CN104450617A - Application of cell sorting system based on specific recognition of endonuclease - Google Patents
Application of cell sorting system based on specific recognition of endonuclease Download PDFInfo
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Abstract
The invention relates to application of a cell sorting system based on specific recognition of endonuclease. The cell sorting system comprises the following components including first single-chain nucleotide, second single-chain nucleotide, an antibody, a cell sorting medium and restriction endonuclease, wherein the first single-chain nucleotide and second single-chain nucleotide are complemented to each other, and in addition, recognition sites of the restriction endonuclease are contained in complementary regions; the first single-chain nucleotide, the second single-chain nucleotide, the antibody and the cell sorting medium are all modified, the modified first single-chain nucleotide can be connected with the modified antibody, and the modified second single-chain nucleotide can be connected with the modified cell sorting medium. The invention also provides a purpose of a kit for sorting DC cells, NK cells and CIK cells from cell samples. With the adoption of the cell sorting technology, various cells are simultaneously sorted, a great amount of cells and fragile cells can be sorted, and the yield and the survival rate of the cells are high.
Description
Technical field
The present invention relates to biomedicine technical field, specifically, relate to a kind of application of the cell sorting system based on endonuclease specific recognition.
Background technology
Existing cell sorting techniques mainly contains following two kinds:
1. Flow cytometry
Realize containing single celled drop by being separated.Make suspension sample after being combined with monoclonal antibody and fluorescence dye by cell to be measured, under certain gaseous tension, sample enters flow chamber, single-row layout under not celliferous damping fluid parcel.Under the HF oscillation of the piezoquartz of the ultra-high frequency of flow chamber, liquid stream is fractured into uniform drop, and cell to be measured is just included among drop.Fill these drops with plus or minus electric charge, when charged drop is by electric field, deflects under the effect of electric field, then fall among corresponding collector, thus realize cell sorting.
2. immunomagnetic bead technique
Magnetic bead surfaces bag, by the immunoreactive antibody of tool, carries out specific binding with the antigen of cell surface; Under the latter is placed in powerful magnetic field, will separate with unconjugated cell; Depart from meeting disappearing magnetism immediately behind magnetic field, so just can screen or remove marked cell, thus reach the object filtering out positive or negative cell.Its schema as shown in Figure 1.
Above technology has the following disadvantages: although Flow cytometry sorting can obtain object cell, 1. cell must be able to be marked by fluorescence molecule; 2. the unicellular of suspension can only be detected; 3. assorting room is gentle not, large to cell damage; 4. possibility contamination of cells; 5. the sorting of bulk sample is unfavorable for.Although immunological magnetic bead sorting technology can obtain object cell by positive and negative two kinds of screenings, 1. cannot complete " once combining " and be separated various kinds of cell; If 2. need to screen multiple object cell inside a sample, need repeatedly to combine and wash-out, the cytoactive obtained is poor, yield is low; 3. repeatedly sorting, cost is high; 4. object cell can not be separated completely with magnetic bead, is unfavorable for follow-up study.
At present about little to cell damage, can the various kinds of cell of sorting simultaneously, acquisition object cell thoroughly can be separated with carrier, the technology being applicable to a large amount of and fragile cell sorting have not been reported.
Summary of the invention
The object of the invention is, for deficiency of the prior art, to provide the application of the cell sorting system based on endonuclease specific recognition.
Of the present invention again one object be that the application of the test kit of sorting DC cell, NK cell and CIK cell is provided.
For realizing above-mentioned first object, the technical scheme that the present invention takes is:
Based on the application of cell sorting system in sorting cells of endonuclease specific recognition, described separation system comprises following component: the first single-stranded nucleotide, the second single-stranded nucleotide, antibody, cell sorting medium, restriction enzyme; The first described single-stranded nucleotide and the complementation of the second single-stranded nucleotide, and complementary region contains the recognition site of described restriction enzyme; The first described single-stranded nucleotide, the second single-stranded nucleotide, antibody, cell sorting medium are all modified, the first single-stranded nucleotide after modification can be connected with the antibody after modification, and the second single-stranded nucleotide after modification can be connected with the cell sorting medium after modification.
