细胞保存液提高中枢神经系统白血病检出率的实验研究
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摘要
目的:探讨细胞保存液能否延长白血病细胞的存活时间,提高细胞存活率,从而提高中枢神经系统白血病的检出率。方法:在有细胞保存液或无细胞保存液的脑脊液上清中,分别加入Kasumi白血病细胞株,在不同时间点比较2组细胞的存活情况和生物学特性改变,研究方法包括细胞沉渣甩片镜检、细胞计数法、台盼蓝拒染法、CCK-8法和流式细胞术等,并以荧光定量RT-PCR检测肿瘤融合基因AML-ETO的表达。结果:在不同的时间点(8和24 h),已添加细胞保存液的脑脊液中细胞的存活状态和分子生物学特征,明显优于普通脑脊液的保存效果。结论:细胞保存液能有效提高白血病细胞的存活时间、存活率,从而提高中枢神经系统白血病检出率。
Objective: To investigate whether cell preservation solution can prolong the survival time of leukemia cells and increase the survival rate, so as to improve the detection rate of central nervous system leukemia. Methods: Kasumi cells were added into cerebrospinal fluid(CSF) supernatant with or without cell preservation solution to compare cell viability and biological characteristics at different time point. Wright Giemsa staining was used to compare cell morphology; cell counting, CCK-8 method, and trypan blue staining were used to compare the cell number, and flow cytometry was used to compare the cell viability. The expression of AML-ETO tumor fusion gene was detected by fluorescence quantitative RT-PCR. Results: At different time points(8 h and 24 h), the survival, molecular biological characteristics and RT-PCR result of the cells in CSF with cell preservation solution were significantly better than those in normal cerebrospinal fluid. Conclusion: Cell preservation solution can effectively improve the survival time and survival rate of leukemic cells, thereby increase the detection rate of CNS leukemia.
Objective: To investigate whether cell preservation solution can prolong the survival time of leukemia cells and increase the survival rate, so as to improve the detection rate of central nervous system leukemia. Methods: Kasumi cells were added into cerebrospinal fluid(CSF) supernatant with or without cell preservation solution to compare cell viability and biological characteristics at different time point. Wright Giemsa staining was used to compare cell morphology; cell counting, CCK-8 method, and trypan blue staining were used to compare the cell number, and flow cytometry was used to compare the cell viability. The expression of AML-ETO tumor fusion gene was detected by fluorescence quantitative RT-PCR. Results: At different time points(8 h and 24 h), the survival, molecular biological characteristics and RT-PCR result of the cells in CSF with cell preservation solution were significantly better than those in normal cerebrospinal fluid. Conclusion: Cell preservation solution can effectively improve the survival time and survival rate of leukemic cells, thereby increase the detection rate of CNS leukemia.
引文
1 de Graaf MT,van den Broek PD,Kraan J,et al.Addition of serumcontaining medium to cerebrospinal fluid prevents cellular loss over time.J Neurol,2011;258(8):1507-1512.
2 Steele RW,Marmer DJ,O'Brien MD,et al.Leukocyte survival in cerebrospinal fluid.J Clin Microbiol,1986;23(5):965-966.
3 Chow G,Schmidley JW.Lysis of erythrocytes and leukocytes in traumatic lumbar punctures.Arch Neurol,1984;41(10):1084-1085.
4 Robier C,Quehenberger F,Neubauer M,et al.The cytospin technique improves the detection of calcium pyrophosphate crystals in synovial fluid samples with a low leukocyte count.Rheumatol Int,2014;34(6):773-776.
5 Strober W.Trypan blue exclusion test of cell viability.Curr Protoc Immunol,2015;111:A3.B.1-3.
6 Dux R,Kindler-Rohrborn A,Annas M,et al.A standardized protocol for flow cytometric analysis of cells isolated from cerebrospinal fluid.J Neurol Sci,1994;121(1):74-78.
7杨文钰,王慧君,陈玉梅等.脑脊液流式细胞学检测技术在急性淋巴细胞白血病儿童并发中枢神经系统白血病中的诊断价值.中国实验血液学杂志,2012;20(1):38-42.
8邵化敏,唐宇宏,姜鹏君等.葛根总黄酮对不同急性髓系白血病细胞株增殖抑制的影响.中国实验血液学杂志,2010;18(2):296-299.
9 Li Y,Wang Y,Zhou Y,et al.Cooperative effect of chidamide and chemotherapeutic drugs induce apoptosis by DNA damage accumulation and repair defects in acute myeloid leukemia stem and progenitor cells.Clin Epigenetics,2017;9:83.
10 Beillard E,Pallisgaard N,van der Velden VH,et al.Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using'real-time'quantitative reversetranscriptase polymerase chain reaction(RQ-PCR)-a Europe against cancer program.Leukemia,2003;17(12):2474-2486.
