CN1142270C - Method of separating megakaryo archeocyte - Google Patents
Method of separating megakaryo archeocyte Download PDFInfo
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- CN1142270C CN1142270C CNB011201509A CN01120150A CN1142270C CN 1142270 C CN1142270 C CN 1142270C CN B011201509 A CNB011201509 A CN B011201509A CN 01120150 A CN01120150 A CN 01120150A CN 1142270 C CN1142270 C CN 1142270C
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- Prior art keywords
- cell
- lin
- separating
- bead separation
- antibody
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Abstract
The present invention comprises the following steps: a mixture (which comprises CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, CD41 and Glycophorin A) of a specificity monoclonal antibody and an immunomagnetic bead separation system are used for removing cells whose differentiation direction is determined; an IL-6 receptor antibody and the immunomagnetic bead separation system are used for separating an unexpression IL-6 receptor, namely that an Lin<->/CD126<-> cell is obtained through a two-step immunomagnetic bead separation method. Invitro directional inducing differentiation experiments indicate that megakaryocytes can be efficiently induced and differentiated in a directional mode by the cell. The cell is an optimal precursor cell which leads megakaryocytes to be differentiated at present.
Description
Technical field
The present invention relates to a kind of method of utilizing the immunomagnetic beads two-step approach to separate megakaryoblast.
Reach at present domestic all simple employing separation of C D34 in the world
+Cell is induced the precursor cell of differentiation as megalokaryocyte, and not seeing has the report that separates the megakaryoblast method.
Hemopoietic stem cell (HSC) is kept body hematopoiesis by means of the ability of its powerful self and polyphyly differentiation and is in a relative constant state, and continues all one's life.Therefore, HSC is considered to carry out hemopoietic tissue and transplants the key component that the back rebuilds long-term hematopoiesis and immunologic function, and is optimal target cell in the gene therapy of some disease.Thereby how effectively isolating hematopoietic stem cells is the focus of hematology research field all the time, and this makes the research of hemopoietic stem cell phenotype seem particularly important.In recent years, the foundation of the discovery of hematopoietic cell sorting technology, hemopoieticgrowth factor and clone, various ex vivo operative techniquies and perfect, make hematopoietic cell be easier to operation and use in the transformation of quantity, performance and function aspects, thereby emerging research and Application Areas have been produced, i.e. the hematopoietic cell engineering.The hematopoietic cell engineering is to utilize the height multiplication capacity and the multidirectional differentiation potential of hematopoietic stem, technology by cell engineering, in in-vitro simulated or intravital hematopoiesis environment of partial simulation or process, hematopoietic stem to separation and purification is carried out amplification in vitro, the directional induction differentiation, function activation and regulation and control, goal gene transfection etc., thereby the hemopoietic progenitor cell that a large amount of at short notice amplifications are early stage and the hemopoietic forebody cell in each stage, and a large amount of red corpuscle of directional induction amplification, grain/scavenger cell, megalokaryocyte/thrombocyte, dendritic cell, the NK cell, functioning cell and immunologically competent cells such as T/B lymphocyte, and can the function of part cell be activated and regulate and control, the defective gene is carried out the target transfection, and hematopoietic cell is treated more extensive and is effectively applied to stem cell transplantation the most at last, the biological immune treatment, fields such as hematopoiesis supportive treatment and gene therapy.The application of hematopoietic cell engineering has two bases, and the one, the separation of precursor cell (because the growth of the meta-bolites of mature cell meeting pair cell in the vitro culture process produces very adverse influence), the 2nd, the application of vitro culture system.The megalokaryocyte vitro culture is the most difficult up to now cell, is the easily Megakaryocytic growth of influence of multiple factor on the one hand, is to be difficult to obtain megakaryoblast on the other hand.