Preferably, the first described single-stranded nucleotide and the second single-stranded nucleotide are partial sequence complementarity or complete complementary.
Preferably, the first described single-stranded nucleotide or the length of the second single-stranded nucleotide are 6bp-1kb.
More preferably, the first described single-stranded nucleotide or the length of the second single-stranded nucleotide are 18bp-50bp.
Preferably, described cell sorting medium is magnetic bead, separator column or polycation nano particle.
More preferably, the first described single-stranded nucleotide is by sulfydryl modification, and the second described single-stranded nucleotide is by biotin modification, and described antibody is modified by maleimide, and described magnetic bead is modified by Streptavidin.
For realizing above-mentioned second object, the technical scheme that the present invention takes is:
The application of test kit in sorting DC cell, NK cell and CIK cell of sorting DC cell, NK cell and CIK cell, described test kit comprises following component: three pairs of single-stranded nucleotide of sulfydryl and biotin modification, CD3, CD56 and HLA-DR antibody, Streptavidin MagneSphere, restriction enzyme BamH I, EcoR I, Hind III.
The complementary region of three pairs of described single-stranded nucleotide comprises the recognition site of restriction enzyme BamH I, EcoRI, Hind III respectively.
Preferably, the sequence of three pairs of described single-stranded nucleotide is respectively as shown in SEQ ID NO.1-SEQ ID NO.6.
The invention still further relates to a kind of method of sorting object cell from cell sample, described method comprises the following steps:
A) single-stranded nucleotide that synthesis is complementary, preparation " magnetic bead/single-stranded nucleotide " and " antibody/single-stranded nucleotide " mixture;
B) in cell sample, add " antibody/single-stranded nucleotide " mixture, form " single-stranded nucleotide/antibody/cell " mixture;
C) " magnetic bead/single-stranded nucleotide " mixture is joined in " single-stranded nucleotide/antibody/cell " mixture, form " magnetic bead/double chain nucleotide/antibody/cell " mixture;
D) by the effect of externally-applied magnetic field, magnetic absorption object cell, removes non-object cell;
E) for different object cells, each double chain nucleotide using different restriction enzyme shearings to contain specific identification sequence, the object cell that successively wash-out is different.
Preferably, step b) in the volume of cell sample, object number of cells in cell sample, antibody mass that object cell is corresponding ratio be: 1mL cell sample: 10
6-10
9individual object cell: the antibody that 5-100 μ g object cell is corresponding.
Preferably, step c) described in the number of " single-stranded nucleotide/antibody/cell " mixture and " magnetic bead/single-stranded nucleotide " mixture than being 1:5-1:100.
Preferably, step b) reaction conditions is 2-8 DEG C, 5-30min.
Preferably, step c) reaction conditions is 15-25 DEG C, 5-30min.
The present invention separately relates to a kind of method of sorting DC cell, NK cell and CIK cell from cell sample, and it comprises the following steps:
A) single-stranded nucleotide of synthesis three to complementation, prepare three kinds " magnetic bead/single-stranded nucleotide " and three kinds of " antibody/single-stranded nucleotide " mixtures, described antibody is respectively CD3, CD56 and HLA-DR antibody, and the sequence of three pairs of described single stranded nucleotide acid fragments is respectively as shown in SEQ ID NO.1-SEQ ID NO.6;
B) in cell sample, add three kinds of " antibody/single-stranded nucleotide " mixtures, form " single-stranded nucleotide/antibody/cell " mixture;
C) three kinds of " magnetic bead/single-stranded nucleotide " mixtures are joined in " single-stranded nucleotide/antibody/cell " mixture, form " magnetic bead/double chain nucleotide/antibody/cell " mixture;
D) by the effect of externally-applied magnetic field, the cell obtaining and be combined with magnetic bead is separated;
E) for different object cells, restriction enzyme BamH I, EcoR I, Hind III successively wash-out is used respectively.
