WO2018010632A1 - 胎儿有核红细胞的分离纯化方法及试剂盒 - Google Patents

胎儿有核红细胞的分离纯化方法及试剂盒 Download PDF

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WO2018010632A1
WO2018010632A1 PCT/CN2017/092462 CN2017092462W WO2018010632A1 WO 2018010632 A1 WO2018010632 A1 WO 2018010632A1 CN 2017092462 W CN2017092462 W CN 2017092462W WO 2018010632 A1 WO2018010632 A1 WO 2018010632A1
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cells
antibody
separation
blood sample
cell
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French (fr)
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夏庆杰
杨林
杨雪飞
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山东亚大药业有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the invention belongs to the field of cell biology detection, and in particular relates to a method for separating and purifying fetal nucleated red blood cells and a kit.
  • the invention can be applied to prenatal genes or chromatin examinations.
  • Prenatal diagnosis identifies congenital birth defects and genetic defects, helping early intervention to avoid complications and alleviate parental concerns.
  • Fetal cells for prenatal diagnosis of chromosomes and monogenic diseases are usually obtained with amniocentesis, chorionic villus sampling, and umbilical puncture. They are invasive and have a small but inherent risk of abortion (0.5-4%). Patient compliance is poor. In recent years, it has been found that certain components of maternal peripheral blood can be used to detect fetal disease conditions. Fetal cells or DNA from maternal blood in the first trimester are expected to be a non-invasive alternative to current prenatal diagnostic methods.
  • the cell-free DNA fragments in the maternal circulation are small and low in content, which is insufficient to provide complete chromosomal information for diagnosis (eg, aneuploidy), so fetal cells in the maternal circulation are the hope for non-invasive detection of chromosomal abnormalities.
  • its number of cells is very small, especially its cell characteristics are similar to those of maternal cells, and it is difficult to distinguish it from maternal cells.
  • the main limiting factor for non-invasive prenatal diagnosis is the lack of effective techniques for isolating and purifying fetal cells in maternal peripheral blood.
  • Prenatal diagnosis using fetal blood cells from pregnant women has no risk to the fetus and is painless and acceptable to pregnant women.
  • Promotion, research in recent years is very active, is the development direction of prenatal diagnosis of genetic diseases. Therefore, establishing a non-invasive prenatal diagnosis method and reducing the risk of prenatal diagnosis is a major issue in prenatal diagnosis.
  • the presence of fetal cells in the peripheral blood of pregnant women has been confirmed by many research institutes at home and abroad.
  • the fetal cells in the peripheral blood of pregnant women include Nucleated red blood cells (NRBC), fetal lymphocytes, fetal trophoblasts and granulocytes. It can be seen that fetal cells entering the maternal circulation through the placental barrier are a mixed population. Separation of target cells suitable for prenatal diagnosis from maternal peripheral blood is a necessary condition for performing a non-invasive prenatal diagnosis.
  • NRBC Nucleated red blood cells
  • fetal lymphocytes fetal lymphocytes
  • fetal trophoblasts fetal trophoblasts
  • granulocytes granulocytes
  • NRBC has the following advantages: (1) It can occur early in pregnancy and has a short life span (not more than 90 days) in maternal peripheral blood, which is convenient for early diagnosis and is not interfered with by previous pregnancy; (2) It can express special antigen components for easy enrichment; (3) It can produce special hemoglobin reaction for easy identification; (4) Carry all fetal genetic information (genome) for genetics and molecular biology analysis. Most domestic and foreign scholars use various monoclonal antibodies against fetal cell surface specific antigens to separate and purify fetal cells by flow cytometry. However, most of these NRBCs are maternal, and the proportion of fetal NRBC is very low.
  • the present invention provides a method for separating and purifying fetal nucleated red blood cells and a kit.
  • the present invention provides:
  • a method for separating and purifying fetal nucleated red blood cells comprising the steps of:
  • the nucleated red blood cells in the peripheral venous blood samples of pregnant women are subjected to lysis treatment;
  • the cell-immobilized blood sample is subjected to cell separation to obtain fetal nucleated red blood cells.
  • the cells in the blood sample were negatively screened by labeling magnetic beads with CD45 antibody to remove non-nucleated red blood cells.
  • the first positive screening was performed by labeling magnetic beads with CD71 antibody.
  • the eluted cells were again positively screened with HBG1 antibody-labeled magnetic beads.
  • a fluorescently labeled CD71 antibody and a fluorescently labeled HBG1 antibody are added to the negatively screened blood sample, wherein the two antibodies have different fluorescent colors.
  • the step of centrifuging the blood sample to obtain a crude blood cell before performing the lysis treatment and
  • the blood sample is centrifuged after the lysis treatment to obtain a crude nucleated cell step.
  • a kit for isolating and purifying fetal nucleated red blood cells from a peripheral blood sample of a pregnant woman comprising:
  • CD45 antibody labeled magnetic beads
  • HBG1 antibody labeled magnetic beads
  • the invention has the following advantages and positive effects:
  • the invention provides a method and a kit for rapidly separating nucleated red blood cells (NRBC) and purified fetal nucleated red blood cells (FNRBC) from peripheral blood of pregnant women.
  • NRBC nucleated red blood cells
  • FNRBC purified fetal nucleated red blood cells
  • the present invention obtains nucleated cells in peripheral blood by selectively lysing non-nuclear red blood cells instead of density gradient centrifugation, thereby avoiding a large loss of nucleated cells caused by density gradient centrifugation.
  • the invention uses the cell immobilizing reagent to immobilize the nucleated cells obtained after the lysis, so that the cells avoid fragmentation, thereby further enduring subsequent separation and washing operations, and further reducing the loss of target cells caused by the previous separation method. Increases the efficiency and quantity of target cells.
  • the present invention adopts CD71 antibody and HBG1 antibody for double positive screening, which improves the purity of the isolated target cells, and lays a good foundation for subsequent analysis.
