WO2019223406A1 - 利用红细胞dna损伤信号预测血液病转归的方法及其应用 - Google Patents

利用红细胞dna损伤信号预测血液病转归的方法及其应用 Download PDF

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WO2019223406A1
WO2019223406A1 PCT/CN2019/078279 CN2019078279W WO2019223406A1 WO 2019223406 A1 WO2019223406 A1 WO 2019223406A1 CN 2019078279 W CN2019078279 W CN 2019078279W WO 2019223406 A1 WO2019223406 A1 WO 2019223406A1
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dna damage
outcome
red blood
experimental group
blood
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French (fr)
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高晓飞
张清
葛建萍
楼赛
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西湖大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/011—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells with lysing, e.g. of erythrocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

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  • the invention relates to the field of biomedicine, in particular to a method for predicting the outcome of hematological diseases by using a red blood cell DNA damage signal and its application, and has extensive application in clinical medicine.
  • Hematological diseases also called hematopoietic system diseases, refer to diseases that originate in the hematopoietic system, or diseases that affect the hematopoietic system with abnormal changes in blood.
  • the hematopoietic system includes blood, bone marrow mononuclear cells, macrophage systems, and Lymphatic tissue.
  • all diseases that involve the pathogenesis and physiology of the hematopoietic system and are mainly manifested belong to the category of hematological diseases. In the clinic, hematological diseases can be divided into red blood cell disease, white blood cell disease, bleeding and thrombotic diseases.
  • the common diseases are leukemia, aplastic anemia, myelodysplastic syndrome, thrombocytopenia, and multiple myeloma. , Lymphoma, and hemophilia.
  • many of the predisposing factors for hematological diseases are refractory diseases.
  • Acute leukemia is a malignant clonal disease of hematopoietic stem cells.
  • abnormal blast cells and immature cells (leukemia cells) in the bone marrow multiply, accumulate in the bone marrow and inhibit normal blood formation.
  • Infiltration of extramedullary organs such as liver, spleen, and lymph nodes.
  • AL can generally be divided into two categories: acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • the incidence of AML in China is about 1.62 / 100,000, while ALL is about 0.69 / 100,000. More common in adults with AML and more common in children with ALL. If acute leukemia is not treated, the average survival time is only about 3 months, and the short-term patient will die even a few days after diagnosis.
  • the current diagnosis of acute leukemia is to eliminate as many leukemia cell populations as possible and control the massive proliferation of leukemia cells, and relieve various clinical manifestations caused by leukemia infiltration in order to achieve complete remission.
  • the diagnosis and treatment methods are also increasingly developed, and the diagnosis and risk stratification of acute leukemia are mainly determined based on the results of cell morphology, immunology, cytogenetics, and molecular typing.
  • due to the high cost of testing many patients only Performing a simple morphological examination is detrimental to patient diagnosis and improvement of long-term prognosis.
  • the purpose of the present invention is to provide a method for predicting the outcome of hematological diseases by using DNA damage signals of red blood cells and to predict the outcome of hematological diseases by quickly judging the DNA damage signals in early damaged red blood cells. And the advantage of high sensitivity.
  • the purpose of the present invention is to provide a method for predicting the outcome of hematological diseases by using a red blood cell DNA damage signal, and to predict the outcome of a hematological disease by screening specific red blood cells and detecting specific red blood cell damage signals.
  • the purpose of the present invention is to provide a method for predicting the outcome of hematological diseases by using a red blood cell DNA damage signal, and a blood sample taken from a patient's peripheral blood. Efficiency while reducing patient suffering.
  • the purpose of the present invention is to provide a method for predicting the outcome of hematological diseases by using a red blood cell DNA damage signal, and the method is particularly suitable for predicting the outcome of acute leukemia, and has wide clinical application.
  • the present invention provides a method for predicting the outcome of hematological diseases by using a red blood cell DNA damage signal, including the following steps:
  • S1 Prepare blood sample: Obtain an anticoagulant sample from a patient with a blood disease, and fix the anticoagulant sample in a 1% -4% paraformaldehyde solution at room temperature in a 1% volume ratio, and fix 10-20 After a few minutes, a fixative containing a blood sample was formed;
  • S3 staining the second experimental group: adding the second experimental group to Human FcR binding, incubating at 4 degrees Celsius for 15-20 minutes, and then staining the second experimental group with a dyeing buffer at room temperature and dark environment to obtain The third experimental group, wherein the staining buffer is a combination of a RNase-containing staining reagent and a nucleic acid stain, and staining is performed for 15-30 minutes, and the RNase-containing staining reagent is a CD235a labeled with a first marker and a second CD47 labeled with a marker, the nucleic acid stain is 1 ⁇ PBS containing Hoechst33342 and Thiazole orange, wherein the first marker is different from the second marker;
  • the staining buffer is a combination of a RNase-containing staining reagent and a nucleic acid stain, and staining is performed for 15-30 minutes
  • the RNase-containing staining reagent is a CD235a labeled with a
  • the step S6 further includes:
  • step S62 selecting a cell population with a CD47 expression level of TOP5% based on step S62;
  • step S63 On the basis of step S62, select a Hoechst33342 and Thiazole orange double positive cell population, record the percentage of the population of Thiazole orange and Hoechst33342 double positive in the cell population with CD47 expression of TOP5%, and obtain the damage signal.