Preferably, step b) in the volume of cell sample, object number of cells in cell sample, antibody mass that object cell is corresponding ratio be: 1mL cell sample: 10
6-10
9individual object cell: the antibody that 5-100 μ g object cell is corresponding.
Preferably, step c) described in the number of " single-stranded nucleotide/antibody/cell " mixture and " magnetic bead/single-stranded nucleotide " mixture than being 1:5-1:100.
Preferably, step b) reaction conditions is 2-8 DEG C, 5-30min.
Preferably, step c) reaction conditions is 15-25 DEG C, 5-30min.
Herein, described " cell sorting medium " comprises all media for sorting cells, as long as the medium that can reach the object of sorting is all fine, such as magnetic bead, separator column, polycation nano particle etc. other can reach the medium of sorting object.The modification mode of the first described single-stranded nucleotide, the second single-stranded nucleotide, antibody, cell sorting medium is not limited to the mode of above content record, and any mode that these molecules can be made to combine is all acceptable.The invention has the advantages that:
The present invention introduces two species specificity recognition mechanisms (restriction enzyme is to the specific recognition of the specific recognition of Double-stranded nucleotide sequence, antibody and antigen) on the basis of regular growth sorting technology, realize primary sorption again separating for several times obtain multiple object product.
Cell sorting techniques of the present invention can make up the deficiencies in the prior art, and to compare cell damage little with Flow Cytometry; Compared with immunomagnetic bead technique, can simultaneously sorting various kinds of cell, and the object cell obtained can be separated with cell sorting medium, thus cell can be used for sorting again.Technology of the present invention can carry out the sorting of a large amount of and fragile cell, cell yield and survival rate high.
Technology of the present invention is on cell sorting basis, can be used as the supporting technology in cell therapy, for cell therapy provides high purity, great-hearted cell, also can be used for detecting analysis field, detection of nucleic acids equipment conbined usage with trace, carries out detection by quantitative to different target compounds.It is that the fields such as clinical diagnosis, cellular segregation, detection, cell therapy bring glad tidings.
Accompanying drawing explanation
Fig. 1 is principle and the schema of immunological magnetic bead sorting technology.
Fig. 2 is principle and the schema of the cell sorting system that the present invention is based on endonuclease specific recognition.
Fig. 3 is single-stranded nucleotide with antibody by Thiol/Malemide in conjunction with schematic diagram.Ab: antibody; Oligo: single-stranded nucleotide; SH-: sulfydryl.
Fig. 4 is the NK cell flow cytometry detected result of sorting of the present invention.
Fig. 5 is the CIK cell Flow cytometry result of sorting of the present invention.
Fig. 6 is the DC cell flow cytometry detected result of sorting of the present invention.
Fig. 7 is form after the NK cell cultures of two kinds of method sortings.A. the cellular form of selected by flow cytometry apoptosis, the cellular form of B. sorting of the present invention.
Embodiment
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.
The present invention is based on the cell sorting system sorting schema of endonuclease specific recognition as shown in Figure 2, step comprises:
1. select different antibody to different object cell surface marker, synthesize two complementary single stranded oligonucleotides (about 20bp), single stranded oligonucleotide is end modified Thiol (sulfydryl), and another single stranded oligonucleotide is end modified Biotin (vitamin H).
2. the cell sorting dielectric surface that two single stranded oligonucleotides wrap quilt with the antibody modified by Maleimide (maleimide) and Streptavidin (Streptavidin) respectively reacts, and forms " cell sorting medium/single-stranded nucleotide " and " antibody/single-stranded nucleotide " two kinds of structures respectively.
3. in cell sample, add " antibody/single-stranded nucleotide " mixture, antibody by the surface markers on identifying purpose cell, and and Cell binding, formed " single-stranded nucleotide/antibody/cell " structure.
