成功最灵敏特异的指标。3,该方法灵敏度高、特异性强、标本需要量少、适应范围广,具有很好的临床应用价值。
Objective Using microsatellites genetic polymorphism , polymerase chain reaction and capillary electrophoresis to establish quantitative detection of chimerism by multiple PCR amplification of short tandem repeats markers and capillary electrophoresis with fluorescence detection for 16 STR locus after Allogeneic hematopoietic stem cell transplantation(Allo-HSCT). The chimerism examination were made for 30 dyas after Bone marrow transplantation(BMT) combined with peripheral blood stem cells transplantation(PBSCT) or singel PBSCT while the chimerism was continuous detected within 30 days after Unrelated Cord blood transplantation(UCBT) to judge transplants implantation. At the same time, the evaluation of diseases and prediction of diseases outcome were made by chimerism examination long-term after Allo-HSCT and observe its practical clinical application value by comparing with other detection means.
Method To build the STR typing system by fluorescence labeled multiplex-PCR and capillary electrophoresis technique for personal identification. Amplified fluorescence labeled multiplex-STR with PowerPlex16 personal identification kits from Promage corporation. Specific str locus contains fifteen gene locus such as D3S1358、TH01、D21S11、D18S51、PentaE、D5S818、D13S317、D7S820、D16S539、CSF1PO、PentaD、vWA、D8S1179、TPOX、FGA and one sexual gene locus Amelogenin. The amplified products were electrophoreticed by 3100 genetic analyzer from ABI corporation, and data analysis by GenScan analysis software. At the same time, under the basement of qualitative detection, quantitative detection of chimerism were investigated according to the Proportion of the Peak areas corresponding to DNA signal after electrophoresis. The method was used to detected chimerism at different time after Allo-HSCT for 44 hematologic malignancies patients who received Allo-HSCT in Anhui provincial hospital between 2006-2007. In the all 44 hematologic malignancies patients, chimerism was detected at 30 days to judge grafts implantation after Allo-HSCT for 33 BMT combined with PBSCT and 2 singel PBSCT. While in the 9 UCBT, chimerism was detected four times within 30 days after Allo-HSCT, and compared with peripheral hemogram to observe the rule of grafts implantation or predict the transplantation effect in advance. The grafts implantation permanently and diseases outcome were predicted by the continuous tracking of chimerism detection after Allo-HSCT.
Result The chimerism can be detected in all the transplantation recipients. 35 Allo-HSCT obtained hematopoietic reconstitution successfully. 34 PBSCT/BMT transplantation recipients formed FDC at 30 days after Allo-HSCT, 1 63 year old patient received decreased intensity PBSCT and formed MC at 30 days and FDC at 90 days after Allo-HSCT. The chimerism can be detected in all the UCBT recipients, 7 patients who obtained hematopoietic reconstitution successfully have FDC in the peripheral blood hemopoietic cells.other 2 patients formed MC at 7 days after Allo-HSCT and whose hematopoietic reconstitution were failed.it is failed to find donor at the chimerism at 30 days after Allo-HSCT, In the tracking follow-up, 7 bone marrow relapse have the sign of donor decreased in the chimerism while 2 out of bone marrow relapse were formed FDC all the time.
conclusion 1.The STR typing system by fluorescence labeled multiplex-PCR and capillary electrophoresis technique can dudge graft accurately at early time after Allo-HSCT. The method can can predict graft implantation permanently and disease prognosis by Chimerism track after Allo-HSCT, provid reliable experimental basis for clinical treatment. 2. Detect the Chimerism continuously within 30 days after UCBT can provide the information of graft implantation, Graft can be detected as early as 7 day after UCBT while peripheral hemogram could not recover and have a good coherence with implantation index ANC, the method can reveal UCBT engraftment kinetics and evaluate UCBT engraftment sensitivity and specificity 3.The method has high sensitivity and specificity and have a good coherence with other detection method , the sampling quantity was low and clinical application value was good.
引文
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44) Budowle B ,Chakraborty R ,Giusti AM ,et al. Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE. Am J Hum Genet ,1991 ,48 (1) :137-144.
45)孙琪云,艾辉胜,姚波等. STR-PCR定量检测供受者嵌合体方法的建立及临床应用[J ] .解放军医学杂志,2001 ,26 (1) :13-15.
1. Lane TA. Umbilical cord blood grafts for hematopoietic transplantation in adults: a cup half empty or half full? Transfusion, 2005, 45(6):1027-1034.
2. Lane TA. Umbilical cord blood grafts for hematopoietic transplantation in adults: a cup half empty or half full? Transfusion, 2005, 45(6):1027-1034.
3. Laughlin MJ, Barker J, Bambach B, et al. Hematopoietic engraftment and survival in adult recipients of umbilical cord blood from unrelated donors. N Engl J Med, 2001, 344(24):1815-1822.
4. Brunstein CG, Wagner JE. Umbilicial cord blood transplantation and banking. Annu Rev Med, 2006,57:403-417.
5.汪赤宇,谢毅.脐血造血祖细胞培养及T淋巴细胞亚群的研究.中华血液学杂志,1994, 15(4):178-180.