  • 0.1-0.15 ⁇ g of the first marker-labeled CD235a, 0.03-0.1 ⁇ g of the second marker-labeled CD47, 0.014-0.08 nmol Hoechst33342, and 0.04-0.2 ⁇ g are used for 1 ul of blood samples.
  • step S7 is included: quickly and effectively comparing changes in the proportion of the patient's own DNA damage signal.
  • step S3 the first marker is APC, and the second marker is PE.
  • the number of records of the flow cytometer is 100,000, the recording time is 120s, and the sample flow rate is 30 microliters / minute.
  • the blood sample is bone marrow or peripheral blood
  • the volume of the blood sample is 1-3 ul
  • the low temperature environment is 4 ° C.
  • the centrifugation time is 4-6 minutes.
  • the present invention provides an application of using red blood cell DNA damage signals to predict the outcome of blood diseases, characterized in that the red blood cell DNA damage signals according to any one of claims 1 to 5 are used to predict blood disease outcomes.
  • the method was applied to predict the outcome of acute leukemia.
  • FIGS. 1 to 4 are schematic diagrams of an experimental process of a method for predicting a blood disease outcome by using a red blood cell DNA damage signal according to the present invention.
  • FIG. 5 to FIG. 10 are schematic diagrams of flow cytometry analysis diagrams generated by the method for predicting a blood disease outcome using the red blood cell DNA damage signal according to the present invention.
  • FIG. 11 is a graph of experimental data in an application of the method for predicting a blood disease outcome using the red blood cell DNA damage signal according to the present invention.
  • FIGS. 5 to 10 the reading order in FIGS. 5 to 10 is from the upper left figure to the lower right figure in a “Z” manner.
  • the term “a” should be understood as “at least one” or “one or more”, that is, in one embodiment, the number of one element can be one, and in other embodiments, the number of The number may be plural, and the term “a” cannot be understood as a limitation on the number.
  • Outcome refers to the metastasis and development of the disease.
  • development of the disease can be divided into four phases: incubation period, prodromal period, obvious symptoms period, and outcome period, and the outcome of the disease can be divided into healing, death, lingering, sequelae, and recurrence.
  • there are three possible outcomes of the disease complete recovery, incomplete recovery and death.
  • Red blood cell DNA damage signal Mature red blood cells have no nucleus. If the erythrocytes mature earlier, the chromatid or the centromere fragment of the chromosome, or the entire chromosome is lost due to spindle damage. DNA nucleic acid fragments are still left in the cytoplasm, that is, these residual DNA signals will form one or several regular secondary nuclei in the mature erythrocyte cytoplasm. These secondary nuclei are the residual DNA damage signals.
  • CD235a Blood group glycoprotein A, is a single transmembrane glycoprotein, expressed on mature red blood cells and red blood cell precursors, is a special marker protein on the surface of red blood cells.
  • CD47 also known as integrin associated protein (IAP), is a member of the immunoglobulin superfamily. CD47 is widely expressed on the surface of cells and can interact with signal regulatory protein alpha (SIRP ⁇ ) and platelet reactive protein. (thrombospondin-1, TSP1) and integrins interact to mediate a series of responses such as apoptosis, proliferation, and immunity. Studies have shown that leukemia cells produce more CD47 protein than CD47, and CD47 Protein is involved in the life cycle of red blood cells.
  • SIRP ⁇ signal regulatory protein alpha
  • TSP1 platelet reactive protein
  • integrins interact to mediate a series of responses such as apoptosis, proliferation, and immunity. Studies have shown that leukemia cells produce more CD47 protein than CD47, and CD47 Protein is involved in the life cycle of red blood cells.
  • PBS Abbreviation for phosphate buffered saline (phosphate buffered saline), which is generally used as a solvent and functions as a dissolution protection reagent.
  • APC Allophycocyanin, a phycobiliprotein (pigment) protein isolated from blue-green algae. Like other phycobiliproteins, APC is fluorescent, with very high light absorption and high quantum efficiency.
  • PE Polyethylene
  • PE is a thermoplastic resin made by the polymerization of ethylene.
  • Hoechs33342 can be excited at about 350nm UV light, all emit blue indigo fluorescence at 461nm, the maximum emission of unbound dye is between 510 and 540nm, can be excited by xenon-argon lamp or mercury-argon lamp or ultraviolet laser The Stokes shift between the excitation light and the emitted light is huge, so it can be used for multi-dye multi-color fluorescent staining.
  • Thiazole orange is a chemical substance with the chemical formula C26H24N2O3S2.
  • Human FcR Binding Also known as Human Fc Block, it can effectively prevent non-specific binding of Fc receptors on the cell surface and reduce background interference to obtain more accurate results.
  • red blood cells Since mature red blood cells are non-nucleated, this means that the presence of nucleated red blood cells in the peripheral blood of adults is a pathological phenomenon. If a large number of young or young red blood cells appear in the adult peripheral blood, it means that the red blood cell line in the bone marrow has a significant proliferation.
