WO2016086861A1 - 基于核酸内切酶特异性识别的细胞分选系统、方法及用途 - Google Patents

基于核酸内切酶特异性识别的细胞分选系统、方法及用途 Download PDF

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WO2016086861A1
WO2016086861A1 PCT/CN2015/096216 CN2015096216W WO2016086861A1 WO 2016086861 A1 WO2016086861 A1 WO 2016086861A1 CN 2015096216 W CN2015096216 W CN 2015096216W WO 2016086861 A1 WO2016086861 A1 WO 2016086861A1
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cell
cells
stranded nucleotide
antibody
stranded
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蓝田
盖茨J
郑敦武
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赛业（苏州）生物科技有限公司
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Priority claimed from CN201410737627.5A external-priority patent/CN104450617B/zh
Priority claimed from CN201410735762.6A external-priority patent/CN104498595B/zh
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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

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  • the present invention relates to the field of biomedical technology, and in particular to a cell sorting system, method and use based on endonuclease specific recognition.
  • the cells to be tested are combined with the monoclonal antibody and the fluorescent dye to prepare a suspended specimen, and the sample enters the flow chamber under a certain gas pressure, and is arranged in a single row under the buffer containing no cells.
  • the liquid flow breaks into uniform droplets, and the cells to be tested are contained in the droplets.
  • These droplets are charged with a positive or negative charge, and when the charged droplets pass through the electric field, they are deflected by the electric field and then fall into the corresponding collector, thereby achieving cell sorting.
  • the surface of the magnetic beads is coated with an immunoreactive antibody that specifically binds to the antigen on the cell surface; the latter is placed under a strong magnetic field and separated from the unbound cells; the magnetic field disappears immediately after the magnetic field is removed, thus
  • the labeled cells can be screened or removed for the purpose of screening for positive or negative cells.
  • the flow chart is shown in Figure 1.
  • the flow cell separation technique can sort the target cells, 1 cell must be labeled by fluorescent molecules; 2 can only detect suspended single cells; 3 sorting process is not mild enough, and the cells are greatly damaged; 4 may contaminate cells; 5 is not conducive to the sorting of large volume samples.
  • the immunomagnetic bead sorting technology can obtain the target cells through both positive and negative screening, 1 can not complete the "one-time binding" to separate a variety of cells; 2 if it is necessary to screen multiple target cells from one sample, multiple combinations and Elution, the obtained cell activity is poor, the yield is low; 3 sorting, the cost is high; 4 cells and magnetic beads can not be completely separated, which is not conducive to subsequent research.
  • the technology for small cell damage, simultaneous sorting of a variety of cells, and the obtained target cells can be completely separated from the carrier, and techniques suitable for sorting large and fragile cells have not been reported.
  • the object of the present invention is to provide a cell sorting system based on endonuclease-specific recognition for the deficiencies in the prior art.
  • Another object of the present invention is to provide a kit for sorting DC cells, NK cells, and CIK cells.
  • a fourth object of the present invention is to provide a method for sorting DC cells, NK cells and CIK cells from a cell sample.
  • a fifth object of the present invention is to provide the use of the aforementioned cell sorting system based on endonuclease-specific recognition in sorting cells.
  • a sixth object of the present invention is to provide a kit for sorting DC cells, NK cells and CIK cells as described above for sorting DC cells, NK cells and CIK cells.
  • a cell sorting system based on endonuclease-specific recognition comprising the following components: a first single-stranded nucleotide, a second single-stranded nucleotide, an antibody, a cell sorting medium, a restriction enzyme; the first single-stranded nucleotide and the second single-stranded nucleotide are complementary, and the complementary region comprises a recognition site of the restriction enzyme; the first single-stranded nucleus
  • the nucleotide, the second single-stranded nucleotide, the antibody, and the cell sorting medium are all modified, and the modified first single-stranded nucleotide can be linked to the modified antibody, and the modified second single-stranded nucleotide It can be linked to the modified cell sorting medium.
  • first single stranded nucleotide and the second single stranded nucleotide are complementary or fully complementary to a partial sequence.
  • the first single stranded nucleotide or the second single stranded nucleotide is 6 bp to 1 kb in length.
  • the first single stranded nucleotide or the second single stranded nucleotide is from 18 bp to 50 bp in length.
  • the cell sorting medium is a magnetic bead, a separation column or a polycationic nanoparticle.
  • the first single-stranded nucleotide is modified by a thiol group
  • the second single-stranded nucleotide is modified by biotin
  • the antibody is modified with maleimide
  • the magnetic beads were modified with streptavidin.