  • the invention provides a method for separating and purifying fetal nucleated red blood cells, which comprises the steps of: lysing non-nuclear red blood cells in a peripheral blood sample of a pregnant woman; adding a cell immobilizing reagent to the lysed blood sample; And performing cell separation on the cell-immobilized blood sample to obtain fetal nucleated red blood cells.
  • the method of the invention is carried out on a peripheral venous blood sample of a pregnant woman who is pregnant for 15-21 weeks (e.g., 16-18 weeks).
  • the cells in the blood sample are negatively screened with a CD45 antibody-labeled magnetic bead to remove non-nucleated red blood cells.
  • non-nucleated red blood cells refer to cells other than nucleated red blood cells in the cell-immobilized blood sample, including, for example, white blood cells, lymphocytes, bone marrow stromal cells, and the like.
  • the method further comprises: treating the blood sample with the CD71 antibody labeled magnetic beads for initial screening; eluting the cells on the primary positive magnetic beads with the antibody eluating solution; The HBG1 antibody-labeled magnetic beads were again positively screened for the eluted cells.
  • the purpose of the primary positive screening is to screen cells expressing CD71, such as maternal nucleated red blood cells, fetal nucleated red blood cells, and the like.
  • the purpose of the re-screening is to purify fetal nucleated red blood cells expressing HBG1.
  • CD45 antibody labeled magnetic beads, CD71 antibody labeled magnetic beads and HBG1 used in the present invention Commercially available products can be used for antibody-labeled magnetic beads.
  • the negative screening, the primary positive screening, and the positive positive screening can all be performed using techniques known in the art.
  • the antibody eluate is an alkaline buffer solution having a pH of 11.5 or more.
  • a 0.1 N aqueous sodium hydroxide solution or a 0.1 N sodium hydroxide PBS solution or a 0.1 N potassium hydroxide aqueous solution or a 0.1 N potassium hydroxide PBS solution.
  • a PBS solution is preferred because it has a strong buffering ability compared to an aqueous solution.
  • the blood sample is further processed by adding a fluorescently labeled CD71 antibody and a fluorescently labeled HBG1 antibody to the negatively screened blood sample, wherein the two antibodies have different fluorescent colors (for example Using FITC-labeled rabbit anti-human CD71 IgG and Cy3-labeled rabbit anti-human HBG1 IgG), fluorescently labeled cells were separated by flow cytometry, and two fluorescently labeled cells were simultaneously collected.
  • the means for the lysis treatment is: adding an organic solvent to the peripheral venous blood sample, preferably the organic solvent is methanol, ethanol, ethylene glycol, acetone, DMSO, glycerin; or adding hypotonic a salt solution, including a hydrochloride such as KCl, NaCl, NH 4 Cl, a sulfate such as K 2 SO 4 , Na 2 SO 4 , (NH 4 ) 2 SO 4 , such as K 3 PO 4 , Na 3 PO 4 , (NH 4 ) 3 PO 4 and other phosphates, such as KNO 3 , NaNO 3 , NH 4 NO 3 and other nitrates; or added sodium, potassium, ammonium organic acid salts (such as oxalate, acetate, citrate ); or add distilled water.
  • a hydrochloride such as KCl, NaCl, NH 4 Cl
  • a sulfate such as K 2 SO 4 , Na 2 SO 4 , (NH
  • the lysing treatment is carried out using a hypotonic saline solution.
  • Hypotonic saline solution available commercially include erythrocyte lysate; unilateral or self-made articles, for example, aqueous NH 4 Cl or NH 4 Cl in PBS solution.
  • NH 4 Cl in PBS is preferred because the cleavage is relatively complete and mild.
  • the lysing treatment is carried out using a 0.7-1.5 (w/v)% NH 4 Cl solution in PBS as a lysate. If the concentration of the lysate is too low, the nucleated red blood cell lysis is slow and the lysis is incomplete; if the concentration is too high, nucleated cell lysis is caused. A more preferable concentration is about 1 (w/v)%.
  • the lysate is used in an amount of 1 ml to 4 ml of the lysate relative to 1 ml of the peripheral venous blood sample of the pregnant woman.
  • the method of the present invention further comprises the steps of: centrifuging the blood sample to obtain a crude blood cell before performing the lysis treatment, and centrifuging the blood sample after the lysing treatment The step of obtaining a crude nucleated cell.
  • the centrifugation of the blood sample before the lysis treatment is carried out using a centrifugal force of 200 g to 500 g. If the centrifugal force is too low, it takes a long time, and the stratification effect is poor; if the centrifugal force is too high, the nucleated cells will be deformed, lumped, and agglomerated.
  • the cell immobilizing agent is a 1-3 (v/v)% glutaraldehyde solution prepared in PBS, and a more preferable concentration of the solution is 1.6 (v/v)%.
  • Other useful immobilizing reagents are 6-12 (v/v)% formaldehyde in PBS, a more preferred concentration of 8 (v/v)%; 0.5-5 (v/v)% paraformaldehyde in PBS formulation. A more preferred concentration thereof is 1 (v/v)%.
  • the cell immobilizing agent is used in an amount of 0.5 to 3 ml of the immobilizing agent, more preferably 1 ml of the immobilizing agent, relative to the solid matter in the lysed blood sample.
  • the method of the present invention comprises: taking 3-5 ml of peripheral venous blood of a pregnant woman with a pregnancy period of 15-21 weeks in a heparin anticoagulation blood collection tube, centrifuging the plasma, and adding an appropriate amount to the blood cell sediment.
  • Fetal NRBC Fetal NRBC.
  • the invention also provides a kit for separating and purifying fetal nucleated red blood cells from peripheral blood samples of pregnant women, which comprises: red blood cell lysate; cell immobilization reagent; and CD45 antibody labeled magnetic beads.