4. join in separation system by " cell sorting medium/single-stranded nucleotide " structure subsequently, two single-stranded nucleotide, by base pair complementarity, form " cell sorting medium/double chain nucleotide/antibody/cell " structure.
5. when described cell sorting medium is magnetic bead, then by the effect of externally-applied magnetic field, object cell by magnetic absorption, thus will remove non-object cell; When described cell sorting medium is other medium beyond magnetic bead, then use the method for corresponding this medium of absorption to remove non-object cell.
6. for different object cells, each different restriction enzymes is used to shear Nucleotide containing specific identification sequence, can object cell that successively wash-out is different, complete once to combine being separated multiple different object cell.
Wherein, in above step, also first " antibody/single-stranded nucleotide " mixture and " cell sorting medium/single-stranded nucleotide " can be combined, join in cell sample subsequently again.
Embodiment 1
The cell therapy of taking as the leading factor with immune cell therapy has very large effect in cancer therapy, and the major cell types used in immune cell therapy has: DC cell, NK cell and CIK cell.These cells are because of culture condition difference, so needs carry out cultivating one's ability obtaining good expanding effect after separating again.The object of the present embodiment is these the three kinds of cells in flash liberation blood, and concrete separation scheme is as follows:
1 experiment material
1. CD3, CD56 and HLA-DR antibody, is purchased from Biolegend company;
2. the single stranded nucleotide acid fragment of sulfydryl and biotin modification, synthesizes in Jin Weizhi company;
3. endonuclease BamH I, EcoR I, Hind III, be purchased from NEB company;
4. Streptavidin MagneSphere, is purchased from Dongguan City Han Nuo Bioisystech Co., Ltd;
5. cell culture medium X-VIVO
tM15Chemically Defined, Serum-free HematopoieticCell Medium, is purchased from Lonza company of the U.S.;
6. elution buffer (reaction system of restriction endonuclease and the general Buffer of NEB company endonuclease);
7. human peripheral blood mononuclear cell, extracting method is as follows:
A collector peripheral blood sample 10mL, with the PBS dilute sample of 2-4 times of volume; 15mL lymphocyte separation medium is added in 50mL centrifuge tube;
The blood sample of dilution slowly joins on lymphocyte separation medium by b, makes blood sample remain on lymphocyte separation medium upper strata as far as possible;
C room temperature × 400g × 30min, collects the PBMC cell of cloud and mist layer;
The resuspended PBMC cell of PBS that d 40mL is aseptic, room temperature × 200g × 10min, removes supernatant;
The aseptic PBS of e 40mL is once resuspended, and room temperature × 200g × 10min is frozen with the resuspended packing of frozen storing liquid.
2 experimental techniques
CD3, CD5 and HLA-DR antibody (antibody of various cell surface is as table 1) combines from different single stranded nucleotide acid fragments respectively, be combined with different magnetic beads (nucleotide sequence and endonuclease are as table 2) from the single stranded nucleotide sequence of its pairing, specific endonuclease is configured in elution buffer simultaneously.
The single-stranded nucleotide of biotin modification and the magnetic bead association reaction condition of Streptavidin bag quilt are: the two is blended in 37 DEG C and reacts 30min.
The antibodies that the single-stranded nucleotide of sulfydryl modification and maleimide are modified reacts the Imject Maleimide-Activated Mariculture KLH and Kit test kit using Thermo company.Concrete principle is: the Malemide of sulfo-SMCC linking agent modified has the characteristic of combining closely with sulfydryl, and very stable; The amino of the join dependency antibody protein of antibody and single-stranded nucleotide and the SO of sulfo-SMCC linking agent
3 -interaction; Malemide can interact with the single-stranded nucleotide with sulfydryl and form stable thioether bond (see Fig. 3).