Background technology
At present mostly clinical method of separating HSC with the laboratory is to carry out at the CD34 molecule, and mostly be and utilize carrier magnetic bead method isolated cell, so-called immunomagnetic beads method is to utilize carrier magnetic bead binding immunoassay aglucon (antibody, antigen etc.) constitute tripping device, utilize antigen to realize separating of pair cell with the characteristic that antibodies specific combination and magnetic bead can effectively be adsorbed by magnetic field, and select the target cell difference of absorption to have negative the selection and the positive dual mode of selecting according to it, the target cell that collection is adsorbed by magnetic field is called positive the selection, collects the target cell of not adsorbed by magnetic field and is called negative the selection; At present to HSC separation mostly be positive and select, promptly utilize CD34 monoclonal antibody and cell to hatch and then utilize to wrap to be resisted and hatch by two of magnetic bead, the cell of staying like this in the magnetic field is CD34
+Cell; In recent years for showing with in vitro study in the body of HSC, it is specificity marker (Osawa M that the stem cell that can rebuild long-term hematopoiesis and immunologic function is not expressed, Hanada K, Hamada H, et al.Long-term lymphohematopoietic reconstitution by a single CD34
-low/ negitivehematoloietic stem cell.Science, 1996,173:242-245).The CD126 molecule is the acceptor (IL-6R) of IL-6, studies show that CD34
+Cell can be divided into two cell subsets, expresses IL-6R and does not express IL-6R, IL-6R
+Cell can be stimulated to form grain-scavenger cell, lymphocyte and granulocyte, and IL-6R
-Cell can form red system when being given IL-6/sIL-6R and macronucleus is a cell, and therefore provable megakaryoblast is present in IL-6R
-In the cell mass, we utilize These characteristics to adopt isolating method of two step of immunomagnetic beads to set up a kind of separation method of megakaryoblast.Promptly not expressing according to progenitor cell is the characteristics of specificity marker, utilizes the negative method of selecting to express and is the cell removal of specific antigens, utilizes CD126 molecule (IL-6R) to carry out feminine gender again and select, and has so just obtained Lin
-/ CD126
-Cell.
Summary of the invention
Content of the present invention is unexposed to be delivered, and those skilled in the art can not obtain separation method of the present invention according to existing technology deduction as not spending creative work at all.
The purpose of this invention is to provide a kind of method of separating megakaryoblast, so that external can efficiently inducing differentiates megalokaryocyte to be applied to basis and clinical.
Of the present invention being used to is the isolating monoclonal antibody combination of negative cells: CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, CD41 and Glycophorin A, said components and separating effect are closely related, and all maturations of having included grain system, monokaryon, red system, macronucleus system and lymphoid lineage cell substantially are specificity marker.By the cell after the negative selection of this antibody combination is Lin
-Cell; Utilize the CD126 molecule to carry out the negative selection of immunomagnetic beads again, promptly obtain Lin
-/ CD126
-Cell.
By to the separation of Cord blood mononuclearcell and utilize this method isolated cells directional induction in vitro differentiation, flow cytometry analysis studies show that the isolated cell of the present invention can differentiate megalokaryocyte (CD41 external efficiently inducing
+/ CD61
+Cell).Induce that the shared ratio of megalokaryocyte can reach (cell isolation method is in the past induced only and differentiated about 30%) more than 60% after 14 days.
With embodiment the present invention is further elaborated below.
Four, description of drawings (seeing the Figure of description page or leaf)
Figure of description is depicted as: Lin
-/ CD126
-Cells in vitro is to megalokaryocyte directional induction noble cells phenotype analytical flow cytometer detected result.A, induce differentiation before; B, induce differentiation after.
Five, embodimentSeparation one method of embodiment 1. Cord blood mononuclearcells
1. the bleeding of the umbilicus of anticoagulant heparin (from Yongding Lu hospital) is in 1: 1 ratio and PBS mixing, again by 4: 1 with
0.5% methylcellulose gum mixing, room temperature left standstill 30 minutes, treated that the red corpuscle natural subsidence is clearly demarcated to boundary.
2. the sucking-off supernatant places the 50ml centrifuge tube, centrifugal 5 minutes of 20 ℃, 1500rpm.
3. abandon supernatant, add the 5mlPBS re-suspended cell.
4. in the 10ml centrifuge tube, add the 5ml lymphocyte separation medium earlier, slowly add the 5ml cell again along tube wall
Suspension, centrifugal 20 minutes of 20 ℃, 1500rpm are isolated mononuclearcell.
5. collect the interface mononuclearcell, wash with PBS.