11 Gabert J,Beillard E,van der Velden VH,et al.Standardization and quality control studies of'real-time'quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia-a Europe Against Cancer program.Leukemia,2003;17(12):2318-2357.
12 Zhang L,Zhou Y,Chen K,et al.The pan-Bcl2 inhibitor AT101activates the intrinsic apoptotic pathway and causes DNA damage in acute myeloid leukemia stem-like cells.Target Oncol,2017;12(5):677-687.
13 Banker DE,Radich J,Becker A,et al.The t(8;21)translocation is not consistently associated with high Bcl-2 expression in de novo acute myeloid leukemias of adults.Clin Cancer Res,1998;4(12):3051-3062.
14常城,郭搏,张琳等.全反式维甲酸联合猪胆酸钠对K562和Kasumi-1细胞系生物活性抑制作用.中国实验血液学杂志,2013;21(4):879-885.
15 Veerman AJ,Huismans L,van Zantwijk I.Storage of cerebrospinal fluid samples at room temperature.Acta Cytol,1985;29(2):188-189.
16 Bienzle D,McDonnell JJ,Stanton JB.Analysis of cerebrospinal fluid from dogs and cats after 24 and 48 hours of storage.J Am Vet Med Assoc,2000;216(11):1761-1764.
17 Bellino C,Miniscalco B,Bertone I,et al.Analysis of cerebrospinal fluid from cattle with central nervous system disorders after storage for 24 hours with autologous serum.BMC Vet Res,2015;11:201.
2 Steele RW,Marmer DJ,O'Brien MD,et al.Leukocyte survival in cerebrospinal fluid.J Clin Microbiol,1986;23(5):965-966.
3 Chow G,Schmidley JW.Lysis of erythrocytes and leukocytes in traumatic lumbar punctures.Arch Neurol,1984;41(10):1084-1085.
4 Robier C,Quehenberger F,Neubauer M,et al.The cytospin technique improves the detection of calcium pyrophosphate crystals in synovial fluid samples with a low leukocyte count.Rheumatol Int,2014;34(6):773-776.
5 Strober W.Trypan blue exclusion test of cell viability.Curr Protoc Immunol,2015;111:A3.B.1-3.
6 Dux R,Kindler-Rohrborn A,Annas M,et al.A standardized protocol for flow cytometric analysis of cells isolated from cerebrospinal fluid.J Neurol Sci,1994;121(1):74-78.
7杨文钰,王慧君,陈玉梅等.脑脊液流式细胞学检测技术在急性淋巴细胞白血病儿童并发中枢神经系统白血病中的诊断价值.中国实验血液学杂志,2012;20(1):38-42.
8邵化敏,唐宇宏,姜鹏君等.葛根总黄酮对不同急性髓系白血病细胞株增殖抑制的影响.中国实验血液学杂志,2010;18(2):296-299.
9 Li Y,Wang Y,Zhou Y,et al.Cooperative effect of chidamide and chemotherapeutic drugs induce apoptosis by DNA damage accumulation and repair defects in acute myeloid leukemia stem and progenitor cells.Clin Epigenetics,2017;9:83.
10 Beillard E,Pallisgaard N,van der Velden VH,et al.Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using'real-time'quantitative reversetranscriptase polymerase chain reaction(RQ-PCR)-a Europe against cancer program.Leukemia,2003;17(12):2474-2486.
11 Gabert J,Beillard E,van der Velden VH,et al.Standardization and quality control studies of'real-time'quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia-a Europe Against Cancer program.Leukemia,2003;17(12):2318-2357.
12 Zhang L,Zhou Y,Chen K,et al.The pan-Bcl2 inhibitor AT101activates the intrinsic apoptotic pathway and causes DNA damage in acute myeloid leukemia stem-like cells.Target Oncol,2017;12(5):677-687.
13 Banker DE,Radich J,Becker A,et al.The t(8;21)translocation is not consistently associated with high Bcl-2 expression in de novo acute myeloid leukemias of adults.Clin Cancer Res,1998;4(12):3051-3062.
14常城,郭搏,张琳等.全反式维甲酸联合猪胆酸钠对K562和Kasumi-1细胞系生物活性抑制作用.中国实验血液学杂志,2013;21(4):879-885.
15 Veerman AJ,Huismans L,van Zantwijk I.Storage of cerebrospinal fluid samples at room temperature.Acta Cytol,1985;29(2):188-189.
16 Bienzle D,McDonnell JJ,Stanton JB.Analysis of cerebrospinal fluid from dogs and cats after 24 and 48 hours of storage.J Am Vet Med Assoc,2000;216(11):1761-1764.
17 Bellino C,Miniscalco B,Bertone I,et al.Analysis of cerebrospinal fluid from cattle with central nervous system disorders after storage for 24 hours with autologous serum.BMC Vet Res,2015;11:201.