  • red blood cells in the bone marrow may be proliferative anemia, and if a large number of primary red blood cells and early red blood cells appear in adult peripheral blood, it means that immature red blood cells in the bone marrow are abnormally proliferated and released into the blood, it may be red blood disease or red leukemia If red blood cells can be seen at all stages of development in adult peripheral blood, and immature granulocytes and megakaryocytes can be seen, it may be extramedullary hematopoietic. In particular, for malignant diseases of the hematopoietic system, the bone marrow is filled with a large number of leukemia cells to release early red blood cells, and the nucleated red blood cells are mainly young and young.
  • the method provided by the present invention for predicting the outcome of hematological diseases by using DNA damage signals of red blood cells selects specific red blood cells by flow cytometry, and uses a nucleic acid dye to analyze the nucleic acid signals in the specific red blood cells, that is, red blood cells. Based on the rapid and effective comparison of changes in the proportion of patients' own DNA damage signals, an efficient, rapid and highly sensitive RBCs damage detection system is established to predict the outcome of patients with hematological diseases.
  • the method for predicting blood outcome using red blood cell DNA damage signals is hereinafter referred to as the outcome prediction method, and includes the following steps:
  • S1 Prepare blood samples: Obtain anticoagulant samples from patients with hematological diseases, and fix the anticoagulant samples in a 1% -4% paraformaldehyde solution at room temperature in a 1% volume ratio, and fix 10-20 After a few minutes, a fixed solution containing blood samples was formed and stored in a low temperature environment for a long time.
  • S3 staining the second experimental group: adding the second experimental group to Human FcR binding, incubating at 4 degrees Celsius for 15-20 minutes, and then staining the second experimental group with a dyeing buffer at room temperature and dark environment to obtain The third experimental group, wherein the staining buffer is a combination of a RNase-containing staining reagent and a nucleic acid stain, and staining is performed for 15-30 minutes, and the RNase-containing staining reagent is a CD235a labeled with a first marker and a second CD47 labeled with a marker, the nucleic acid stain was 1 ⁇ PBS containing Hoechst33342 and Thiazoleorange.
  • the staining buffer is a combination of a RNase-containing staining reagent and a nucleic acid stain, and staining is performed for 15-30 minutes
  • the RNase-containing staining reagent is a CD235a labeled with a first marker and a second CD47 labeled
  • S4 Resuspend the second control group: resuspend the second control group with 1 * PBS to obtain a third control group.
  • S5 detecting the third experimental group and the third control group by a flow method: placing the third experimental group and the third control group in a flow cytometer for detection, and obtaining an analysis result.
  • step S62 selecting a cell population with a CD47 expression level of TOP5% based on step S61;
  • step S63 On the basis of step S62, select a Hoechst33342 and Thiazole orange double-positive cell population, record the percentage of the Hoechst33342 and Thiazole orange double-yang population in the cell population with a CD47 expression of TOP5%, and obtain an injury signal.
  • CD235 was selected because it is a unique marker on the surface of red blood cells, so that the red blood cells in the bleeding sample were screened, and then the red blood cells were selected to have a CD47 expression level of TOP 5% Population, that is, the fresher red blood cells in the blood were selected.
  • the anticoagulant sample is bone marrow or peripheral blood
  • the low-temperature environment is preferably 4 ° C. This temperature has the best preservation effect.
  • the present invention only needs to obtain a small amount of A blood sample is sufficient, which greatly improves the testing efficiency and reduces the patient's pain. Generally, only 1-3ul micro blood samples are required.
  • step S2 the variable factors for preparing the control group and the experimental group should be controlled to be consistent, that is, the control group and the experimental group should be prepared for experiments in the same environment to This method ensures the interference of unnecessary interference factors.
  • step S3 Human FcRBinding is added according to the standard of 2 ⁇ l-15 ⁇ L / test.
  • the first marker in the staining buffer is different from the second marker to distinguish the CD235a and
  • the CD47 is corresponding to different wavelength ranges when detecting different markers.
  • APC-labeled CD235a and PE-labeled CD47 are used. The types of the markers are different. Affected.
  • a CytoFLEX LX flow cytometer whose type of the flow cytometer is Beckman Coulter is used.
  • the related parameters of the flow analyzer are as follows:
  • Acquisition parameters 100,000 records, recording time 120s, sample flow rate 30 microliters / minute;
  • CD235a-APC is Red 660/10
  • CD47-PE is Yellow 585/42
  • Hoechst33342 is UV 450/45
  • Thiazole orange is Blue 525/40.
  • step S5 the result obtained by the flow cytometer analysis is shown in FIG. 6.
  • CD235a labeled with the first marker can be clearly seen, and the CD235a-positive cell population is obtained.
  • the expression level of CD47 was in a TOP 5% cell population, and in this CD47 expression level was in a TOP 5% cell population, a Hoechst33342 and Thiazole orange range-positive cell population was obtained, and finally a DNA damage signal was calculated.
  • the medical staff can quickly and effectively compare the change in the proportion of the patient's own DNA damage signal, and then predict the outcome of the patient's blood disease.
  • experimental data will be used as experimental support for the outcome prediction method below, but those familiar with the technology should understand that the experimental data exists only as a specific embodiment, and the experimental data can be analogized in a proportional manner. .