  • the cell immobilizing agent is preferably a PBS solution of 1-3 (v/v)% glutaraldehyde.
  • the kit further comprises: a CD71 antibody-labeled magnetic bead, and an HBG1 antibody-labeled magnetic bead.
  • the kit further comprises: FITC-labeled rabbit anti-human CD71 IgG, and Cy3-labeled rabbit anti-human HBG1 IgG.
  • Example 1 Isolation and purification of fetal NRBC
  • a 1 ml 3% glutaraldehyde solution in PBS was added and the cells were fixed for 10 minutes.
  • the cells were washed twice with 10 ml of physiological saline, resuspended in 1 ml of physiological saline, and added with 10E+8 CD45 antibody-labeled magnetic beads (Miltenyl biotech, serial number 130-045-801), and thoroughly mixed for 15 minutes on a magnetic separation rack.
  • the cells were separated, and the liquid fraction was transferred to a test tube containing 10E+6 CD71 antibody-labeled magnetic beads (Miltenyl biotech, SEQ ID NO: 130-046-201), thoroughly mixed for 15 minutes, and the cells were separated on a magnetic separation rack and discarded.
  • the liquid portion is thoroughly washed with physiological saline to obtain a crude nucleated red blood cell extract of the fetus.
  • the isolated cells were washed with 1 ml of antibody eluate (5% (w/v) BSA, 20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 11.5), and the cells were separated by magnetic separator to transfer the liquid portion.
  • 3 ml of peripheral venous blood of pregnant women with 16-18 weeks of pregnancy was taken from heparin anticoagulated blood collection tubes, centrifuged at 500 g for 5 minutes to remove plasma, and blood cell pellet was added to 8 ml PBS prepared 1% NH 4 Cl solution, mixed, and placed in an ice bath. In minutes, the red blood cells were lysed, centrifuged at 500 g for 5 minutes, and the supernatant was centrifuged, and the pellet was shaken to resuspend the cells. The cells were fixed by adding 1 ml of a 3% glutaraldehyde solution in PBS for 10 minutes.
  • the cells were washed twice with 10 ml of physiological saline, resuspended in 1 ml of physiological saline, and added with 10E+8 CD45 antibody-labeled magnetic beads (Miltenyl biotech, serial number 130-045-801), and thoroughly mixed for 15 minutes on a magnetic separation rack.
  • the cells were separated, and the liquid fraction was transferred to a test tube containing 10E+6 CD71 antibody-labeled magnetic beads (Miltenyl biotech, SEQ ID NO: 130-046-201), thoroughly mixed for 15 minutes, and the cells were separated on a magnetic separation rack and discarded.
  • the liquid portion is thoroughly washed with physiological saline to obtain a crude nucleated red blood cell extract of the fetus.
  • the isolated cells were resuspended in 1 ml, and FITC fluorescently labeled anti-CD71 antibody (Miltenyl biotech, SEQ ID NO: 130-098-779, mouse anti-human IgG, 1:350 dilution), anti-HBG1 antibody (abcam, ab137096, Rabbit anti-human IgG, diluted 1:500) and Cy3-labeled fluorescent secondary antibody (abcam, ab6939, goat anti-rabbit, 1:1200 dilution), the cells were incubated for 30 minutes at 4 ° C in the dark, and washed well with normal saline.
  • FITC fluorescently labeled anti-CD71 antibody Miltenyl biotech, SEQ ID NO: 130-098-779, mouse anti-human IgG, 1:350 dilution
  • anti-HBG1 antibody abcam, ab137096, Rabbit anti-human IgG, diluted 1:500
  • Cy3-labeled fluorescent secondary antibody abcam
  • Example 2 The same procedure as in Example 1 was carried out except that the cleavage step was not carried out.
  • peripheral venous blood of pregnant women aged 16-18 weeks was taken in heparin anticoagulated blood collection tubes, centrifuged at 500 g for 5 minutes to remove plasma, blood cell pellet was added with 1 ml of PBS and the cells were resuspended, and then 3 ml of PBS was added.
  • the cells were fixed in % glutaraldehyde solution for 10 minutes.
  • the cells were washed twice with 10 ml of physiological saline, resuspended in 1 ml of physiological saline, and added with 10E+8 CD45 antibody-labeled magnetic beads (Miltenyl biotech, serial number 130-045-801), and thoroughly mixed for 15 minutes on a magnetic separation rack.
  • the cells were separated, and the liquid fraction was transferred to a test tube containing 10E+6 CD71 antibody-labeled magnetic beads (Miltenyl biotech, SEQ ID NO: 130-046-201), thoroughly mixed for 15 minutes, and the cells were separated on a magnetic separation rack and discarded.
  • the liquid portion is thoroughly washed with physiological saline to obtain a crude nucleated red blood cell extract of the fetus.
  • the isolated cells were washed with 1 ml of antibody eluate, separated by magnetic separator, and the liquid fraction was transferred to a new centrifuge tube, washed with 10 ml of physiological saline, and then added with anti-HBG1 antibody (abcam, ab137096, rabbit).
  • peripheral venous blood of pregnant women in the period of 16-18 weeks of pregnancy was taken from heparin anticoagulated blood collection tubes, centrifuged at 500 g for 5 minutes to remove plasma, and blood cell pellet was added to 8 ml of PBS prepared 1% NH 4 Cl solution, and mixed, ice. After standing in the bath for 10 minutes, the red blood cells were lysed, centrifuged at 500 g for 5 minutes, and the supernatant was centrifuged, and the pellet was shaken to resuspend the cells.
  • the cells were washed twice with 10 ml of physiological saline, resuspended in 1 ml of physiological saline, and added with 10E+8 CD45 antibody-labeled magnetic beads (Miltenyl biotech, serial number 130-045-801), and thoroughly mixed for 15 minutes on a magnetic separation rack.