  • a method of sorting a target cell from a cell sample comprising the steps of:
  • the ratio of the volume of the cell sample in step b), the number of cells of interest in the cell sample, and the mass of the antibody corresponding to the cell of interest is: 1 mL of cell sample: 10 6 -10 9 cells of interest: 5 - 100 ⁇ g of cell of interest antibody.
  • the ratio of the "single-stranded nucleotide/antibody/cell” complex to the "magnetic bead/single-stranded nucleotide” complex described in step c) is from 1:5 to 1:100.
  • reaction conditions of step b) are 2-8 ° C, 5-30 min.
  • reaction conditions of step c) are 15-25 ° C, 5-30 min.
  • kits for sorting DC cells, NK cells and CIK cells comprising the following components: thiol and biotin modified three pairs of single-stranded nucleotides, CD3, CD56 and HLA-DR antibodies, chains Mycotoxin magnetic beads, restriction endonucleases BamH I, EcoR I, Hind III.
  • sequences of the three pairs of single-stranded nucleotides are shown in SEQ ID NO. 1 - SEQ ID NO. 6, respectively.
  • a method of sorting DC cells, NK cells, and CIK cells from a cell sample comprising the steps of:
  • the ratio of the volume of the cell sample in step b), the number of cells of interest in the cell sample, and the mass of the antibody corresponding to the cell of interest is: 1 mL of cell sample: 10 6 -10 9 cells of interest: 5 - 100 ⁇ g of cell of interest antibody.
  • the ratio of the "single-stranded nucleotide/antibody/cell” complex to the "magnetic bead/single-stranded nucleotide” complex described in step c) is from 1:5 to 1:100.
  • reaction conditions of step b) are 2-8 ° C, 5-30 min.
  • reaction conditions of step c) are 15-25 ° C, 5-30 min.
  • the "cell sorting medium” includes all media used for sorting cells, such as magnetic beads, separation columns, polycationic nanoparticles, and the like, which can achieve sorting purposes.
  • the manner of modifying the first single-stranded nucleotide, the second single-stranded nucleotide, the antibody, and the cell sorting medium is not limited to the manner described above, and any manner in which these molecules can be combined is acceptable. .
  • the invention introduces two specific recognition mechanisms based on the conventional cell sorting technology (specific recognition of double-stranded nucleotide sequence by restriction endonuclease, specific recognition of antibody and antigen), and one adsorption is realized. Multiple separations yield multiple desired products.
  • the cell sorting technology of the invention can make up for the deficiencies of the prior art, and has less damage to cells than the flow cell technology; compared with the immunomagnetic bead technology, a plurality of cells can be sorted simultaneously, and the obtained target cells can be The cell sorting medium is separated so that the cells can be used for sorting again.
  • the technique of the present invention allows for the sorting of large and fragile cells with high cell yield and survival.
  • the technology of the invention can be used as a supporting technology in cell therapy on the basis of cell sorting, and provides high-purity and viable cells for cell therapy, and can also be used in the field of detection and analysis, and is used in combination with a trace amount of nucleic acid detecting equipment.
  • the target is quantitatively tested. It brings good news to the fields of clinical diagnosis, cell separation, detection, and cell therapy.
  • Figure 1 is the principle and flow chart of the immunomagnetic bead sorting technique.
  • FIG. 2 is a schematic diagram and a flow chart of a cell sorting system based on endonuclease-specific recognition of the present invention.
  • Figure 3 is a schematic representation of the binding of single-stranded nucleotides to antibodies via Thiol/Malemide.
  • Ab antibody; Oligo: single-stranded nucleotide; SH-: thiol.
  • Figure 4 is a graph showing the results of flow cytometry detection of sorted NK cells of the present invention.
  • Figure 5 is a graph showing the results of flow cytometry of the sorted CIK cells of the present invention.
  • Figure 6 is a graph showing the results of DC cell flow cytometry assay of the present invention.
  • Figure 7 is a post-culture morphology of NK cells sorted by two methods.
  • FIG. 2 The flow chart of the sorting system of the cell sorting system based on the specific recognition of the endonuclease of the present invention is shown in FIG. 2, and the steps are as follows:
  • the "cell sorting medium / single-stranded nucleotide” structure is added to the separation system, and the two single-stranded nucleotides are paired by base pairing to form "cell sorting medium / double-stranded nucleotide / antibody / Cell” structure.
  • the target cell When the cell sorting medium is a magnetic bead, the target cell will be magnetically adsorbed by the action of an external magnetic field, thereby removing non-target cells; when the cell sorting medium is a medium other than magnetic beads Then, the corresponding method of adsorbing the medium is used to remove non-target cells.
  • different target cells can be eluted one by one, and a plurality of different cells of interest can be separated by one binding.