  • a 10 ul patient anticoagulant sample was selected and fixed in a 1% to 4% paraformaldehyde solution at a volume ratio of 1%, that is, the volume of the paraformaldehyde solution was 1000ul and fixed at room temperature for 10 It can be stored at 4 °C for a long time after -20min.
  • the anticoagulant sample is added to the paraformaldehyde solution in a volume ratio of 1: 100 to obtain a fixed solution containing a blood sample. Take out a fixed solution containing a certain volume of blood sample and perform subsequent staining and labeling operations.
  • the volume ratio of the blood sample and the staining buffer is controlled to 1: 200, and the staining buffer corresponding to 1 ul of blood sample contains 0.1-0.15. ⁇ g APC-labeled CD235a, 0.03-0.1 ⁇ g PE-labeled CD47, 0.014-0.08 nmol Hoechst33342, and 0.04-0.2 ⁇ g Thiazole orange 1 ⁇ PBS.
  • the outcome prediction method provided by the present invention uses only 1 ul of a micro blood sample for each measurement, and the antibody incubation time is 20-30 minutes, and the detection time of a single sample is about 1 minute.
  • a blood sample requires 5 ml of blood sample, and its detection speed is about 1 hour.
  • the outcome prediction method has a small amount of detection, which has high detection effect, good specificity, and high sensitivity. China has very important practical significance.
  • the method for predicting blood outcome using the red blood cell DNA damage signal provided by the present invention is particularly suitable for predicting the outcome of acute leukemia.
  • Specific clinical data has been provided as support, which is explained as follows:
  • the above table is a graph of the data obtained by treating patients with ALL with different treatment options. As shown in the figure, on November 24, 2017, the ALL patient was treated with dasatinib, and the ALL patient was diagnosed as a central relapse ALL. (Ph positive), the peripheral blood of the patient was extracted and tested by the outcome prediction method, and the red blood cell DNA damage of the patient was as high as 90.03%.
  • the present invention is not limited to the above-mentioned preferred embodiments.
  • anyone can derive other various forms of products under the inspiration of the present invention, but regardless of any changes in its shape or structure, all of them have the same or similar The similar technical solutions all fall within the protection scope of the present invention.

