免疫抑制治疗不增加再生障碍性贫血骨髓造血细胞遗传不稳定性
摘要
【目的】
研究再生障碍性贫血(AA)患者骨髓造血细胞遗传不稳定性及强烈免疫抑制治疗(IST)对于从发生晚期克隆性血液学异常的影响。
【对象和方法】
采用彗星试验评价骨髓造血细胞遗传不稳定性,以彗星尾部DNA百分含量(TDNA%)、尾长(TL)、尾矩(TM)、Olive尾矩(OTM)和彗星细胞率(Comet%)作为分析参数,分别检测IST治疗前后从患者骨髓造血细胞,并与18例对照组结果比较。
【结果】
91例初治AA患者TDNA、TL、TM、OTM、Comet%分别为:5.02±3.95、11.25±7.19、1.70±2.02、1.50±1.38、16.84±13.65明显高于对照组(p＜0.05);58例重症AA(SAA)与33例非重症AA(NSAA)患者彗星试验各项参数比较均无差别(p＞0.05),二组患者分别与对照组比较,各彗星参数仍明显为高(p＜0.05)。53例SAA治疗后3月患者TDNA、TL、TM、OTM、Comet%分别为:4.43±3.64、10.37±7.49、1.37±1.61、1.29±1.37、20.17±21.17,30例SAA治疗后6月患者TDNA、TL、TM、OTM、Comet%分别为:3.71±3.32、10.04±7.18、1.15±1.80、1.12±1.29、18.47±19.02,与58例IST治疗前SAA患者彗星参数结果比较均无差异(p＞0.05)。18例SAA患者IST治疗前后均进行了彗星试验检测,彗星参数比较无统计学差异(p＞0.05)。
【结论】
AA患者骨髓造血细胞存在遗传不稳定,免疫抑制治疗本身并不增加SAA患者细胞遗传不稳定性。提示免疫抑制治疗与AA发生晚期克隆性血液学异常可能无关。
Objective
To investigate the genetic instabilities of bone marrow hematopoietic cells from patients with aplastic anemia and evaluate the impact of IST in aplastic anemia evolving into clonal hematologic disorders.
Methods
Comet assay were used to evaluate the genetic instabilities of hematopoietic cells and the percent of DNA in the comet tail (TDNA%), tail length(TL), tail moment(TM) olive tail moment(OTM) and the rate of comet cells (Comet%) were selected as analyzing parameters. Bone marrow hematopoietic cells from patients with aplastic anemia examined with comet assay before and after IST separately, and the results were compared with those from 18 controls.
Result
Comet parameters from 91 patients with aplastic anemia including TDNA, TL, TM, OTM and Comet% were 5.02±3.95, 11.25±7.19, 1.70±2.02, 1.50±1.38 and 16.84±13.65 separately, all of which were obviously superior to those from control group respectively (p<0.05). The results were remained notablely higher when separately compared the comet parameters of SAA and NSAA with those of control group (p<0.05) , and there were no differences of the examined comet parameters of 58 SAA from those of 33 NSAA patients (p>0.05) .The parameters TDNA, TL, TM, OTM and Comet% were 4.43±3.64, 10.37±7.49, 1.37±1.61, 1.29±1.37 and 20.17±21.17 respectively 3-months later after IST in 53 SAA patients and those were 3.71±3.32, 10.04±7.18,1.15±1.80, 1.12±1.29 and 18.47±19.02 respectively 6-months later after IST in 30 SAA patients , which had no difference from those of 58 patients before IST (p>0.05) .There was no statistical difference in comet parameters of 18 SAA patients before and after IST (p>0.05) .
Conclusions
Bone marrow hematopoietic cells of aplastic anemia had intrinsic nature of genetic instabilities which were not seemed to be increased by IST. It may be impossible of IST being crucial in aplastic anemia evolving into clonal hematologic disorders.