  • the "antibody/single-stranded nucleotide” complex may also be first combined with "cell sorting medium/single-stranded nucleotide” and then added to the cell sample.
  • the main cell types used in immune cell therapy are: DC cells, NK cells, and CIK cells. Since these cells have different culture conditions, they need to be separated and cultured to obtain a good amplification effect.
  • the purpose of this example is to separate the three cells in the blood at a time.
  • the specific separation scheme is as follows:
  • peripheral blood mononuclear cells of 7 people were extracted as follows:
  • CD3, CD5 and HLA-DR antibodies (antibody on various cell surfaces are shown in Table 1) bind to different single-stranded nucleotide fragments, respectively, and the single-stranded nucleotide sequence paired with it binds to different magnetic beads (nucleoside The acid sequence and endonuclease are shown in Table 2), and the specific endonuclease is disposed in the elution buffer.
  • biotin-modified single-stranded nucleotide was bound to the streptavidin-coated magnetic beads by the reaction conditions: the two were mixed and reacted at 37 ° C for 30 min.
  • the thiol-modified single-stranded nucleotide was reacted with a maleimide-modified antibody using the Imject Maleimide-Activated Mariculture KLH and Kit kit from Thermo.
  • the specific principle is that malemide modified by sulfo-SMCC cross-linking has the characteristics of tight binding to sulfhydryl groups and is very stable; the attachment of antibodies to single-stranded nucleotides depends on the amino group of antibody protein and SO 3 of sulfo-SMCC cross-linking agent.
  • Malemide can interact with a single-stranded nucleotide with a thiol group to form a stable thioether bond (see Figure 3).
  • the ratio of single-stranded nucleotide / antibody / cell ratio is 50:1, reaction at room temperature for 5 min;
  • HLA-DR corresponding endonuclease eluate is added to elute the DC cells
  • CD56 corresponding endonuclease eluate to elute CD56+ and CD3- cells and CD56+ cells
  • the ratio of the volume of the cell sample in step 1, the number of cells in the cell sample, and the mass of the antibody corresponding to the target cell can be in the following range: 1 mL of cell sample: 10 6 -10 9 cells of interest: 5-100 ⁇ g
  • the antibody corresponding to the target cell; the number ratio of the "single-stranded nucleotide/antibody/cell” complex described in step 2 to the "magnetic bead/single-stranded nucleotide” complex may be in the following range 1:5- 1:100;
  • Step 1 reaction conditions can be in the following range: 2-8 ° C, 5-30 min;
  • Step 2 reaction conditions can be in the following range: 15-25 ° C, 5-30 min.
  • NK cells were isolated from human peripheral blood mononuclear cells by flow cytometry and the sorting method of Example 1, respectively, and cultured to observe cell morphology.
  • Inoculation of cells A cell suspension of an appropriate concentration is inoculated into a 96-well plate in a volume of 100 ⁇ L to 200 ⁇ L/well, and 10 replicate wells are provided. In addition, blank control wells were set with the culture solution.
  • NK cells sorted by flow cytometry form a small part of uniform colonies, while the present invention is a majority of uniform colonies.
  • the colony yield of the cells selected by the present invention is significantly higher than that of flow cytometry.
  • the cell activity absorbance value (0.4941) obtained by flow cytometry sorting was significantly lower than that of the sorting method (0.798) of the present invention, indicating that the cell activity obtained by the sorting method of the present invention was high.
  • the cell sorting method of the present invention is more gentle than the flow cell sorting technique, has less cell damage, and is capable of performing a large number of cell sorting; and the conventional immunomagnetic bead sorting technique
  • a plurality of cells of interest can be sorted and combined, and the cells of interest can be selectively eluted as needed, and the obtained cells are separated from the cell sorting medium (such as magnetic beads).