  • the cells were separated, and the liquid fraction was transferred to a test tube containing 10E+6 CD71 antibody-labeled magnetic beads (Miltenyl biotech, SEQ ID NO: 130-046-201), thoroughly mixed for 15 minutes, and the cells were separated on a magnetic separation rack and discarded.
  • the liquid portion is thoroughly washed with physiological saline to obtain a crude nucleated red blood cell extract of the fetus.
  • the separated cells were washed with 1 ml of antibody eluate, the cells were separated by magnetic separator, the liquid fraction was transferred to a new centrifuge tube, and 10 ml of physiological saline was added for washing, and then anti-HBG1 antibody and 10E+6 antibody were added.
  • the magnetic beads were thoroughly mixed for 15 minutes, the cells were separated on a magnetic separation rack, the liquid portion was discarded, and the purified fetal NRBC was obtained after being thoroughly washed with physiological saline.
  • Example 2 The same procedure as in Example 1 was carried out except that the cleavage step and the immobilization step were not carried out.
  • peripheral venous blood of pregnant women in the period of 16-18 weeks of pregnancy was taken from heparin anticoagulated blood collection tube, centrifuged at 500 g for 5 minutes to remove plasma, and the cell pellet was resuspended in 1 ml of physiological saline, and 10E+8 CD45 antibody was added to mark the magnetic field.
  • the separated cells were washed with 1 ml of antibody eluate, the cells were separated by magnetic separator, the liquid fraction was transferred to a new centrifuge tube, and 10 ml of physiological saline was added for washing, and then anti-HBG1 antibody and 10E+6 antibody were added.
  • the magnetic beads were thoroughly mixed for 15 minutes, the cells were separated on a magnetic separation rack, the liquid portion was discarded, and the purified fetal NRBC was obtained after being thoroughly washed with physiological saline.
  • Example 2 The same procedure as in Example 1 was carried out except that the cleavage step, the immobilization step and the second positive screening step were not carried out.
  • peripheral venous blood of pregnant women in the period of 16-18 weeks of pregnancy was taken from heparin anticoagulated blood collection tube, centrifuged at 500 g for 5 minutes to remove plasma, and the cell pellet was resuspended in 1 ml of physiological saline, and 10E+8 CD45 antibody was added to mark the magnetic field.