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Abstract

一种利用红细胞DNA损伤信号预测血液病转归的方法，包括以下步骤：S1：准备血样本；S2：准备对照组和实验组；S3：实验组标志物及核酸染色；S4：重悬对照组；S5：流式方法检测；S6：获取结果；以及S7：分析结果。该方法通过快速判断早期受损红细胞中的DNA损伤信号的方式预测血液病的转归情况，具有特异性强以及灵敏度高的优势。

Description

利用红细胞DNA损伤信号预测血液病转归的方法及其应用 技术领域
本发明涉及生物医药领域，特别涉及一种利用红细胞DNA损伤信号预测血液病转归的方法及其应用，在临床医学上有着广泛的应用。
背景技术
血液病，又可称之为造血系统疾病，指的是原发于造血系统的疾病，或影响造血系统伴发血液异常改变的疾病，其中造血系统包括血液、骨髓单核、巨噬细胞系统和淋巴组织。概言之，凡是涉及到造血系统病理、生理，并以其为主要表现的疾病，都属于血液病的范畴。在临床上，血液病又可分为红细胞疾病、白细胞疾病、出血和血栓性疾病，具体来讲，常见的有白血病、再生障碍性贫血、骨髓增生异常综合症、血小板减少症、多发性骨髓瘤、淋巴瘤以及血友病等。然而，血液系统疾病的诱发因素极多又多半是难治性疾病。
以急性白血病为例，急性白血病(acute leukemia，AL)是造血干细胞的恶性克隆性疾病，发病时骨髓中异常的原始细胞及幼稚细胞(白血病细胞)大量增殖，蓄积于骨髓并抑制正常造血，广泛浸润肝、脾、淋巴结等髓外脏器。表现为贫血、出血、感染和浸润等征象。根据受累的细胞类型，AL通常可以分为急性淋巴细胞白血病(acute lymphoblastic leukemia，ALL)和急性髓细胞白血病(acute myeloid leukemia，AML)两大类。我国AML的发病率约为1.62/10万，而ALL则约为0.69/10万。成人以AML多见，儿童以ALL多见。急性白血病若不经特殊治疗，平均生存期仅3个月左右，短者甚至在诊断数天后即死亡。
目前针对急性白血病的诊断就是尽可能多地消灭白血病细胞群体和控制白血病细胞的大量增生，解除因白血病浸润而引起的各种临床表现，以期获得完全缓解，随着医疗水平的进步，急性白血病的诊断治疗方法也日益发展完善，而针对急性白血病的诊断和危险分层主要依据细胞形态学、免疫学、细胞遗传学和分子学分型的检测结果来确定，但由于检测费用昂贵，不少患者只进行简单的形态学检查，这对于患者诊疗和远期预后的改善是不利的。当然目前市面上还有通过酶反应速率法、反射免疫法检测乳酸脱氢酶(lactate dehydrogenase,LDH)及血清铁蛋白(serum ferritin,SF)水平预测急性白血病患者病情发展及预后的方法以及通过RT-PCR方法监测血细胞磷酸酶(SHP-1)基因和原癌基因c-kit的表达来作为白血病转归的指标，但是这些转化预测方法的特异性差，灵敏度低，步骤繁琐，检测周期长，依旧无法很好地被适用于临床的治疗和诊断。
发明内容
本发明的目的在于提供一利用红细胞DNA损伤信号预测血液病转归的方法及其应用，通过快速判断早期受损的红细胞中的DNA损伤信号的方式预测血液病的转归情况，具有特异性强以及灵敏度高的优势。
本发明的目的在于提供一利用红细胞DNA损伤信号预测血液病转归的方法及其应用，通过筛选特定红细胞并进行特定红细胞损伤信号检测的方式，预测血液病的转归，具有步骤方法简单，检测周期短的优势。
本发明的目的在于提供一利用红细胞DNA损伤信号预测血液病转归的方法及其应用，检测血样本取自患者的外周血，检测过程对患者伤害小，且可实现微量检测，以在提高检测效率的同时减少病患的痛苦。
本发明的目的在于提供一利用红细胞DNA损伤信号预测血液病转归的方法及其应用，该方法特别适用于急性白血病的转归预测，在临床上有着广泛的应用。
为了实现以上任一发明目的，本发明提供一利用红细胞DNA损伤信号预测血液病转归的方法，包括以下步骤:
S1:准备血样本：获取血液疾病患者的抗凝血样本，按照1％体积比将所述抗凝血样本在室温环境下固定于1％-4％的多聚甲醛溶液当中，固定10-20分钟后，形成含有血样本的固定液；
S2:准备对照组和实验组：分别取用相同体积的固定液设置为第一对照组和第一实验组，所述第一对照组和所述第一实验组均在室温环境下300-400g离心,弃上清液，随后用1*PBS重悬清洗2-3次，得到处理过后的第二对照组和处理过后的第二实验组；
S3：染色第二实验组：取所述第二实验组加入Human FcR Binding，在4摄氏度下孵育15-20分钟，随后使用染色缓冲剂在室温避光环境下染色所述第二实验组，得到第三实验组，其中所述染色缓冲剂为含RNase的染色试剂以及核酸染色剂的组合，染色15-30分钟，其中所述含RNase的染色试剂为含第一标记物标记的CD235a以及第二标记物标记的CD47，所述核酸染色剂为含Hoechst33342和Thiazole orange的1×PBS，其中所述第一标记物不同于所述第二标记物；
S4：重悬第二对照组：使用1*PBS重悬所述第二对照组，得到第三对照组；
S5：流式方法检测所述第三实验组以及所述第三对照组：将所述第三实验组和所述第三对照组放置于流式细胞仪中检测，得到分析结果；以及
S6：分析所述分析结果。
在一些实施例中，所述步骤S6进一步包括；
S61：选择含有所述CD235a的阳性细胞群体；
S62：在步骤S62的基础上选择CD47表达量处于TOP5％细胞群体；以及
S63：在步骤S62的基础上选择Hoechst33342和Thiazole orange双阳性的细胞群体，记录CD47表达量为TOP5％的细胞群体中Thiazole orange和Hoechst33342双阳的群体百分比，得到损伤信号。