The major surfaces mark of the various cell of table 1.
| Cell type | Surface markers |
| NK cell | CD3-、CD56+ |
| DC cell | HLA-DR+ |
| CIK cell | CD3+、CD56+ |
Table 2. nucleotide sequence and endonuclease
Experiment flow is as follows:
1. joined respectively in human peripheral blood mononuclear cell by three kinds of antibody/single-stranded nucleotide, additional proportion is human peripheral volume: object number of cells: the antibody mass that object cell is corresponding=1mL human peripheral: 10
8individual object cell: the antibody that 50 μ g object cells are corresponding.Use and carry out resuspended, mixing containing 0.5%HAS, react 10min on ice, obtain single-stranded nucleotide/antibody/cell;
2. add magnetic bead/single-stranded nucleotide, it is 50:1, normal-temperature reaction 5min with the number ratio of single-stranded nucleotide/antibody/cell;
3. put into magnetic frame to be separated, isolate all can with the magnetic bead of Cell binding;
4. cultivate after unconjugated cell harvesting as CIK cell;
5., in the magnetic bead combined, add the endonuclease elutriant that HLA-DR is corresponding, DC cell is eluted;
6. add the endonuclease elutriant that CD56 is corresponding again, by CD56+ and the cell of the cell of CD3-and CD56+ elute;
7. add the endonuclease elutriant that CD3 is corresponding, by CD3+ and the cell of the cell of CD56+ and CD3+ elute;
8. by elute cell centrifugation, collection, by PBS buffer solution for cleaning 2 times;
9. by be separated after different cells with after corresponding culture medium culturing 10d, identify separation results with flow cytometer.
It should be noted that: step 1. in the volume of cell sample, object number of cells in cell sample, antibody mass that object cell is corresponding ratio can in following scope: 1mL cell sample: 10
6-10
9individual object cell: the antibody that 5-100 μ g object cell is corresponding; Step 2. described in the number ratio of " single-stranded nucleotide/antibody/cell " mixture and " magnetic bead/single-stranded nucleotide " mixture can at following scope 1:5-1:100; Step 1. reaction conditions can in following scope: 2-8 DEG C, 5-30min; Step 2. reaction conditions can in following scope: 15-25 DEG C, 5-30min.
2 experimental results
Flow cytometry result as Figure 4-Figure 6.Data analysis: as can be seen from upflowing result, the NK cell yield using the inventive method sorting to obtain is 97.4%, and heteroproteose cell content is low, and NK cell obtains good purifying; CIK cell yield is 63.6%, although heteroproteose cell is more, can obtain purifying in culturing process; DC cell yield is 5.4%, is more than 3 times of ordinary stream cell instrument (1.7%) sorting.
Embodiment 2
1 experimental technique
1. from human peripheral blood mononuclear cell, isolate NK cell with the sorting method of flow cytometer and embodiment 1 respectively, cultivate, observation of cell form.
2. mtt assay detects the survival rate of cell, and step is as follows:
A) inoculating cell: by the cell suspension of proper concn, according to the volume in 100 μ L/ holes, μ L ~ 200, is seeded to 96 orifice plates, if 10 multiple holes.In addition, blank control wells is set with nutrient solution.
B) culturing cell: 5%CO
2, hatch culturing cell for 37 DEG C.
C) colour generation: after cultivation, adds the MTT solution (5mg/mL) of nutrient solution volume 10% in hand-hole, hatches 2 ~ 4h.
D) stop cultivating, after centrifugal (250g × 4min), discard supernatant.
E) every hole adds 100 μ L DMSO, is positioned over vibration 5 ~ 10min on plate shaker, crystallisate is fully melted.By dimethyl sulfoxide (DMSO), zeroing hole is set; Control wells blank well returns to zero.
F) colorimetric: select 550nm wavelength, enzyme linked immunological monitor measures each hole absorbance value.
2 experimental results
Cultivate the cellular form after 4d as shown in Figure 7, the survival ratio of cell is as shown in table 3.Data analysis: the NK cell of selected by flow cytometry apoptosis forms the homogeneous colony of small portion, and the present invention is then most of homogeneous colony, and sorting cultured cells colony yield of the present invention is apparently higher than the cell of selected by flow cytometry apoptosis; The cytoactive light absorption value (0.4941) that selected by flow cytometry apoptosis obtains, is starkly lower than with sorting method of the present invention (0.798), illustrates that the cytoactive that sorting method of the present invention obtains is high.