研究再生障碍性贫血(AA)患者骨髓造血细胞遗传不稳定性及强烈免疫抑制治疗(IST)对于从发生晚期克隆性血液学异常的影响。
【对象和方法】
采用彗星试验评价骨髓造血细胞遗传不稳定性,以彗星尾部DNA百分含量(TDNA%)、尾长(TL)、尾矩(TM)、Olive尾矩(OTM)和彗星细胞率(Comet%)作为分析参数,分别检测IST治疗前后从患者骨髓造血细胞,并与18例对照组结果比较。
【结果】
91例初治AA患者TDNA、TL、TM、OTM、Comet%分别为:5.02±3.95、11.25±7.19、1.70±2.02、1.50±1.38、16.84±13.65明显高于对照组(p＜0.05);58例重症AA(SAA)与33例非重症AA(NSAA)患者彗星试验各项参数比较均无差别(p＞0.05),二组患者分别与对照组比较,各彗星参数仍明显为高(p＜0.05)。53例SAA治疗后3月患者TDNA、TL、TM、OTM、Comet%分别为:4.43±3.64、10.37±7.49、1.37±1.61、1.29±1.37、20.17±21.17,30例SAA治疗后6月患者TDNA、TL、TM、OTM、Comet%分别为:3.71±3.32、10.04±7.18、1.15±1.80、1.12±1.29、18.47±19.02,与58例IST治疗前SAA患者彗星参数结果比较均无差异(p＞0.05)。18例SAA患者IST治疗前后均进行了彗星试验检测,彗星参数比较无统计学差异(p＞0.05)。
【结论】
AA患者骨髓造血细胞存在遗传不稳定,免疫抑制治疗本身并不增加SAA患者细胞遗传不稳定性。提示免疫抑制治疗与AA发生晚期克隆性血液学异常可能无关。
Objective
To investigate the genetic instabilities of bone marrow hematopoietic cells from patients with aplastic anemia and evaluate the impact of IST in aplastic anemia evolving into clonal hematologic disorders.
Methods
Comet assay were used to evaluate the genetic instabilities of hematopoietic cells and the percent of DNA in the comet tail (TDNA%), tail length(TL), tail moment(TM) olive tail moment(OTM) and the rate of comet cells (Comet%) were selected as analyzing parameters. Bone marrow hematopoietic cells from patients with aplastic anemia examined with comet assay before and after IST separately, and the results were compared with those from 18 controls.
Result
Comet parameters from 91 patients with aplastic anemia including TDNA, TL, TM, OTM and Comet% were 5.02±3.95, 11.25±7.19, 1.70±2.02, 1.50±1.38 and 16.84±13.65 separately, all of which were obviously superior to those from control group respectively (p<0.05). The results were remained notablely higher when separately compared the comet parameters of SAA and NSAA with those of control group (p<0.05) , and there were no differences of the examined comet parameters of 58 SAA from those of 33 NSAA patients (p>0.05) .The parameters TDNA, TL, TM, OTM and Comet% were 4.43±3.64, 10.37±7.49, 1.37±1.61, 1.29±1.37 and 20.17±21.17 respectively 3-months later after IST in 53 SAA patients and those were 3.71±3.32, 10.04±7.18,1.15±1.80, 1.12±1.29 and 18.47±19.02 respectively 6-months later after IST in 30 SAA patients , which had no difference from those of 58 patients before IST (p>0.05) .There was no statistical difference in comet parameters of 18 SAA patients before and after IST (p>0.05) .
Conclusions
Bone marrow hematopoietic cells of aplastic anemia had intrinsic nature of genetic instabilities which were not seemed to be increased by IST. It may be impossible of IST being crucial in aplastic anemia evolving into clonal hematologic disorders.
引文
1 Marsh JC,Ball SE,Darbyshire P,et al.Guidelines for the diagnosis and management of acquired aplastic anaemia.Br J Haematol.2003,123(5):782-801.