6. with PBS suspension cell counting, standby.Annotate: above operation is all in strict accordance with aseptic technique rules two. the result
Can from Cord blood, isolate (8.465 ± 4.04) * 10 through aforesaid method
7Individual mononuclearcell.It is monoclonal antibody specific combination and immunomagnetic beads segregative line feminine gender (Lin that embodiment 2. utilizes
-) cell one, method () immunomagnetic beads mark
1 gets isolating Cord blood mononuclearcell 1 * 10
8, be diluted to 1ml with PBS, add 100 μ l
The cocktail mixtures of antibodies (comprise CD2, CD3, CD14, CD16, CD19, CD24,
CD56, CD66b, CD41 and GlycophorinA), mix, hatched on ice 30 fens
Clock or incubated at room 15 minutes.
The 2PBS washed cell.
3 every ml cells add 60 μ l magnetic beads, mixing.
4 hatched 30 minutes or incubated at room 15 minutes on ice.(2) separating step:
1 pillar is installed:
1) magnet stand is vertically placed.
2) all operations all carries out under aseptic condition.
3) from sterile bag, take out pillar, note not running into the joint of pillar.
4) pillar is installed in the magnetic field, notices that joint can not contact magnetic bead and magnet stand.
5) take out three joint pistons, and syringe needle is connected on the piston.
The preparation of 2 pillars (note: in the initialize of pillar, wash and the application of sample process in whenever all do not allow
Pillar stream is empty)
1) magnet and pillar are installed, are removed vertical stopper.
2) three joint pistons are set, make liquid enter pillar from the side junction.
3) syringe of a sterilization is filled PBS, remove bubble, be connected to the side of three joints.
4) piston on the pushing syringe slowly is pressed onto PBS in the pillar and reaches stainless steel-based up to PBS
More than the face, do not allow bubble enter mesh,, flick post jamb, can get rid of gas as bubble is arranged in the post
Bubble.
5) wash post: a waste liquid cylinder is put in the syringe needle lower end, remove on the syringe needle lid, add slow from styletable
Towards liquid (PBS that contains 5%FBS), rotate three joint pistons, make damping fluid flow to pin from pillar
Head.
6) continue to add damping fluid, up to the liquid of collecting 3 times of column volumes.When liquid level just in post
When matrix was above, rotory piston stoped medium to continue to flow out from post.
3 isolated cells
1) upper end from pillar adds sample.
2) rotory piston makes medium enter collection tube by syringe needle, adds the 8ml damping fluid and washes post (all collecting).
3) collect effusive cell and be Lin
-Cell.Two results
By aforesaid method isolated Lin from Cord blood
-The ratio that cell accounts for mononuclearcell is (0.66 ± 0.331) %; The cell absolute number is: (5.59 ± 2.79) * 10
5Embodiment 3.Lin
-/ CD126
-The separation one of cell. method
1. get CD126 antibody (anti-IL-8 6 receptor antibodies) working fluid 50 μ l (Backman Coulter)
Mix with 50 μ l immunomagnetic beadses, mixing was hatched 30 minutes for 4 ℃.
2. with isolating Lin
-Cell and said mixture mixing were hatched 30 minutes for 4 ℃.
3. the mixture with cell and antibody places in the 5ml separator tube, and fully mixing places Backman
Room temperature leaves standstill on the Coulter magnet stand, up to magnetic bead be affixed on magnetic field one side, in vitro liquid limpid till.
In magnetic field gently with the liquid sucking-off, centrifugal collecting cell is CD126
-/ Lin
-Cell.
5. add PBS mixing washed cell.Two. the result
CD126
-/ Lin
-Cell accounts for Lin
-The ratio of cell is: 49.05 ± 7.07%, and the cell absolute number is: (2.74 ± 0.40) * 10
5Embodiment 4.Lin
-/ CD126
-Cells in vitro is to megalokaryocyte directional induction differentiation one. method
1. cell cultures:
Contain SCF 100ng/ml in IMDM (Gibco company) substratum, TPO 4U/ml, IL-6 100ng/ml,
SIL-6R 200ng/ml, 2%BSA, Regular Insulin 10 μ g/ml, Transferrins,iron complexes 200 μ g/ml, 2-mercapto
Base ethanol 0.01mmol/L, L-glutamine 2mg/ml, folic acid 50ng/ml, vitamins B
12
50ng/ml。Lin
-/ CD126
-Cell inoculation concentration is 2 * 10
4/ ml is at 37 ℃, 5%CO
2, 5%O
2,
90%N
2Cultivate under the condition, half amount is changed liquid weekly, and per 3 days additional above-mentioned substances once.The 7th day and
Collect the part cell in 14 days and be used for cell counting and megalokaryocyte phenotype analytical.