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Abstract

提供一种胎儿有核红细胞的分离纯化方法，其特征在于包括如下步骤：对孕妇外周静脉血样中的无核红细胞进行裂解处理；向经裂解处理过的血样中添加细胞固定化试剂；以及对经细胞固定化的血样进行细胞分离，以获得胎儿有核红细胞。还提供从孕妇外周静脉血样中分离纯化胎儿有核红细胞的试剂盒。所述方法提高了分离靶细胞的得率和纯度，为后续的各种分析奠定了良好的基础。

Description

胎儿有核红细胞的分离纯化方法及试剂盒 技术领域
本发明属于细胞生物学检测领域，具体而言涉及一种胎儿有核红细胞的分离纯化方法及试剂盒。本发明可以应用于产前基因或染色质检查。
背景技术
随着现代医学发展和医疗技术的进步，医疗卫生水平的发展，感染性疾病已经得到了较好的控制，人们的平均寿命不断延长，而遗传性疾病(基因病或染色体病)由于在医学上尚无有效的治疗办法，在严重威胁人们健康的疾病中地位越来越重要，给家庭和社会带来了极大的精神负担和经济损失，因而进行产前诊断从而避免患儿出生成为控制遗传性疾病发生，降低人群遗传负荷，提高遗传素质的重要途径。
产前诊断能够识别先天性的出生缺陷及遗传缺陷，有助于早期介入从而避免并发症并减轻父母的担忧。用于染色体和单基因病的产前诊断的胎儿细胞通常采用羊膜穿刺术、绒膜绒毛取样和脐穿刺获得，为有创取材，带有虽小而固有的流产的危险(0.5-4％)，患者依从性较差。近年来发现母体外周血中的某些成分可以用来检测胎儿疾病状况，来自孕早期母体血液中的胎儿细胞或DNA有望成为目前的产前诊断方法的无创伤性替代方案。但母体循环中的无细胞DNA片段小，含量低，不足以提供用于诊断的完整的染色体信息(例如，异倍体)，因而母体循环中的胎儿细胞是用于无创检测染色体异常的希望所在，但其细胞数量很少，尤其是其细胞特性又与母体细胞相近，难以与母体细胞截然区分。目前无创产前诊断的主要限制因素是缺乏有效的分离纯化母体外周血中胎儿细胞的技术。利用孕妇外周血胎儿细胞进行产前诊断因对于胎儿没有任何风险，对孕妇无痛苦，易被接受、 推广，近年来研究十分活跃，是遗传病产前诊断的发展方向。因此建立无创性产前诊断取材方法，降低产前诊断的风险是产前诊断工作的一项重大课题。
孕妇外周血存在胎儿细胞已被国内外许多研究所证实。孕妇外周血中胎儿细胞包括胎儿有核红细胞(Nucleated red blood cell、NRBC)、胎儿淋巴细胞、胎儿滋养细胞及粒细胞，可见通过胎盘屏障进入母体循环的胎儿细胞是一个混合群体。将适宜于进行产前诊断的靶细胞从母体外周血中分离出来是实施无创伤性产前诊断的必要条件。目前国内外学者的研究集中在胎儿有核红细胞上，并一致认为胎儿有核红细胞是最佳的靶细胞。原因是NRBC具有如下可资利用的优点：(1)可在妊娠早期出现，且在母体外周血中的寿命短(不超过90天)，便于早期诊断且不被前次妊娠干扰；(2)可表达出特殊的抗原成分，便于富集；(3)可发生特殊的血红蛋白反应，便于鉴定；(4)携带胎儿全部遗传信息(基因组)，便于遗传学和分子生物学分析。国内外学者大多利用各种胎儿细胞表面特异抗原的单克隆抗体，经流式细胞计数仪分离纯化胎儿细胞，但这些NRBC绝大多数为母源性，胎源性NRBC比例很低。有研究表明，母体外周血单核细胞经过CD45标记磁珠负筛选，剩余细胞再采用CD71抗体标记的磁珠正筛选能够获得一定量的胎儿有核红细胞，但已有方法无论在得率或者胎儿有核红细胞纯度方面都有不足。Reading等报道用1.083和1.090g/ml两种不同密度梯度的Percoll分离液对母体外周血单个核细胞进行分离，经FISH和γ珠蛋白单抗标记后进行FACS证实，高密度梯度的分离液可显著提高胎儿细胞的产量，但得率也有不足。
综上所述，目前国内外关于这方面的研究都存在胎儿细胞富集、分离的数量少，纯度不够，操作过程复杂，并且应用于产前诊断的疾病范围非常有限，使得该项工作没有实质性的突破。因此建立一套稳定可靠、操作简便、富集力强、分离纯度高的富集和分离胎儿有核红细胞的新方法，并可用于产前多种遗传性疾病的诊断，具有重要现实意义。
发明内容
为解决上述现有技术中所存在的问题，本发明提供了胎儿有核红细胞的分离纯化方法及试剂盒。
具体而言，本发明提供了：
(1)一种胎儿有核红细胞的分离纯化方法，其特征在于包括如下步骤：
对孕妇外周静脉血样中的无核红细胞进行裂解处理；
向经裂解处理过的血样中添加细胞固定化试剂；以及
对经细胞固定化的血样进行细胞分离，以获得胎儿有核红细胞。
(2)根据(1)所述的分离纯化方法，其中所述细胞分离步骤中，
先采用CD45抗体标记磁珠对血样中细胞进行负筛选，以将非有核红细胞除去。
(3)根据(2)所述的分离纯化方法，其中在进行负筛选后，还包括对血样进行如下处理：
以CD71抗体标记磁珠进行初次正筛选，
用抗体洗脱液将初次正筛选磁珠上的细胞洗脱下来，以及
以HBG1抗体标记磁珠对洗脱下的细胞进行再次正筛选。
(4)根据(3)所述的分离纯化方法，其中所述的抗体洗脱液为：pH为11.5以上的碱性缓冲溶液。
(5)根据(2)所述的分离纯化方法，其中在进行负筛选后，还包括对血样进行如下处理：
向经过负筛选后的血样中加入荧光标记的CD71抗体和荧光标记的HBG1抗体，其中两种抗体的荧光颜色不同，
采用流式细胞分离技术，对经荧光标记的细胞进行分离，以及
收集同时显示两种荧光标记的细胞。
(6)根据(1)所述的分离纯化方法，其中所述裂解处理的手段为：向所述外周静脉血样中添加有机溶剂；或添加低渗盐溶液；或者添加蒸馏水。
(7)根据(6)所述的分离纯化方法，其中采用低渗盐溶液进 行所述的裂解处理。
(8)根据(7)所述的分离纯化方法，其中还包括
在进行裂解处理前对血样进行离心以获得血细胞粗品的步骤，以及
在裂解处理后对血样进行离心以获得有核细胞粗品的步骤。
(9)根据(7)所述的分离纯化方法，其中所述裂解液为PBS配制的0.7-1.5(w/v)％NH4Cl溶液；其用量为：相对于1ml的血样添加1ml-4ml的该裂解液。