  • the cell sorting medium such as magnetic beads

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Abstract

本发明公开了一种基于核酸内切酶特异性识别的细胞分选系统，所述分选系统包含以下组分：第一单链核苷酸、第二单链核苷酸、抗体、细胞分选介质和限制性内切酶。所述第一单链核苷酸和第二单链核苷酸互补，并且互补区域含有所述限制性内切酶的识别位点。所述第一单链核苷酸、第二单链核苷酸、抗体和细胞分选介质均被修饰，修饰后的第一单链核苷酸可与修饰后的抗体相连接，修饰后的第二单链核苷酸可与修饰后的细胞分选介质相连接。本发明还公开了利用上述系统分选细胞的方法及其应用，从细胞样品中分选DC细胞、NK细胞和CIK细胞的试剂盒及其方法。

Description

基于核酸内切酶特异性识别的细胞分选系统、方法及用途 技术领域
本发明涉及生物医药技术领域，具体地说，涉及一种基于核酸内切酶特异性识别的细胞分选系统、方法及用途。
背景技术
现有的细胞分选技术主要有以下两种：
①流式细胞分离技术
通过分离含有单细胞的液滴而实现的。将待测细胞与单克隆抗体和荧光染料结合后制成悬浮标本，在一定气体压力下样品进入流动室，在不含细胞的缓冲液包裹下单行排列。在流动室的超高频的压电晶体的高频振荡下，液流断裂为均匀的液滴，待测细胞就包含在液滴之中。将这些液滴充上正或负电荷，当带电液滴通过电场，在电场的作用下发生偏转，然后落入相应的收集器之中，从而实现细胞分选。
②免疫磁珠技术
磁珠表面包被具免疫反应性的抗体，与细胞表面的抗原进行特异性结合；后者置于强大的磁场下，就会与未结合的细胞分开；脱离磁场后会立即消失磁性，这样就可以筛选或去除所标记的细胞，从而达到筛选出阳性或阴性细胞的目的。其流程图如图1所示。
以上技术存在以下不足：流式细胞分离技术虽然可以分选得到目的细胞，但是①细胞必须能被荧光分子标记；②只能检测悬浮的单细胞；③分选过程不够温和，对细胞伤害大；④可能污染细胞；⑤不利于大体积样品的分选。免疫磁珠分选技术虽然可以通过正、负两种筛选获得目的细胞，但是①无法完成“一次结合”分离多种细胞；②如果需要从一个样品里面筛选多个目的细胞，需要多次结合和洗脱，得到的细胞活性差、得率低；③多次分选，成本高；④目的细胞与磁珠不能完全分离，不利于后续研究。
目前关于对细胞伤害小、可同时分选多种细胞、获得的目的细胞能够与载体彻底分离，适用于大量及脆弱细胞分选的技术还未见报道。
发明内容
本发明的目的是针对现有技术中的不足，提供一种基于核酸内切酶特异性识别的细胞分选系统。
本发明的再一的目的是，提供一种从细胞样品中分选目的细胞的方法。
本发明的另一的目的是，提供一种分选DC细胞、NK细胞和CIK细胞的试剂盒。
本发明的第四个目的是，提供一种从细胞样品中分选DC细胞、NK细胞和CIK细胞的方法。
本发明的第五个目的是，提供前述的一种基于核酸内切酶特异性识别的细胞分选系统在分选细胞中的应用。
本发明的第六个目的是，提供前述的一种分选DC细胞、NK细胞和CIK细胞的试剂盒在分选DC细胞、NK细胞和CIK细胞中的应用。
为实现上述第一个目的，本发明采取的技术方案是：
一种基于核酸内切酶特异性识别的细胞分选系统，所述的分选系统包含以下组分：第一单链核苷酸、第二单链核苷酸、抗体、细胞分选介质、限制性内切酶；所述的第一单链核苷酸和第二单链核苷酸互补，且互补区域含有所述限制性内切酶的识别位点；所述的第一单链核苷酸、第二单链核苷酸、抗体、细胞分选介质均被修饰，修饰后的第一单链核苷酸可与修饰后的抗体相连接，修饰后的第二单链核苷酸可与修饰后的细胞分选介质相连接。
其中，所述的第一单链核苷酸和第二单链核苷酸为部分序列互补或完全互补。
优选地，所述的第一单链核苷酸或第二单链核苷酸的长度为6bp-1kb。
更优选地，所述的第一单链核苷酸或第二单链核苷酸的长度为18bp-50bp。
优选地，所述的细胞分选介质是磁珠、分离柱或聚阳离子纳米颗粒。
更优选地，所述的第一单链核苷酸被巯基修饰，所述的第二单链核苷酸被生物素修饰，所述的抗体被顺丁烯二酰亚胺修饰，所述的磁珠被链霉亲和素修饰。
为实现上述第二个目的，本发明采取的技术方案是：