在一些实施例中，对应1ul的血样本使用0.1-0.15μg所述第一标记物标记的CD235a、0.03-0.1μg所述第二标记物标记的CD47、0.014-0.08nmol Hoechst33342和0.04-0.2μg Thiazole orange的1×PBS。
在一些实施例中，包括步骤S7:快速有效的对比患者自身DNA损伤信号比例的变化。
在一些实施例中，所述步骤S3中，所述第一标记物为APC，所述第二标记物为PE。
在一些实施例中，所述流式细胞仪的记录个数为100000个、记录时间120s、样本流速30微升/分钟。
在一些实施例中，所述血样本为骨髓或者外周血，所述血样本的体积为1-3ul，所述低温环境为4℃。
在一些实施例中，所述步骤S2和所述步骤4中，离心时间为4-6分钟。
根据本发明的另一方面，本发明提供一利用红细胞DNA损伤信号预测血液病转归的应用，其特征在于，采用权利要求1到5任一所述的用红细胞DNA损伤信号预测血液病转归的方法，被应用于预测急性白血病的转归情况。
附图说明
图1到图4是根据本发明的利用红细胞DNA损伤信号预测血液病转归的方法的实验过程示意图。
图5到图10是根据本发明的所述利用红细胞DNA损伤信号预测血液病转归的方法产生的流式分析图的示意图。
图11是根据本发明的所述利用红细胞DNA损伤信号预测血液病转归方法在应用中的实验数据图。
其中，附图5到图10中阅读顺序为从左上图以“Z”字方式到右下图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员所获得的所有其他实施例，都属于本发明保护的范围。
本领域技术人员应理解的是，在本发明的揭露中，术语“纵向”、“横向”、“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”“内”、“外”等指示的方位或位置关系是基于附图所示的方位或位置关系，其仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此上述术语不能理解为对本发明的限制。
可以理解的是，术语“一”应理解为“至少一”或“一个或多个”，即在一个实施例中，一个元件的数量可以为一个，而在另外的实施例中，该元件的数量可以为多个，术语“一”不能理解为对数量的限制。
在介绍本发明的具体实施内容之前，先对该实施内容中涉及到的名词进行简要的说明介绍：
转归：是指疾病的转移和发展的意思。一般而言，疾病的发展过程常可分为四期：潜伏期、前驱期、症状明显期、转归期，而疾病的转归又可分为痊愈、死亡、缠绵、后遗以及复发。或者可以说，疾病的转归有完全恢复健康，不完全恢复健康以及死亡三种情况。
红细胞DNA损伤信号：成熟的红细胞是没有细胞核的，如果在红细胞发育成熟的前期，染色单体或染色体的无着丝点断片，或因纺锤体受损而丢失整个染色体，在细胞分裂后期，部分DNA核酸片段仍然遗留在细胞质中，也就是说，这些残留的DNA信号会在成熟的红细胞胞质内单独形成一个或几个规则的次核，这些次核即为残留的DNA损伤信号。
CD235a：血型糖蛋白A，是一种单跨膜糖蛋白，表达于成熟红细胞和红系前体细胞，为红细胞表面特殊的标记蛋白。
CD47：也叫做整合素相关蛋白(integrin associated protein，IAP)，是免疫球蛋白超家族成员，CD47广泛地表达于细胞的表面，可与信号调节蛋白α(Signal regulatory proteinα,SIRPα)、血小板反应蛋白(thrombospondin-1,TSP1)以及整合素(integrins)相互作用，介导凋亡、增殖、免疫等一系列的反应，研究表明，白血病细胞比起正常细胞来，会生成更多的CD47蛋白，CD47蛋白与红细胞生存周期有关。
PBS：磷酸缓冲盐溶液(phosphate buffer saline)的缩写,一般作为溶剂，起溶解保护试剂的作用。
APC：别藻蓝蛋白(Allophycocyanin)，是一种蓝绿色水藻中分离出来的藻胆(色素)蛋白。与其它藻胆蛋白一样，APC具有荧光性，伴随着非常高的光 吸收和高量子效率。
PE：聚乙烯(polyethylene，简称PE)是乙烯经聚合制得的一种热塑性树脂。
Hoechs33342：可在350nm左右的紫外光激发，都在461nm处发出蓝靛色荧光，未被结合的染料最大发射光在510到540nm之间，可被氙-氩灯或水银-氩灯或紫外激光激发，激发光和发射光之间的斯托克斯位移巨大，故可用于多染料多颜色荧光染色。
Thiazole orange：噻唑橙是一种化学物质，化学式是C26H24N2O3S2。
Human FcR Binding：或者称作Human Fc Block，可以有效阻止细胞表面Fc受体的非特异结合，降低背景干扰获得更准确的结果。
由于成熟的红细胞是无核的，这就意味着成人外周血中出现有核红细胞均属病理现象，如果成人外周血内出现大量的晚幼红细胞或中幼红细胞，意味着骨髓中红细胞系增生明显活跃，则有可能是增生性贫血，而如果成人外周血中出现大量的原红细胞、早幼红细胞的话，意味着骨髓中幼稚红细胞异常增生并释放入血，则有可能是红血病或者红白血病，如果成人外周血中各个发育阶段的红细胞都可以见到，并且可以见到幼稚粒细胞及巨核细胞，则有可能是髓外造血。特别地，针对造血系统恶性疾病来说，骨髓充满大量白血病细胞使幼红细胞提前释放，有核红细胞以中幼晚幼为主。
本发明提供的利用红细胞DNA损伤信号预测血液病转归的方法，基于CD235a和CD47标记蛋白组合，通过流式方法筛选出特定红细胞，使用核酸染料分析所述特定红细胞内的核酸信号，即为红细胞的DNA损伤信号，通过快速有效的对比患者自身DNA损伤信号比例的变化，建立高效、快速、高敏感的RBCs损伤检测体系，进而预测血液病患者的转归情况。