2 Dincol G,Aktan M,Diz-K(u｜¨)c(u｜¨)kkaya R,et al.Treatment of acquired severe aplastic anemia with antilymphocyte globulin,cyclosporin A,methyprednisolone,and granulocyte colony-stimulating factor.Am J Hematol.2007,82(9):783-786.
3 Schrezenmeier H,Hertenstein B,Wagner B,A pathogenetic link between aplastic anemia and paroxysmal nocturnal hemoglobinuria is suggested by a high frequency of aplastic anemia patients with a deficiency of phosphatidylinositol glycan anchored proteins.Exp Hematol.1995,23(1):81-87.
4 Socié G,Rosenfeld S,Frickhofen N,et,al.Late clonal diseases of treated aplastic anemia.Semin Hematol.2000,37(1):91-101.
5 Incidence of aplastic anemia:the relevance of diagnostic criteria.By the International Agranulocytosis and Aplastic Anemia Study.Blood.1987,70(6):1718-1721.
6 British Committee for Standards in Haematology(BCSH) General Haematology Task Force.Guidelines for the diagnosis and anagement of acquired aplastic anaemia.BrJ Haematol.2003,123:782-801.
7 Banath JP,Fushiki M,Olive PL.Rejoining of DNA single and double-strand breads in human white blood cells exposed to ionizing radiation.Int J Radiat Biol.1998,73(6):649-660.
8 陈玮琳.快速敏感检测DNA断裂的方法-彗星试验.国外医学卫生学分册.1998,25(2):101-104.
9 何玮,唐永煌,梁旭竞等.慢性乙型肝炎合并肝癌患者淋巴细胞的彗星试验分析.中国病理生理杂志.2005,21(4):820-821.
10 乔琰,鲁志松,姚汉超.彗星试验分析指标的进展和应用.卫生毒理学杂 志.2004,18(3):190-192.
11 Ueda H,Tashiro S,Kojima S,et al.Instability of chromosome 7 in colony forming cells of patients with aplastic anemia.Int J Hematol.1999,70(1):13-19.
12 杨利丽 李如江 耿秀芳.中药对再障染色体损伤的修复作用.中国优生与遗传杂志.2003,11(1):55-56.
13 魏旭东,黄明蓝,张伟等.再生障碍性贫血患者DNA损伤修复活性检测.河南医科大学学报.1994,29(3):258-260.
14 Socié G,Henry-Amar M,Bacigalupo A,et al.Malignant tumors occurring after treatment of aplastic anemia.European Bone Marrow Transplantation-Severe Aplastic Anaemia Working Party.N Engl J Med.1993,329(16):1152-1157.
15 Brodsky RA,Sensenbrenner LL,Jones RJ.Complete remission in severe aplastic anemia after high-dose cyclophosphamide without bone marrow transplantation.Blood.1996;87(2):491-494.
16 Brodsky RA,Jones RJ.High-Dose Cyclophosphamide for Treament of Aplastic Anemia.Ann Intern Med.2002,137(6):549-550.
17 De Planque MM,Bacigalupo A,W(u｜¨)rsch A,et al.Long-term follow-up of severe aplastic anaemia patients treated with antithymocyte globulin.Severe Aplastic Anaemia Working Party of the European Cooperative Group for Bone Marrow Transplantation(EBMT).Br J Haematol.1989,73(1):121-126.
[1]田云,卢向阳,易克,等.单细胞凝胶电泳技术[J].生命的化学.2004,24(1):77-78.
[2]陈玮琳.快速敏感检测ONA断裂的方法-彗星试验[J].国外医学卫生学分册.1998,25(2):101-104.
[3]Halliwell B,Aruoma OI.DNA damage by oxygen-derived species:its mechanism and measurement in mammalian systems[J].FEBS Lett.1991,281(1-2):9-19.
[4]Aslan M, Horoz M, Kocyigit A, et al. Lymphocyte DNA damage and oxidative stress in patients with iron deficiency anemia[J]. Mutat. Res. 2006, 601:144 -149.