一种从细胞样品中分选目的细胞的方法，所述的方法包括以下步骤：
a)合成互补的单链核苷酸，制备“磁珠/单链核苷酸”和“抗体/单链核苷酸”复合物；
b)在细胞样品中加入“抗体/单链核苷酸”复合物，形成“单链核苷酸/抗体/细胞”复合物；
c)将“磁珠/单链核苷酸”复合物加入到“单链核苷酸/抗体/细胞”复合物中，形成“磁珠/双链核苷酸/抗体/细胞”复合物；
d)通过外加磁场的作用，磁性吸附目的细胞，去除非目的细胞；
e)针对不同的目的细胞，每次使用不同的限制性内切酶剪切含有特定识别序列的双链核苷酸，逐次洗脱不同的目的细胞。
优选地，步骤b)中细胞样品的体积、细胞样品中目的细胞个数、目的细胞对应的抗体质量的比例为：1mL细胞样品∶106-109个目的细胞∶5-100μg目的细胞对应的抗体。
优选地，步骤c)中所述的“单链核苷酸/抗体/细胞”复合物与“磁珠/单链核苷酸”复合物的个数比为1:5-1:100。
优选地，步骤b)反应条件为2-8℃，5-30min。
优选地，步骤c)反应条件为15-25℃，5-30min。
为实现上述第三个目的，本发明采取的技术方案是：
一种分选DC细胞、NK细胞和CIK细胞的试剂盒，所述的试剂盒包含以下组分：巯基与生物素修饰的三对单链核苷酸，CD3、CD56和HLA-DR抗体，链霉亲和素磁珠，限制性内切酶BamH I、EcoR I、Hind III。
优选地，所述的三对单链核苷酸的序列分别如SEQ ID NO.1-SEQ ID NO.6所示。
为实现上述第四个目的，本发明采取的技术方案是：
一种从细胞样品中分选DC细胞、NK细胞和CIK细胞的方法，它包括以下步骤：
a)合成三对互补的单链核苷酸，制备三种“磁珠/单链核苷酸”和三种“抗体/单链核苷酸”复合物，所述的抗体分别为CD3、CD56和HLA-DR抗体，所述的三对单链核苷酸片段的序列分别如SEQ ID NO.1-SEQ ID NO.6所示；
b)在细胞样品中加入三种“抗体/单链核苷酸”复合物，形成“单链核苷酸/抗体/细胞”复合物；
c)将三种“磁珠/单链核苷酸”复合物加入到“单链核苷酸/抗体/细胞”复合物中，形成“磁珠/双链核苷酸/抗体/细胞”复合物；
d)通过外加磁场的作用，分离获得与磁珠结合的细胞；
e)针对不同的目的细胞，分别使用限制性内切酶BamH I、EcoR I、Hind III逐次洗脱。
优选地，步骤b)中细胞样品的体积、细胞样品中目的细胞个数、目的细胞对应的抗体质量的比例为：1mL细胞样品∶106-109个目的细胞∶5-100μg目的细胞对应的抗体。
优选地，步骤c)中所述的“单链核苷酸/抗体/细胞”复合物与“磁珠/单链核苷酸”复合物的个数比为1:5-1:100。
优选地，步骤b)反应条件为2-8℃，5-30min。
优选地，步骤c)反应条件为15-25℃，5-30min。
本文中，所述的“细胞分选介质”包含所有用于分选细胞的介质，例如磁珠、分离柱、聚阳离子纳米颗粒等其他可以达到分选目的的介质。所述的第一单链核苷酸、第二单链核苷酸、抗体、细胞分选介质的修饰方式并不限于以上内容记载的方式，任何可以使这些分子结合的方式都是可以接受的。
本发明优点在于：
本发明在常规细胞分选技术的基础上引入了两种特异性识别机制(限制性内切酶对双链核苷酸序列的特异性识别、抗体与抗原的特异性识别)，实现一次吸附再多次分离得到多种目的产物。
本发明的细胞分选技术可以弥补现有技术的不足，与流式细胞技术相比对细胞伤害小；与免疫磁珠技术相比，可同时分选多种细胞，且获得的目的细胞能够与细胞分选介质分离，因而细胞可用于再次分选。本发明的技术可进行大量及脆弱细胞的分选，细胞得率和存活率高。
本发明的技术在细胞分选基础上，可作为细胞治疗中的配套技术，为细胞治疗提供高纯度、有活力的细胞，也可用于检测分析领域，与微量的核酸检测设备联合使用，对不同的目标物进行定量检测。其为临床诊断、细胞分离、检测、细胞疗法等领域带来福音。
附图说明
图1是免疫磁珠分选技术的原理与流程图。
图2是本发明基于核酸内切酶特异性识别的细胞分选系统的原理与流程图。
图3是单链核苷酸与抗体通过Thiol/Malemide结合示意图。Ab：抗体；Oligo：单链核苷酸；SH-：巯基。
图4是本发明分选的NK细胞流式细胞术检测结果。
图5是本发明分选的CIK细胞流式细胞术检测结果。