6. Nauta AJ, Kruisselbrink AB, Lurvink E, et al. Enhanced engraftment of umbilical cord blood-derived stem cells in NOD/SCID mice by cotransplantation of a second unrelated cord blood unit. Exp Hematol, 2005, 33(10):1249-1256.
7.张丽萍,沈柏均,候怀水,等.混合脐血移植后造血及免疫重建.中华器官移植杂志,2003,24(4):205-208.
8.鞠秀丽,沈柏均,刘星霞,等.混合脐血移植研究.山东医药,2001,41(11):9-10.
9. [罗幼平4] Barker JN, Weisdorf DJ, Defor TE, et al. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood, 2005, 105(3):1343-1347.
10. Lima MD, John LSS, Wieder ED, et al. Double-chimaerism after transplantation of two human leucocyte antigen mismatched, unrelated cord blood units. Br J Haematol , 2002, 119(3):773-776.
11. Ooi J, Iseki T, Takahashi S, et al. Unrelated cord blood transplantation for adult patients with de novo acute myeloid leukemia. Blood, 2004,103(2):489-491.
12. Majhail NS, Brunstein CG, Wagner JE, et al. Double umbilical cord blood transplantation. Curr Opin Immunol, 2006, 18(5):571-575.
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14. Kang HJ, Kho SH, Jang MK, et al. Early engraftment kinetics of two units cord blood transplantation. Bone Marrow Transplantation, 2006,38(3):197-201.
15. Kim DW, Chung YJ, GyuKim T , et al. Cotransplantation of third-party mesenchymal stromal cells can alleviate single-donor predominance and increase engraftment from double cord transplantation. Blood, 2004, 103(5):1941-1948.
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42)孙琪云,艾辉胜,姚波等,STR-PCR定量检测供受者嵌合体方法的建立及临床应用。解放军医学杂志,2001 26(1):13-15。
43)李素霞,达万明,高春记等,STR-PCR对造血干细胞移植后植入证据的检测,中国试验血液学杂志,2005,13(1):25-29.
44) Budowle B ,Chakraborty R ,Giusti AM ,et al. Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE. Am J Hum Genet ,1991 ,48 (1) :137-144.
45)孙琪云,艾辉胜,姚波等. STR-PCR定量检测供受者嵌合体方法的建立及临床应用[J ] .解放军医学杂志,2001 ,26 (1) :13-15.
1. Lane TA. Umbilical cord blood grafts for hematopoietic transplantation in adults: a cup half empty or half full? Transfusion, 2005, 45(6):1027-1034.
2. Lane TA. Umbilical cord blood grafts for hematopoietic transplantation in adults: a cup half empty or half full? Transfusion, 2005, 45(6):1027-1034.
3. Laughlin MJ, Barker J, Bambach B, et al. Hematopoietic engraftment and survival in adult recipients of umbilical cord blood from unrelated donors. N Engl J Med, 2001, 344(24):1815-1822.
4. Brunstein CG, Wagner JE. Umbilicial cord blood transplantation and banking. Annu Rev Med, 2006,57:403-417.
5.汪赤宇,谢毅.脐血造血祖细胞培养及T淋巴细胞亚群的研究.中华血液学杂志,1994, 15(4):178-180.
6. Nauta AJ, Kruisselbrink AB, Lurvink E, et al. Enhanced engraftment of umbilical cord blood-derived stem cells in NOD/SCID mice by cotransplantation of a second unrelated cord blood unit. Exp Hematol, 2005, 33(10):1249-1256.
7.张丽萍,沈柏均,候怀水,等.混合脐血移植后造血及免疫重建.中华器官移植杂志,2003,24(4):205-208.
8.鞠秀丽,沈柏均,刘星霞,等.混合脐血移植研究.山东医药,2001,41(11):9-10.
9. [罗幼平4] Barker JN, Weisdorf DJ, Defor TE, et al. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood, 2005, 105(3):1343-1347.
10. Lima MD, John LSS, Wieder ED, et al. Double-chimaerism after transplantation of two human leucocyte antigen mismatched, unrelated cord blood units. Br J Haematol , 2002, 119(3):773-776.
11. Ooi J, Iseki T, Takahashi S, et al. Unrelated cord blood transplantation for adult patients with de novo acute myeloid leukemia. Blood, 2004,103(2):489-491.
12. Majhail NS, Brunstein CG, Wagner JE, et al. Double umbilical cord blood transplantation. Curr Opin Immunol, 2006, 18(5):571-575.
13. Barker JN, Weisdorf DJ, Defor TE, et al. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood, 2005, 105(3):1343-1347.
14. Kang HJ, Kho SH, Jang MK, et al. Early engraftment kinetics of two units cord blood transplantation. Bone Marrow Transplantation, 2006,38(3):197-201.
15. Kim DW, Chung YJ, GyuKim T , et al. Cotransplantation of third-party mesenchymal stromal cells can alleviate single-donor predominance and increase engraftment from double cord transplantation. Blood, 2004, 103(5):1941-1948.