[5]Seyed A. M-N, Nemati A, Tiraihi T. Evaluation of DNA damage in leukocytes of G6PD-deficient Iranian newborns (Mediterranean variant) using comet assay[J]. Mutat. Res. 2004, 568: 179-185.
[6]Maluf SW, Erdtmann B. Genomic instability in Down syndrome and Fanconi anemia assessed by micronucleus analysis and single-cell gel electrophoresis[J]. Cancer Genet Cytogenet. 2001, 124(1): 71 — 75.
[7]Djuzenova CS, Rothfuss A, Oppitz U, et al. Response to X-irradiation of Fanconi anemia homozygous and heterozygous cells assessed by the single-cell gel electrophoresis (comet) assay[J]. Lab Invest. 2001, 81 (2):185-192.
[8]Djuzenova CS, Flentje. M. Characterization of Fanconi anemia fibroblasts in terms of clonogenic survival and DNA damage assessed by the Comet assay[J].Med Sci Monit. 2002, 8(10): BR421-430.
[9]Bacova G,Gabelova A, Babusikova O, et al. The single cell gel electrophoresis:a potential tool for DNA analysis of the patients with hematological malignancies[J]. Neoplasma. 1998, 45(6): 349—359.
[10]Novotna B, Neuwirtova R, Blazkova V, et, al. The comet assay: principles of_the method and clinical utilization[J].Cas Lek Cesk. 2001, 140(24):761-766.
[11]Tuck A,Smith S, Larcom L, et al. Chronic lymphocytic leukemia lymphocytes lack the capacity to repair UVC-induced lesions[J]. Mutat Res. 2000,459:73-80.
[12]Czechowska A, Poplawski T, Drzewoski J, et al. Imatinib (STI571) induces DNA damage in BCR/ABL-expressing leukemic cells but not in normal lymphocytes[J].Chem Biol Interact. 2005, 152: 139-150.
[13]Gloc E,Warszawski M,Mlynarski W,et al.TEL/JAK2 tyrosine kinase inhibits DNA repair in the presence of amifostine[J].Acta Biochim Polon.2002,49(1):121-128.
[14]Blasiak J,Gloc E,Drzewoski J,et al.Free radical scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells[J].Mutat Res.2003,535(1):25-34.
[15]黄成垠,吴雅萍,邢宪英,等.彗星分析技术对难治性白血病的早期诊断价值[J].中国实验诊断学.2000,4(3):139-140.
[16]黄成垠,薛重重,郑晓丽,等.急性白血病化疗后“凋亡彗星”的检测及临床意义[J].临床血液学杂志.2000,13(5):204-206.
[17]Spanswick VJ,Craddock C,Sekhar M,et al.Repair of DNA interstand crosslinks as a mechanism of clinical resistance to melphalan in multiple myeloma[J].Blood.2002,100(1):224-229.
[18]Qing Chen,Van der Sluis PC,Boulware D,et al.The FA/BRCA pathway is involved in melphalan-induced DNA interstrand cross-link repair and accounts for melphalan resistance in multiple myeloma cells[J].Blood.2005,106(2):698-705.
[19]Yamauchi T,Kawai Y,Ueda T.Enhanced DNA excision repair in CCRF-CEM cells resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea,quantitated using the single cell gel electrophoresis(Comet) assay[J].Biochem Pharmacol.2003,66:939-946.
[20]Majsterek I,Blasiak J,Mlynarski W,et al.Does the bcr/abl-mediated increase in the efficacy of DNA repair play a role in the drug resistance of cancer cells?[J].Cell Biol Int.2002,26(4):363-370.
[21]Majsterek I,Sliwinski T,Poplawski T,et al.Imatinib mesylate(STI571)abrogates the resistance to doxorubicin in human K562 chronic myeloid leukemia cells by inhibition of BCR/ABL kinase-mediated DNA repair[J].Mutat Res.2006,603:74-82.