The activity of the cell that table 3.MTT method detects
Above embodiment shows: cell sorting method of the present invention is compared with fluidic cell sorting technology, and sepn process is gentleer, little to cell injury, and can carry out a large amount of cell sorting; Compared with traditional immunological magnetic bead sorting technology, can realize once in conjunction with the multiple object cell of sorting, can also selective elution object cell as required, and the cell obtained is separate with cell sorting medium (as magnetic bead).On the whole, improve cell survival rate and yield, save time and cost.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (9)
1. based on the application of cell sorting system in sorting cells of endonuclease specific recognition, it is characterized in that, described separation system comprises following component: the first single-stranded nucleotide, the second single-stranded nucleotide, antibody, cell sorting medium, restriction enzyme; The first described single-stranded nucleotide and the complementation of the second single-stranded nucleotide, and complementary region contains the recognition site of described restriction enzyme; The first described single-stranded nucleotide, the second single-stranded nucleotide, antibody, cell sorting medium are all modified, the first single-stranded nucleotide after modification can be connected with the antibody after modification, and the second single-stranded nucleotide after modification can be connected with the cell sorting medium after modification.
2. application according to claim 1, is characterized in that, the first described single-stranded nucleotide and the second single-stranded nucleotide are partial sequence complementarity or complete complementary.
3. application according to claim 1, is characterized in that, the first described single-stranded nucleotide or the length of the second single-stranded nucleotide are 6bp-1kb.
4. application according to claim 3, is characterized in that, the first described single-stranded nucleotide or the length of the second single-stranded nucleotide are 18bp-50bp.
5. application according to claim 1, is characterized in that, described cell sorting medium is magnetic bead, separator column or polycation nano particle.
6. application according to claim 5, it is characterized in that, the first described single-stranded nucleotide is by sulfydryl modification, and the second described single-stranded nucleotide is by biotin modification, described antibody is modified by maleimide, and described magnetic bead is modified by Streptavidin.
7. the application of test kit in sorting DC cell, NK cell and CIK cell of sorting DC cell, NK cell and CIK cell, it is characterized in that, described test kit comprises following component: three pairs of single-stranded nucleotide of sulfydryl and biotin modification, CD3, CD56 and HLA-DR antibody, Streptavidin MagneSphere, restriction enzyme BamH I, EcoR I, Hind III.
8. application according to claim 7, is characterized in that, the complementary region of three pairs of described single-stranded nucleotide comprises the recognition site of restriction enzyme BamH I, EcoR I, Hind III respectively.
9. application according to claim 8, is characterized in that, the sequence of three pairs of described single-stranded nucleotide is respectively as shown in SEQ ID NO.1-SEQ ID NO.6.
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| CN105087788A (en) * | 2015-08-03 | 2015-11-25 | 上海白泽医疗器械有限公司 | Immunomagnetic bead for sorting human cells and preparation and cutting-off method of immunomagnetic bead |
| WO2016086861A1 (en) * | 2014-12-05 | 2016-06-09 | 赛业（苏州）生物科技有限公司 | Cell sorting system and method based on endonuclease specific recognition, and applications thereof |
| CN114807031A (en) * | 2022-05-13 | 2022-07-29 | 山东赛恩福干细胞工程集团有限公司 | Construction method of human peripheral blood immune cell bank and stem cell bank |
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| CN101165173A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for carrying cell sorting by using gold magnetism particles |
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| WO2016086861A1 (en) * | 2014-12-05 | 2016-06-09 | 赛业（苏州）生物科技有限公司 | Cell sorting system and method based on endonuclease specific recognition, and applications thereof |
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| CN105087788B (en) * | 2015-08-03 | 2018-11-23 | 上海白泽医疗器械有限公司 | It is a kind of sort people's cell immunomagnetic beads, its preparation and cutting method |
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