图6是本发明分选的DC细胞流式细胞术检测结果。
图7是两种方法分选的NK细胞培养后形态。A.流式细胞仪分选的细胞形态，B.本发明分选的细胞形态。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
本发明基于核酸内切酶特异性识别的细胞分选系统分选流程图如图2所示，步骤如下：
①选择不同的抗体对不同目的细胞表面标记，合成两个互补的单链寡核苷酸(约20bp)，一个单链寡核苷酸末端修饰有Thiol(巯基)，另一个单链寡核苷酸末端修饰有Biotin(生物素)。
②两个单链寡核苷酸分别与被Maleimide(顺丁烯二酰亚胺)修饰的抗体以及Streptavidin(链霉亲和素)包被的细胞分选介质表面进行反应，分别形成“细胞分选介质/单链核苷酸”和“抗体/单链核苷酸”两种结构。
③在细胞样品中加入“抗体/单链核苷酸”复合物，抗体将识别目的细胞上的表面标记，并与细胞结合，形成“单链核苷酸/抗体/细胞”结构。
④随后将“细胞分选介质/单链核苷酸”结构加入到分离体系中，两条单链核苷酸通过碱基互补配对，形成“细胞分选介质/双链核苷酸/抗体/细胞”结构。
⑤当所述的细胞分选介质为磁珠时，则通过外加磁场的作用，目的细胞将被磁性吸附，从而去除非目的细胞；当所述的细胞分选介质为磁珠以外的其它介质时，则使用相应的吸附该介质的方法以去除非目的细胞。
⑥针对不同的目的细胞，每次使用不同的限制性内切酶来剪切含有特定识别序列的核苷酸，可以逐次洗脱不同的目的细胞，完成一次结合分离多种不同目的细胞。
其中，以上步骤中，也可以先将“抗体/单链核苷酸”复合物与“细胞分选介质/单链核苷酸”结合，随后再加入到细胞样品中。
实施例1
以免疫细胞治疗为主导的细胞治疗在癌症治疗方面有很大的作用，免疫细胞治疗中用到的主要细胞类型有：DC细胞、NK细胞和CIK细胞。这些细胞因培养条件不同，所以需要分开后再进行培养才能取得良好的扩增效果。本实施例的目的是一次分离血液中的这三种细胞，具体的分离方案如下：
1实验材料
①CD3、CD56和HLA-DR抗体，购于Biolegend公司；
②巯基与生物素修饰的单链核苷酸片段，合成于金唯智公司；
③核酸内切酶BamH I、EcoR I、Hind III，购于NEB公司；
④链霉亲和素磁珠，购于东莞市汉诺生物技术有限公司；
⑤细胞培养基X-VIVOTM15 Chemically Defined,Serum-free Hematopoietic Cell Medium，购于美国Lonza公司；
⑥洗脱缓冲液(限制性核酸内切酶的反应体系即NEB公司核酸内切酶的通用Buffer)；
⑦人外周血单核细胞，提取方法如下：
a收集人外周血样品10mL，用2-4倍体积的PBS稀释样品；50mL离心管中加入15mL淋巴细胞分离液；
b将稀释的血液样品缓慢加入到淋巴细胞分离液上，尽量使血液样品保持在淋巴细胞分离液上层；
c室温×400g×30min，收集云雾层的PBMC细胞；
d 40mL无菌的PBS重悬PBMC细胞，室温×200g×10min，去上清；
e 40mL无菌的PBS重悬一次，室温×200g×10min，用冻存液重悬分装冻存。
2实验方法
CD3、CD5和HLA-DR抗体(各种细胞表面的抗体如表1)分别与不同的单链核苷酸片段结合，同时与其配对的单链核苷酸序列与不同的磁珠结合(核苷酸序列及核酸内切酶如表2)，特异性的核酸内切酶配置在洗脱缓冲液中。
生物素修饰的单链核苷酸与链霉亲和素包被的磁珠结合反应条件为：二者混合在37℃反应30min。
巯基修饰的单链核苷酸与顺丁烯二酰亚胺修饰的抗体结合反应使用Thermo公司的Imject Maleimide-Activated Mariculture KLH and Kit试剂盒。具体原理为：sulfo-SMCC交联剂修饰过的Malemide具有与巯基紧密结合的特性， 且很稳定；抗体与单链核苷酸的连接依赖抗体蛋白的氨基与sulfo-SMCC交联剂的SO3 -的相互作用；Malemide可以与带有巯基的单链核苷酸相互作用形成稳定的硫醚键(参见图3)。
表1.各种细胞的主要表面标记
细胞类型 表面标记
NK细胞 CD3-、CD56+
DC细胞 HLA-DR+
CIK细胞 CD3+、CD56+
表2.核苷酸序列及核酸内切酶