2 Dincol G,Aktan M,Diz-K(u｜¨)c(u｜¨)kkaya R,et al.Treatment of acquired severe aplastic anemia with antilymphocyte globulin,cyclosporin A,methyprednisolone,and granulocyte colony-stimulating factor.Am J Hematol.2007,82(9):783-786.
3 Schrezenmeier H,Hertenstein B,Wagner B,A pathogenetic link between aplastic anemia and paroxysmal nocturnal hemoglobinuria is suggested by a high frequency of aplastic anemia patients with a deficiency of phosphatidylinositol glycan anchored proteins.Exp Hematol.1995,23(1):81-87.
4 Socié G,Rosenfeld S,Frickhofen N,et,al.Late clonal diseases of treated aplastic anemia.Semin Hematol.2000,37(1):91-101.
5 Incidence of aplastic anemia:the relevance of diagnostic criteria.By the International Agranulocytosis and Aplastic Anemia Study.Blood.1987,70(6):1718-1721.
6 British Committee for Standards in Haematology(BCSH) General Haematology Task Force.Guidelines for the diagnosis and anagement of acquired aplastic anaemia.BrJ Haematol.2003,123:782-801.
7 Banath JP,Fushiki M,Olive PL.Rejoining of DNA single and double-strand breads in human white blood cells exposed to ionizing radiation.Int J Radiat Biol.1998,73(6):649-660.
8 陈玮琳.快速敏感检测DNA断裂的方法-彗星试验.国外医学卫生学分册.1998,25(2):101-104.
9 何玮,唐永煌,梁旭竞等.慢性乙型肝炎合并肝癌患者淋巴细胞的彗星试验分析.中国病理生理杂志.2005,21(4):820-821.
10 乔琰,鲁志松,姚汉超.彗星试验分析指标的进展和应用.卫生毒理学杂 志.2004,18(3):190-192.
11 Ueda H,Tashiro S,Kojima S,et al.Instability of chromosome 7 in colony forming cells of patients with aplastic anemia.Int J Hematol.1999,70(1):13-19.
12 杨利丽 李如江 耿秀芳.中药对再障染色体损伤的修复作用.中国优生与遗传杂志.2003,11(1):55-56.
13 魏旭东,黄明蓝,张伟等.再生障碍性贫血患者DNA损伤修复活性检测.河南医科大学学报.1994,29(3):258-260.
14 Socié G,Henry-Amar M,Bacigalupo A,et al.Malignant tumors occurring after treatment of aplastic anemia.European Bone Marrow Transplantation-Severe Aplastic Anaemia Working Party.N Engl J Med.1993,329(16):1152-1157.
15 Brodsky RA,Sensenbrenner LL,Jones RJ.Complete remission in severe aplastic anemia after high-dose cyclophosphamide without bone marrow transplantation.Blood.1996;87(2):491-494.
16 Brodsky RA,Jones RJ.High-Dose Cyclophosphamide for Treament of Aplastic Anemia.Ann Intern Med.2002,137(6):549-550.
17 De Planque MM,Bacigalupo A,W(u｜¨)rsch A,et al.Long-term follow-up of severe aplastic anaemia patients treated with antithymocyte globulin.Severe Aplastic Anaemia Working Party of the European Cooperative Group for Bone Marrow Transplantation(EBMT).Br J Haematol.1989,73(1):121-126.
[1]田云,卢向阳,易克,等.单细胞凝胶电泳技术[J].生命的化学.2004,24(1):77-78.
[2]陈玮琳.快速敏感检测ONA断裂的方法-彗星试验[J].国外医学卫生学分册.1998,25(2):101-104.
[3]Halliwell B,Aruoma OI.DNA damage by oxygen-derived species:its mechanism and measurement in mammalian systems[J].FEBS Lett.1991,281(1-2):9-19.
[4]Aslan M, Horoz M, Kocyigit A, et al. Lymphocyte DNA damage and oxidative stress in patients with iron deficiency anemia[J]. Mutat. Res. 2006, 601:144 -149.