具体而言，所述利用红细胞DNA损伤信号预测血液转归的方法，以下简称为转归预测方法，包括以下步骤：
S1：准备血样本：获取血液疾病患者的抗凝血样本，按照1％体积比将所述抗凝血样本在室温环境下固定于1％-4％的多聚甲醛溶液当中，固定10-20分钟后，形成含有血样本的固定液，放置于低温环境下长期保存。
S2：准备对照组和实验组：分别取用一定体积的固定液设置为第一对照组和第一实验组，所述第一对照组和所述第一实验组均在室温环境下300-400g离心5min，弃上清液，随后用1*PBS重悬清洗2-3次，得到处理过后的第二对照组和处理过后的第二实验组。
S3：染色第二实验组：取所述第二实验组加入Human FcR Binding，在4摄氏度下孵育15-20分钟，随后使用染色缓冲剂在室温避光环境下染色所述第二实验组，得到第三实验组，其中所述染色缓冲剂为含RNase的染色试剂以及核酸染色剂的组合，染色15-30分钟，其中所述含RNase的染色试剂为含第一标记物标记的CD235a以及第二标记物标记的CD47，所述核酸染色剂为含Hoechst33342和Thiazoleorange的1×PBS。
S4：重悬第二对照组：使用1*PBS重悬所述第二对照组，得到第三对照组。
S5：流式方法检测所述第三实验组以及所述第三对照组：将所述第三实验组和所述第三对照组放置于流式细胞仪中检测，得到分析结果。
S6：分析所述分析结果:
S61：选择含有所述CD235a的阳性细胞群体；
S62：在步骤S61的基础上选择CD47表达量处于TOP5％细胞群体；以及
S63：在步骤S62的基础上选择Hoechst33342和Thiazole orange双阳性的细胞群体，记录CD47表达量为TOP5％的细胞群体中Hoechst33342和Thiazole  orange双阳的群体百分比，得到损伤信号。
值得一提的是，在本发明的实施例中，选择CD235是因为其是红细胞表面特有的标记物，从而筛选出血样本中的红细胞，然后在红细胞里选择处于CD47表达量为TOP 5％的细胞群体，即选择了血液中较为新鲜的红细胞。
具体而言，在所述步骤S1当中，其中所述抗凝血样本为骨髓或者外周血，并且所述低温环境优选为4℃，此温度保存效果最佳，另外，本发明只需要获取微量的血样本即可，极大程度地提高测试效率以及减轻病患的痛苦，一般而言，只需要1-3ul的微量血样本。
在所述步骤S2当中，准备所述对照组以及所述实验组的可变因素应控制为一致，也就是说，所述对照组以及所述实验组应该在同样的环境下进行实验准备，以此方式确保不必要的干扰因素的干扰。
在所述步骤S3当中，按照2μl-15μL/test的标准加入Human FcR Binding，所述染色缓冲剂中所述第一标记物不同于所述第二标记物，以在检测时区别所述CD235a以及所述CD47，值得一提的是，对应不同标记物在检测时以不同的波长范围为准，在本发明的实施例中，选用APC标记的CD235a以及PE标记的CD47，其标记物的类型不受影响。
在所述步骤S5当中，选用流式细胞仪的型号为Beckman Coulter的CytoFLEX LX流式细胞仪，当然可以不只是用这特定类型的流式分析仪。在本发明的实施例中，所述流式分析仪的相关参数如下：
采集参数：记录个数100000个、记录时间120s、样本流速30微升/分钟；
通道选择：CD235a-APC为Red 660/10、CD47-PE为Yellow 585/42、Hoechst33342为UV 450/45、Thiazole orange为Blue 525/40。
在所述步骤S5当中，所述流式分析仪分析得到的结果如图6所示，在分析 图中可以清楚地看到被所述第一标记物标记的CD235a，并得到CD235a阳性细胞群体内CD47表达量处于TOP5％细胞群体，在该CD47表达量处于TOP5％细胞群体又得到Hoechst33342和Thiazole orange双阳性的细胞群体，最终计算得到DNA的损伤信号。
在所述步骤S6之后，在获取到损伤信号后，医用人员可快速有效的对比患者自身DNA损伤信号比例的变化，进而预判患者血液疾病的转归情况。
另外，以下将以详细的实验数据作为该转归预测方法的实验支持，但熟悉该技术的人应该明白，所述实验数据只是作为一个具体实施例存在，所述实验数据可以比例的方式进行类推。
在该实验例中，选用10ul的患者抗凝血样本，按照1％的体积比固定于1％-4％的多聚甲醛溶液中，即所述多聚甲醛溶液的体积为1000ul，室温固定10-20min后可于4℃长期保存。随后从中取出含1ul血样本的固定液，对应1ul血样本采用200ul的1*PBS重悬对照组样本，200ul的染色缓冲剂重悬所述实验组样本，并且所述实验组样本中包括0.1-0.15μg APC标记的CD235a、0.03-0.1μg PE标记的CD47、0.014-0.08nmol Hoechst33342和0.04-0.2μg Thiazole orange的1×PBS。
也就是说，抗凝血样本以体积比1：100的比例添加至多聚甲醛溶液中,得到含有血样本的固定液。取出含有一定体积的血样本的固定液，进行后续染色标记操作，其中所述血样本和所述染色缓冲剂的体积比控制为1:200，对应1ul血样本的染色缓冲剂中含有0.1-0.15μg APC标记的CD235a、0.03-0.1μg PE标记的CD47、0.014-0.08nmol Hoechst33342和0.04-0.2μg Thiazole orange的1×PBS。