Figure PCTCN2015096216-appb-000001
实验流程如下：
①将三种抗体/单链核苷酸分别加入到人外周血单核细胞中，加入比例为人外周血体积∶目的细胞个数∶目的细胞对应的抗体质量＝1mL人外周血∶108个目的细胞∶50μg目的细胞对应的抗体。使用含有0.5％HAS进行重悬、混匀，冰上反应10min，得到单链核苷酸/抗体/细胞；
②加入磁珠/单链核苷酸，其与单链核苷酸/抗体/细胞的个数比为50:1，常温反应5min；
③放入磁力架中进行分离，分离出所有能与细胞结合的磁珠；
④将未结合的细胞收集后作为CIK细胞进行培养；
⑤在结合的磁珠中，加入HLA-DR对应的核酸内切酶洗脱液，将DC细胞洗脱下来；
⑥再加入CD56对应的核酸内切酶洗脱液，将CD56+且CD3-的细胞和CD56+的细胞洗脱下来；
⑦加入CD3对应的核酸内切酶洗脱液，将CD3+且CD56+的细胞和CD3+ 的细胞洗脱下来；
⑧将洗脱下来的细胞离心、收集，用PBS缓冲液清洗2次；
⑨将分离后的不同细胞用相应的培养基培养10d后，用流式细胞仪来鉴定分选结果。
需要说明的是：步骤①中细胞样品的体积、细胞样品中目的细胞个数、目的细胞对应的抗体质量的比例可以在以下范围：1mL细胞样品∶106-109个目的细胞∶5-100μg目的细胞对应的抗体；步骤②中所述的“单链核苷酸/抗体/细胞”复合物与“磁珠/单链核苷酸”复合物的个数比可以在以下范围1:5-1:100；步骤①反应条件可以在以下范围：2-8℃，5-30min；步骤②反应条件可以在以下范围：15-25℃，5-30min。
2实验结果
流式细胞仪鉴定结果如图4-图6所示。数据分析：从以上流式结果可以看出，使用本发明方法分选得到的NK细胞得率为97.4％，且杂细胞含量低，NK细胞得到了很好的纯化；CIK细胞得率为63.6％，虽然杂细胞较多，但可以在培养过程中得到纯化；DC细胞得率为5.4％，是普通流式细胞仪(1.7％)分选的3倍以上。
实施例2
1实验方法
①分别用流式细胞仪与实施例1的分选方法从人外周血单核细胞中分离出NK细胞，进行培养，观察细胞形态。
②MTT法检测细胞的存活率，步骤如下：
a)接种细胞：将适当浓度的细胞悬液，按照100μL～200μL/孔的体积，接种至96孔板，设10个复孔。另外，用培养液设置空白对照孔。
b)培养细胞：5％CO2，37℃孵育培养细胞。
c)呈色：培养后，加入孔内培养液体积10％的MTT溶液(5mg/mL)，孵育2～4h。
d)终止培养，离心(250g×4min)后弃掉上清。
e)每孔加100μL DMSO，放置于平板摇床上振荡5～10min，使结晶物充分融解。用二甲基亚砜设置调零孔；对照孔用空白孔调零。
f)比色：选择550nm波长，在酶联免疫监测仪上测定各孔光吸收值。
2实验结果
培养4d后的细胞形态如图7所示，细胞的存活比例如表3所示。数据分析：流式细胞仪分选的NK细胞形成小部分均一的集落，而本发明则为大部分均一的集落，本发明分选培养的细胞集落得率明显高于流式细胞仪分选的细胞；流式细胞仪分选得到的细胞活性吸光值(0.4941)，明显低于用本发明的分选方法(0.798)，说明本发明分选方法获得的细胞活性高。
表3.MTT法检测的细胞的活性
Figure PCTCN2015096216-appb-000002
以上实施例表明：本发明的细胞分选方法与流式细胞分选技术相比，分离过程更加温和，对细胞损伤小，且能够进行大量细胞分选；与传统的免疫磁珠分选技术相比，可以实现一次结合分选多种目的细胞，还可以根据需要选择性洗脱目的细胞，且获得的细胞与细胞分选介质(如磁珠)是分开的。总体来说，提高了细胞存活率和得率，节省了时间和成本。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (15)

  1. 一种基于核酸内切酶特异性识别的细胞分选系统，其特征在于，所述的分选系统包含以下组分：第一单链核苷酸、第二单链核苷酸、抗体、细胞分选介质、限制性内切酶；所述的第一单链核苷酸和第二单链核苷酸互补，且互补区域含有所述限制性内切酶的识别位点；所述的第一单链核苷酸、第二单链核苷酸、抗体、细胞分选介质均被修饰，修饰后的第一单链核苷酸可与修饰后的抗体相连接，修饰后的第二单链核苷酸可与修饰后的细胞分选介质相连接。