[5]Seyed A. M-N, Nemati A, Tiraihi T. Evaluation of DNA damage in leukocytes of G6PD-deficient Iranian newborns (Mediterranean variant) using comet assay[J]. Mutat. Res. 2004, 568: 179-185.
[6]Maluf SW, Erdtmann B. Genomic instability in Down syndrome and Fanconi anemia assessed by micronucleus analysis and single-cell gel electrophoresis[J]. Cancer Genet Cytogenet. 2001, 124(1): 71 — 75.
[7]Djuzenova CS, Rothfuss A, Oppitz U, et al. Response to X-irradiation of Fanconi anemia homozygous and heterozygous cells assessed by the single-cell gel electrophoresis (comet) assay[J]. Lab Invest. 2001, 81 (2):185-192.
[8]Djuzenova CS, Flentje. M. Characterization of Fanconi anemia fibroblasts in terms of clonogenic survival and DNA damage assessed by the Comet assay[J].Med Sci Monit. 2002, 8(10): BR421-430.
[9]Bacova G,Gabelova A, Babusikova O, et al. The single cell gel electrophoresis:a potential tool for DNA analysis of the patients with hematological malignancies[J]. Neoplasma. 1998, 45(6): 349—359.
[10]Novotna B, Neuwirtova R, Blazkova V, et, al. The comet assay: principles of_the method and clinical utilization[J].Cas Lek Cesk. 2001, 140(24):761-766.
[11]Tuck A,Smith S, Larcom L, et al. Chronic lymphocytic leukemia lymphocytes lack the capacity to repair UVC-induced lesions[J]. Mutat Res. 2000,459:73-80.
[12]Czechowska A, Poplawski T, Drzewoski J, et al. Imatinib (STI571) induces DNA damage in BCR/ABL-expressing leukemic cells but not in normal lymphocytes[J].Chem Biol Interact. 2005, 152: 139-150.
[13]Gloc E,Warszawski M,Mlynarski W,et al.TEL/JAK2 tyrosine kinase inhibits DNA repair in the presence of amifostine[J].Acta Biochim Polon.2002,49(1):121-128.
[14]Blasiak J,Gloc E,Drzewoski J,et al.Free radical scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells[J].Mutat Res.2003,535(1):25-34.
[15]黄成垠,吴雅萍,邢宪英,等.彗星分析技术对难治性白血病的早期诊断价值[J].中国实验诊断学.2000,4(3):139-140.
[16]黄成垠,薛重重,郑晓丽,等.急性白血病化疗后“凋亡彗星”的检测及临床意义[J].临床血液学杂志.2000,13(5):204-206.
[17]Spanswick VJ,Craddock C,Sekhar M,et al.Repair of DNA interstand crosslinks as a mechanism of clinical resistance to melphalan in multiple myeloma[J].Blood.2002,100(1):224-229.
[18]Qing Chen,Van der Sluis PC,Boulware D,et al.The FA/BRCA pathway is involved in melphalan-induced DNA interstrand cross-link repair and accounts for melphalan resistance in multiple myeloma cells[J].Blood.2005,106(2):698-705.
[19]Yamauchi T,Kawai Y,Ueda T.Enhanced DNA excision repair in CCRF-CEM cells resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea,quantitated using the single cell gel electrophoresis(Comet) assay[J].Biochem Pharmacol.2003,66:939-946.
[20]Majsterek I,Blasiak J,Mlynarski W,et al.Does the bcr/abl-mediated increase in the efficacy of DNA repair play a role in the drug resistance of cancer cells?[J].Cell Biol Int.2002,26(4):363-370.
[21]Majsterek I,Sliwinski T,Poplawski T,et al.Imatinib mesylate(STI571)abrogates the resistance to doxorubicin in human K562 chronic myeloid leukemia cells by inhibition of BCR/ABL kinase-mediated DNA repair[J].Mutat Res.2006,603:74-82.