(10)根据(1)所述的分离纯化方法，其中所述细胞固定化试剂为PBS配制的1-3(v/v)％戊二醛溶液。
(11)根据(10)所述的分离纯化方法，其中所述细胞固定化试剂的用量为：相对于经裂解处理过的血样中的固体物质添加0.5-3ml的该固定化试剂。
(12)一种从孕妇外周静脉血样中分离纯化胎儿有核红细胞的试剂盒，其中包括：
红细胞裂解液；
细胞固定化试剂；以及
CD45抗体标记磁珠。
(13)根据(12)所述的试剂盒，还包括：
CD71抗体标记磁珠；和
HBG1抗体标记磁珠。
(14)根据(12)所述的试剂盒，还包括：
FITC标记的兔抗人CD71 IgG，和
Cy3-标记的兔抗人HBG1 IgG。
本发明与现有技术相比具有以下优点和积极效果：
本发明提供了一种从孕妇外周血中快速分离有核红细胞(NRBC)及纯化胎儿有核红细胞(FNRBC)的方法及试剂盒。本发明采用选择性裂解无核红细胞而非密度梯度离心的方法获得了外周血中的有核细胞，由此避免了由密度梯度离心造成的有核细胞的大量损失。另外， 本发明用细胞固定化试剂对裂解后获得的有核细胞进行了固定化处理，使细胞避免碎裂，从而更加耐受后续的分离及洗涤操作，进一步降低了以往分离方法造成的目标细胞的损失，提高了分离靶细胞的效率和数量。
进一步地，本发明采用CD71抗体和HBG1抗体进行双重正筛选，提高了分离靶细胞的纯度，为后续的各种分析奠定了良好的基础。
具体实施方式
以下通过具体实施方式的描述对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
本发明提供了一种胎儿有核红细胞的分离纯化方法，其特征在于包括如下步骤：对孕妇外周静脉血样中的无核红细胞进行裂解处理；向经裂解处理过的血样中添加细胞固定化试剂；以及对经细胞固定化的血样进行细胞分离，以获得胎儿有核红细胞。
优选地，取怀孕15-21周(例如16-18周)的孕妇的外周静脉血样进行本发明的方法。
优选地，在所述细胞分离步骤中，先采用CD45抗体标记磁珠对血样中细胞进行负筛选，以将非有核红细胞除去。
在本文中，所述非有核红细胞是指所述经细胞固定化的血样中有核红细胞以外的细胞，包括例如白细胞、淋巴细胞、骨髓间质细胞等。
优选地，在进行所述负筛选后，还包括对血样进行如下处理：以CD71抗体标记磁珠进行初次正筛选；用抗体洗脱液将初次正筛选磁珠上的细胞洗脱下来；以及以HBG1抗体标记磁珠对洗脱下的细胞进行再次正筛选。所述初次正筛选的目的是将表达CD71的细胞，如母源的有核红细胞、胎儿有核红细胞等筛选出来。所述再次正筛选的目的是将表达HBG1的胎儿有核红细胞纯化出来。
本发明所用CD45抗体标记磁珠、CD71抗体标记磁珠和HBG1 抗体标记磁珠都可采用市售可得的产品。所述负筛选、初次正筛选和再次正筛选均可采用本领域已知的技术进行。
优选地，在本发明中，所述抗体洗脱液为：pH为11.5以上的碱性缓冲溶液。例如，0.1N的氢氧化钠水溶液或0.1N的氢氧化钠PBS溶液，或0.1N的氢氧化钾水溶液或0.1N的氢氧化钾PBS溶液。其中优选PBS溶液，因为相比于水溶液，其缓冲能力强。
可供选择地，在进行负筛选后，还包括对血样进行如下处理：向经过负筛选后的血样中加入荧光标记的CD71抗体和荧光标记的HBG1抗体，其中两种抗体的荧光颜色不同(例如采用FITC标记的兔抗人CD71 IgG和Cy3-标记的兔抗人HBG1 IgG)，采用流式细胞分离技术，对经荧光标记的细胞进行分离，以及收集同时显示两种荧光标记的细胞。
优选地，在本发明中，所述裂解处理的手段为：向所述外周静脉血样中添加有机溶剂，优选的有机溶剂为甲醇、乙醇、乙二醇、丙酮、DMSO、甘油；或者添加低渗盐溶液，包括诸如KCl、NaCl、NH4Cl等盐酸盐、诸如K2SO4、Na2SO4、(NH4)2SO4等硫酸盐、诸如K3PO4、Na3PO4、(NH4)3PO4等磷酸盐、诸如KNO3、NaNO3、NH4NO3等硝酸盐；或者添加钠、钾、铵的有机酸盐(如草酸盐、乙酸盐、柠檬酸盐)；或者添加蒸馏水。
优选地，在本发明中，采用低渗盐溶液进行所述的裂解处理。可用的低渗盐溶液包括市售红细胞裂解液；或自配单方制品，例如NH4Cl的水溶液，或NH4Cl的PBS溶液。其中优选NH4Cl的PBS溶液，因为裂解较为完全并且较为温和。
更优选地，采用PBS配制的0.7-1.5(w/v)％的NH4Cl溶液作为裂解液进行所述的裂解处理。如果该裂解液的浓度太低，无核红细胞裂解慢，并且裂解不完全；如果浓度过高，则导致有核细胞裂解。更优选的浓度为1(w/v)％左右。该裂解液的用量为：相对于1ml的孕妇外周静脉血样添加1ml-4ml的该裂解液。
优选地，本发明的方法还包括：在进行裂解处理前对血样进行离心以获得血细胞粗品的步骤，以及在裂解处理后对血样进行离心以 获得有核细胞粗品的步骤。其中，在进行裂解处理前对血样进行的离心采用200g-500g的离心力。如果离心力太低，耗时长，分层效果差；如果离心力太高，会导致有核细胞变形、起团，出现团块化。
优选地，在本发明中，所述细胞固定化试剂为PBS配制的1-3(v/v)％戊二醛溶液，该溶液更优选的浓度为1.6(v/v)％。其它可用的固定化试剂为PBS配制的6-12(v/v)％甲醛，其更优选的浓度为8(v/v)％；PBS配制的0.5-5(v/v)％多聚甲醛，其更优选的浓度为1(v/v)％。
优选地，所述细胞固定化试剂的用量为：相对于经裂解处理过的血样中的固体物质添加0.5-3ml的该固定化试剂，更优选添加1ml的该固定化试剂。