  2. 根据权利要求1所述的分选系统，其特征在于，所述的第一单链核苷酸或第二单链核苷酸的长度为6bp-1kb。
  3. 根据权利要求1所述的分选系统，其特征在于，所述的第一单链核苷酸或第二单链核苷酸的长度为18bp-50bp
  4. 根据权利要求1所述的分选系统，其特征在于，所述的细胞分选介质为磁珠、分离柱或聚阳离子纳米颗粒。
  5. 根据权利要求4所述的分选系统，其特征在于，所述的第一单链核苷酸被巯基修饰，所述的第二单链核苷酸被生物素修饰，所述的抗体被顺丁烯二酰亚胺修饰，所述的磁珠被链霉亲和素修饰。
  6. 一种从细胞样品中分选目的细胞的方法，其特征在于，所述的方法包括以下步骤：
    a)合成互补的单链核苷酸，制备“磁珠/单链核苷酸”和“抗体/单链核苷酸”复合物；
    b)在细胞样品中加入“抗体/单链核苷酸”复合物，形成“单链核苷酸/抗体/细胞”复合物；
    c)将“磁珠/单链核苷酸”复合物加入到“单链核苷酸/抗体/细胞”复合物中，形成“磁珠/双链核苷酸/抗体/细胞”复合物；
    d)通过外加磁场的作用，磁性吸附目的细胞，去除非目的细胞；
    e)针对不同的目的细胞，每次使用不同的限制性内切酶剪切含有特定识别序列的双链核苷酸，逐次洗脱不同的目的细胞。
  7. 根据权利要求6所述的方法，其特征在于，步骤b)中细胞样品的体积、细胞样品中目的细胞个数、目的细胞对应的抗体质量的比例为：1mL细胞样品∶106-109个目的细胞∶5-100μg目的细胞对应的抗体；步骤c)中所述的“单链核苷酸/抗体/细胞”复合物与“磁珠/单链核苷酸”复合物的个数比为1:5-1:100。
  8. 一种分选DC细胞、NK细胞和CIK细胞的试剂盒，其特征在于，所述的试剂盒包含以下组分：巯基与生物素修饰的三对单链核苷酸，CD3、CD56和HLA-DR抗体，链霉亲和素磁珠，限制性内切酶BamH I、EcoR I、Hind III。
  9. 根据权利要求8所述的试剂盒，其特征在于，所述的三对单链核苷酸的序列分别如SEQ ID NO.1-SEQ ID NO.6所示。
  10. 一种从细胞样品中分选DC细胞、NK细胞和CIK细胞的方法，其特征在于，它包括以下步骤：
    a)合成三对互补的单链核苷酸，制备三种“磁珠/单链核苷酸”和三种“抗体/单链核苷酸”复合物，所述的抗体分别为CD3、CD56和HLA-DR抗体，所述的三对单链核苷酸片段的序列分别如SEQ ID NO.1-SEQ ID NO.6所示；
    b)在细胞样品中加入三种“抗体/单链核苷酸”复合物，形成“单链核苷酸/抗体/细胞”复合物；
    c)将三种“磁珠/单链核苷酸”复合物加入到“单链核苷酸/抗体/细胞”复合物中，形成“磁珠/双链核苷酸/抗体/细胞”复合物；
    d)通过外加磁场的作用，分离获得与磁珠结合的细胞；
    e)针对不同的目的细胞，分别使用限制性内切酶BamH I、EcoR I、Hind III逐次洗脱。
  11. 根据权利要求10所述的方法，其特征在于，步骤b)中细胞样品的体积、细胞样品中目的细胞个数、目的细胞对应的抗体质量的比例为：1mL细胞样品∶106-109个目的细胞∶5-100μg目的细胞对应的抗体，步骤c)中所述的“单链核苷酸/抗体/细胞”复合物与“磁珠/单链核苷酸”复合物的个数比为1:5-1:100。
  12. 如权利要求1所述的一种基于核酸内切酶特异性识别的细胞分选系统在分选细胞中的应用。
  13. 根据权利要求12所述的用途，其特征在于，第一单链核苷酸和第二单链核苷酸为部分序列互补或完全互补。
  14. 根据权利要求12所述的用途，其特征在于，所述的第一单链核苷酸或第二单链核苷酸的长度为6bp-1kb。
  15. 如权利要求8所述的分选DC细胞、NK细胞和CIK细胞的试剂盒在 分选DC细胞、NK细胞和CIK细胞中的应用。
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CN103800898A (zh) * 2014-03-13 2014-05-21 蔡颖 一种肿瘤特异性杀伤细胞制剂及其制备方法
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