特别值得一提的是，本发明提供的所述转归预测方法在每次测量时只要使用1ul的微量血样本，并且抗体孵育的时间为20-30分钟，单个样本检测时间在 1分钟左右。然而血常规需要血样本5ml，并且其检测速度大概在1个小时，相较于传统的血常规检测方法，所述转归预测方法微量检测，检测效果高，特异性佳，灵敏度高，在临床中有着非常重要的实际意义。
另外，本发明提供的所述利用红细胞DNA损伤信号预测血液转归的方法特别适用于预测急性白血病的转归，已有具体的临床数据作为支持，说明如下：
Figure PCTCN2019078279-appb-000001
以上表格为一ALL患者采用不同治疗方案治疗检测得到的数据图，如图所示，在2017年11月24日，该ALL患者采用达沙替尼进行治疗，并且诊断该ALL患者为中枢复发ALL(Ph阳性)，抽取该患者的外周血通过所述转归预测方法进行检测，得到该患者的红细胞DNA损伤高达90.03％。
在2017年12月25日，当该患者采用MAE方案化疗后19天后，诊断为ALL复发，抽取该患者的骨髓，用上述方法检测其红细胞DNA损伤信号为3.06％。
在2018年2月11日，当该患者诊断为骨髓缓解状态，但bcb/abl仍阳性，PH+，复发，T315I突变阳性，中枢累及时，检测得到其红细胞损伤信号为2.44％。
从表格的数据可以看到，随着治疗的推进，该患者的红细胞损伤信号从最初的90.03％到后面维持在3％以下的数值。
通过数据分析，医用人员可以根据患者的红细胞DNA损伤信号预判血液病的转归情况，特别是急性白血病的转归。
本发明不局限于上述最佳实施方式，任何人在本发明的启示下都可得出其他各种形式的产品，但不论在其形状或结构上作任何变化，凡是具有与本申请相同或相近似的技术方案，均落在本发明的保护范围之内。

Claims (9)

  1. 一利用红细胞DNA损伤信号预测血液病转归的方法，其特征在于，包括以下步骤:
    S1:准备血样本：获取血液疾病患者的抗凝血样本，按照1％体积比将所述抗凝血样本在室温环境下固定于1％-4％的多聚甲醛溶液当中，固定10-20分钟后，形成含有血样本的固定液；
    S2:准备对照组和实验组：分别取用相同体积的固定液设置为第一对照组和第一实验组，所述第一对照组和所述第一实验组均在室温环境下300-400g离心,弃上清液，随后用1*PBS重悬清洗2-3次，得到处理过后的第二对照组和处理过后的第二实验组；
    S3：染色第二实验组：取所述第二实验组加入Human FcR Binding，在4摄氏度下孵育15-20分钟，随后使用染色缓冲剂在室温避光环境下染色所述第二实验组，得到第三实验组，其中所述染色缓冲剂为含RNase的染色试剂以及核酸染色剂的组合，染色15-30分钟，其中所述含RNase的染色试剂为含第一标记物标记的CD235a以及第二标记物标记的CD47，所述核酸染色剂为含Hoechst33342和Thiazole orange的1×PBS，其中所述第一标记物不同于所述第二标记物；
    S4：重悬第二对照组：使用1*PBS重悬所述第二对照组，得到第三对照组；
    S5：流式方法检测所述第三实验组以及所述第三对照组：将所述第三实验组和所述第三对照组放置于流式细胞仪中检测，得到分析结果；以及
    S6：分析所述分析结果。
  2. 根据权利要求1所述的利用红细胞DNA损伤信号预测血液病转归的方法，其特征在于，所述步骤S6进一步包括；
    S61：选择含有所述CD235a的阳性细胞群体；
    S62：在步骤S61的基础上选择CD47表达量处于TOP5％细胞群体；以及
    S63：在步骤S62的基础上选择Hoechst33342和Thiazole orange双阳性的细胞群体，记录CD47表达量为TOP5％的细胞群体中Thiazole orange和Hoechst33342呈现双阳的细胞群体百分比，得到损伤信号。
  3. 根据权利要求2所述的利用红细胞DNA损伤信号预测血液病转归的方法，其特征在于，对应1ul的血样本使用0.1-0.15μg所述第一标记物标记的CD235a、0.03-0.1μg所述第二标记物标记的CD47、0.014-0.08nmol Hoechst33342和0.04-0.2μg Thiazole orange的1×PBS。
  4. 根据权利要求2所述的利用红细胞DNA损伤信号预测血液病转归的方法，其特征在于，包括步骤S7:快速有效的对比患者自身DNA损伤信号比例的变化。
  5. 根据权利要求1到5任一所述的利用红细胞DNA损伤信号预测血液病转归的方法，其特征在于，所述步骤S3中，所述第一标记物为APC，所述第二标记物为PE。
  6. 根据权利要求1到5任一所述的利用红细胞DNA损伤信号预测血液病转归的方法，其特征在于，所述血样本为骨髓或者外周血，所述血样本的体积为1-3ul，所述低温环境为4℃。
  7. 根据权利要求1到5任一所述的利用红细胞DNA损伤信号预测血液病转归的方法，其特征在于，所述流式细胞仪的记录个数为100000个、记录时间120s、样本流速30微升/分钟。
  8. 根据权利要求1到5任一所述的述的利用红细胞DNA损伤信号预测血液病转归的方法，其特征在于，所述步骤S2和所述步骤S4中，离心时间为4-6分钟。
  9. 一利用红细胞DNA损伤信号预测血液病转归的应用，其特征在于，采用权 利要求1到5任一所述的用红细胞DNA损伤信号预测血液病转归的方法，被应用于预测急性白血病的转归情况。
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CN109652369B (zh) * 2019-02-28 2020-01-03 西湖生物医药科技(杭州)有限公司 利用外周血体外制备成熟红细胞的方法及制剂
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