在本发明一个具体的实施方案中，本发明的方法包括：取孕期为15-21周的孕妇的外周静脉血3-5ml于肝素抗凝采血管中，离心去血浆，向血细胞沉淀中加入适量红细胞裂解液，混匀；裂解红细胞后离心去上清，获得并重悬有核细胞；再加入PBS配制的戊二醛溶液固定细胞；经生理盐水充分洗涤后加入CD45抗体标记磁珠进行负筛选，充分混匀再于磁分离器上分离细胞；液体部分转移到含有抗CD71抗体标记磁珠的试管中进行初次正筛选，经生理盐水充分洗涤后获得胎儿有核红细胞粗提物；再用抗体洗脱液洗下细胞，转移到新的离心管中，经生理盐水充分洗涤；加入含有抗HBG1抗体标记磁珠的试管中进行再次正筛选，以进行细胞纯化；经生理盐水充分洗涤后即获得纯化的胎儿NRBC。
本发明还提供了一种从孕妇外周静脉血样中分离纯化胎儿有核红细胞的试剂盒，其中包括：红细胞裂解液；细胞固定化试剂；以及CD45抗体标记磁珠。其中，所述细胞固定化试剂优选为1-3(v/v)％戊二醛的PBS溶液。
优选地，所述的试剂盒还包括：CD71抗体标记磁珠，和HBG1抗体标记磁珠。
优选地，所述的试剂盒还包括：FITC标记的兔抗人CD71 IgG，和Cy3-标记的兔抗人HBG1 IgG。
实施例
除非另有说明，溶液中的百分数均为体积百分数。
实施例1：胎儿NRBC的分离纯化
取孕期为16-18周的孕妇外周静脉血3ml于肝素抗凝采血管中，500g离心5分钟去血浆，血细胞沉淀加入8ml PBS(NaCl 137mmol/L，KCl 2.7mmol/L，Na2HPO4 4.3mmol/L，KH2PO4 1.4mmol/L，pH7.2)配制的1％(w/v)NH4Cl溶液，混匀，冰浴中放置10分钟，裂解红细胞，500g离心5分钟离心去上清，振摇沉淀使细胞重悬。再加入1ml PBS配制的3％戊二醛溶液，固定细胞10分钟。用10ml生理盐水充分洗涤细胞2次，用1ml生理盐水重悬细胞，加入10E+8 CD45抗体标记磁珠(Miltenyl biotech，序列号130-045-801)，充分混匀15分钟，于磁分离架上分离细胞，将液体部分转移至一含有10E+6 CD71抗体标记磁珠(Miltenyl biotech，序列号130-046-201)的试管中，充分混匀15分钟，于磁分离架上分离细胞，弃液体部分，经生理盐水充分洗涤后获得胎儿有核红细胞粗提物。所分离到的细胞用1ml抗体洗脱液(5％(w/v)BSA，20mM Tris-HCl，150mM NaCl，0.05％Tween-20，pH 11.5)洗涤，磁分离器分离细胞，将液体部分转移到新的离心管中，加入10ml生理盐水充分洗涤，再加入抗HBG1抗体(abcam，ab137096，兔抗人IgG，1:800稀释)及10E+6抗体标记磁珠(Miltenyl biotech,Order no130-048-602)，充分混匀15分钟，于磁分离架上分离细胞，弃液体部分，经生理盐水充分洗涤后即获得纯化的胎儿NRBC。收获的细胞数为13.8±5.6个，NRBC纯度(NRBC数目/有核细胞总数)达88.3±9.2％(n＝7)。
实施例2
取孕期为16-18周的孕妇外周静脉血3ml于肝素抗凝采血管中，500g离心5分钟去血浆，血细胞沉淀加入8ml PBS配制的1％NH4Cl溶液，混匀，冰浴中放置10分钟，裂解红细胞，500g离心5分钟离 心去上清，振摇沉淀使细胞重悬。再加入1ml PBS配制的3％戊二醛溶液固定细胞10分钟。用10ml生理盐水充分洗涤细胞2次，用1ml生理盐水重悬细胞，加入10E+8 CD45抗体标记磁珠(Miltenyl biotech，序列号130-045-801)，充分混匀15分钟，于磁分离架上分离细胞，将液体部分转移至一含有10E+6 CD71抗体标记磁珠(Miltenyl biotech，序列号130-046-201)的试管中，充分混匀15分钟，于磁分离架上分离细胞，弃液体部分，经生理盐水充分洗涤后获得胎儿有核红细胞粗提物。所分离到的细胞用1ml重悬，加入FITC荧光标记的抗CD71抗体(Miltenyl biotech，序列号130-098-779，小鼠抗人IgG，1:350稀释)、抗HBG1抗体(abcam，ab137096，兔抗人IgG，1:500稀释)和Cy3-标记的荧光二抗(abcam，ab6939，羊抗兔，1：1200稀释)，4℃黑暗环境下孵育细胞30分钟，生理盐水充分洗涤。经流式细胞仪(BD FACSCalibur全自动多色分析流式细胞仪系统)分选CD71和HBG1均为阳性的细胞，即获得纯化的胎儿NRBC。收获的细胞数为15.1±8.2个，NRBC纯度达89.6±8.3％(n＝3)。
对比例1
按照与实施例1相同的方法进行，不同之处在于不进行裂解步骤。
具体而言，取孕期在16-18周的孕妇外周静脉血3ml于肝素抗凝采血管中，500g离心5分钟去血浆，血细胞沉淀加1ml PBS并使细胞重悬，再加入3ml PBS配制的3％戊二醛溶液固定细胞10分钟。用10ml生理盐水充分洗涤细胞2次，用1ml生理盐水重悬细胞，加入10E+8 CD45抗体标记磁珠(Miltenyl biotech，序列号130-045-801)，充分混匀15分钟，于磁分离架上分离细胞，将液体部分转移至一含有10E+6 CD71抗体标记磁珠(Miltenyl biotech，序列号130-046-201)的试管中，充分混匀15分钟，于磁分离架上分离细胞，弃液体部分，经生理盐水充分洗涤后获得胎儿有核红细胞粗提物。所分离到的细胞用1ml抗体洗脱液洗下洗涤，磁分离器分离细胞，将液体部分转移到新的离心管中，加入10ml生理盐水充分洗涤，再加入抗HBG1抗体(abcam，ab137096，兔抗人IgG，1:800稀释)及10E+6抗体标记磁珠(Miltenyl  biotech,Order no 130-048-602)，充分混匀15分钟，于磁分离架上分离细胞，弃液体部分，经生理盐水充分洗涤后即获得纯化的胎儿NRBC。收获的细胞数为65.5±23.7个，NRBC纯度为21.2±8.6％(n＝5)。
对比例2
按照与实施例1相同的方法进行，不同之处在于不进行固定化步骤。
具体而言，取孕期在16-18周的孕妇外周静脉血3ml于肝素抗凝采血管中，500g离心5分钟去血浆，血细胞沉淀加入8ml PBS配制的1％NH4Cl溶液，混匀，冰浴中放置10分钟，裂解红细胞，500g离心5分钟离心去上清，振摇沉淀使细胞重悬。用10ml生理盐水充分洗涤细胞2次，用1ml生理盐水重悬细胞，加入10E+8 CD45抗体标记磁珠(Miltenyl biotech，序列号130-045-801)，充分混匀15分钟，于磁分离架上分离细胞，将液体部分转移至一含有10E+6 CD71抗体标记磁珠(Miltenyl biotech，序列号130-046-201)的试管中，充分混匀15分钟，于磁分离架上分离细胞，弃液体部分，经生理盐水充分洗涤后获得胎儿有核红细胞粗提物。所分离到的细胞用1ml抗体洗脱液洗下洗涤，磁分离器分离细胞，将液体部分转移到新的离心管中，加入10ml生理盐水充分洗涤，再加入抗HBG1抗体及10E+6抗体标记磁珠，充分混匀15分钟，于磁分离架上分离细胞，弃液体部分，经生理盐水充分洗涤后即获得纯化的胎儿NRBC。收获的细胞数为8.7±4.9个，NRBC纯度为89.2±13.5％(n＝5)。
对比例3
按照与实施例1相同的方法进行，不同之处在于不进行裂解步骤和固定化步骤。
具体而言，取孕期在16-18周的孕妇外周静脉血3ml于肝素抗凝采血管中，500g离心5分钟去血浆，血细胞沉淀用1ml生理盐水重悬细胞，加入10E+8 CD45抗体标记磁珠(Miltenyl biotech，序列号130-045-801)，充分混匀15分钟，于磁分离架上分离细胞，将液体部 分转移至一含有10E+6 CD71抗体标记磁珠(Miltenyl biotech，序列号130-046-201)的试管中，充分混匀15分钟，于磁分离架上分离细胞，弃液体部分，经生理盐水充分洗涤后获得胎儿有核红细胞粗提物。所分离到的细胞用1ml抗体洗脱液洗下洗涤，磁分离器分离细胞，将液体部分转移到新的离心管中，加入10ml生理盐水充分洗涤，再加入抗HBG1抗体及10E+6抗体标记磁珠，充分混匀15分钟，于磁分离架上分离细胞，弃液体部分，经生理盐水充分洗涤后即获得纯化的胎儿NRBC。收获的细胞数为38.4±10.2个，NRBC纯度为25.1±11.6％(n＝5)。
对比例4
按照与实施例1相同的方法进行，不同之处在于不进行裂解步骤、固定化步骤和第二次正筛选步骤。
具体而言，取孕期在16-18周的孕妇外周静脉血3ml于肝素抗凝采血管中，500g离心5分钟去血浆，血细胞沉淀用1ml生理盐水重悬细胞，加入10E+8 CD45抗体标记磁珠(Miltenyl biotech，序列号130-045-801)，充分混匀15分钟，于磁分离架上分离细胞，将液体部分转移至一含有10E+6 CD71抗体标记磁珠(Miltenyl biotech，序列号130-046-201)的试管中，充分混匀15分钟，于磁分离架上分离细胞，弃液体部分，经生理盐水充分洗涤后获得胎儿有核红细胞粗提物。收获的细胞数为361.8±103.5个，NRBC纯度为6.2±5.4％(n＝5)。

Claims (14)

  1. 一种胎儿有核红细胞的分离纯化方法，其特征在于包括如下步骤：
    对孕妇外周静脉血样中的无核红细胞进行裂解处理；
    向经裂解处理过的血样中添加细胞固定化试剂；以及
    对经细胞固定化的血样进行细胞分离，以获得胎儿有核红细胞。
  2. 根据权利要求1所述的分离纯化方法，其中所述细胞分离步骤中，
    先采用CD45抗体标记磁珠对血样中细胞进行负筛选，以将非有核红细胞除去。
  3. 根据权利要求2所述的分离纯化方法，其中在进行负筛选后，还包括对血样进行如下处理：
    以CD71抗体标记磁珠进行初次正筛选，
    用抗体洗脱液将初次正筛选磁珠上的细胞洗脱下来，以及
    以HBG1抗体标记磁珠对洗脱下的细胞进行再次正筛选。
  4. 根据权利要求3所述的分离纯化方法，其中所述的抗体洗脱液为：pH为11.5以上的碱性缓冲溶液。
  5. 根据权利要求2所述的分离纯化方法，其中在进行负筛选后，还包括对血样进行如下处理：
    向经过负筛选后的血样中加入荧光标记的CD71抗体和荧光标记的HBG1抗体，其中两种抗体的荧光颜色不同，
    采用流式细胞分离技术，对经荧光标记的细胞进行分离，以及
    收集同时显示两种荧光标记的细胞。
  6. 根据权利要求1所述的分离纯化方法，其中所述裂解处理的 手段为：向所述外周静脉血样中添加有机溶剂；或添加低渗盐溶液；或者添加蒸馏水。
  7. 根据权利要求6所述的分离纯化方法，其中采用低渗盐溶液进行所述的裂解处理。
  8. 根据权利要求7所述的分离纯化方法，其中还包括
    在进行裂解处理前对血样进行离心以获得血细胞粗品的步骤，以及
    在裂解处理后对血样进行离心以获得有核细胞粗品的步骤。
  9. 根据权利要求7所述的分离纯化方法，其中所述裂解液为PBS配制的0.7-1.5(w/v)％NH4Cl溶液；其用量为：相对于1ml的血样添加1ml-4ml的该裂解液。
  10. 根据权利要求1所述的分离纯化方法，其中所述细胞固定化试剂为PBS配制的1-3(v/v)％戊二醛溶液。
  11. 根据权利要求10所述的分离纯化方法，其中所述细胞固定化试剂的用量为：相对于经裂解处理过的血样中的固体物质添加0.5-3ml的该固定化试剂。
  12. 一种从孕妇外周静脉血样中分离纯化胎儿有核红细胞的试剂盒，其中包括：
    红细胞裂解液；
    细胞固定化试剂；以及
    CD45抗体标记磁珠。
  13. 根据权利要求12所述的试剂盒，还包括：
    CD71抗体标记磁珠；和
    HBG1抗体标记磁珠。
  14. 根据权利要求12所述的试剂盒，还包括：
    FITC标记的兔抗人CD71 IgG，和
    Cy3-标记的兔抗人HBG1 IgG。
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