2. cell counting and cell surface marker analysis
Cell directly with the tally counting, behind the PBS washed cell, is used PE-CD41, FITC-CD61 Dan Ke
Grand antibody adds in the cell suspension of PBS suspension, hatched on ice 30 minutes, and washed cell, streaming is thin
Born of the same parents' instrument detects (Coulter).Establish PE-IgG simultaneously
1And FITC-IgG
1The negative control antibody of mark.Two. the result
Lin
-/ CD126
-Cell is under above-mentioned cell culture condition, and total cellular score can increase 35 ± 2.75 times at the 7th day that cultivates, and total cellular score can increase 94.5 ± 12.96 times in 14 days, and the flow cytometer detected result shows CD41
+/ CD61
+Cell (macronucleus is a cell) ratio can reach 67% (seeing Figure of description).
Claims (1)
1. method of separating megakaryoblast, it is characterized in that utilizing immunomagnetic beads two-step approach isolated cell promptly earlier with being that specific monoclonal antibody comprises that CD2, CD3, CD14, CD16, CD19, CD24, CD41, CD56, CD66b and GlycophorinA and cell to be separated hatch, thereby to remove the cell acquisition Lin to lymph, monokaryon, granulocyte, macronucleus and the differentiation of red system
-Cell is hatched with antibody and the cell of CD126 again, thereby obtains Lin
-/ CD126
-Cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011201509A CN1142270C (en) | 2001-07-09 | 2001-07-09 | Method of separating megakaryo archeocyte |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011201509A CN1142270C (en) | 2001-07-09 | 2001-07-09 | Method of separating megakaryo archeocyte |
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| Publication Number | Publication Date |
|---|---|
| CN1322816A CN1322816A (en) | 2001-11-21 |
| CN1142270C true CN1142270C (en) | 2004-03-17 |
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ID=4663936
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011201509A Expired - Lifetime CN1142270C (en) | 2001-07-09 | 2001-07-09 | Method of separating megakaryo archeocyte |
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|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948802A (en) * | 2010-08-31 | 2011-01-19 | 中国人民解放军第三军医大学第一附属医院 | Method for sorting human articular cartilage CD105+/CD166+ mesenchymal stem cells by using immunomagnetic beads |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2321646A1 (en) * | 2008-08-04 | 2011-05-18 | Synmed Research GmbH | Method for characterizing, in particular for quantifying, molecular markers that are intracellularly absorbed from tissues by blood macrophages that are recirculated from the tissues into the circulatory system |
| CN105255831A (en) * | 2015-11-16 | 2016-01-20 | 广州赛莱拉干细胞科技股份有限公司 | Separation method for megakaryocyte progenitors |
-
2001
- 2001-07-09 CN CNB011201509A patent/CN1142270C/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948802A (en) * | 2010-08-31 | 2011-01-19 | 中国人民解放军第三军医大学第一附属医院 | Method for sorting human articular cartilage CD105+/CD166+ mesenchymal stem cells by using immunomagnetic beads |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1322816A (en) | 2001-11-21 |
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Owner name: BEIJING JIACHENHONG BIOLOGICAL TECHNOLOGY CO., LT Free format text: FORMER OWNER: FEILD FLOOD TRANSFUSION INST., ACADEMY OF MILITARY MEDICINE SCIENCES, PLA Effective date: 20061013 |
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Effective date of registration: 20061013 Address after: 100176 No. 11 Wanyuan street, Beijing economic and Technological Development Zone Patentee after: Beijing Jia Chen Hong Biological Technologies Company Limited Address before: 100850 No. 27 Taiping Road, Beijing, Haidian District Patentee before: Inst. of Field Blood Transfusion, Academy of Military Medical Sciences, PLA |
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Address after: No. 4, Yonglu North Road, Beijing Economic and Technological Development Zone zip code: 100176(Yizhuang) Patentee after: Beijing Jia Chen Hong Biological Technologies Company Limited Address before: No. 11 Wanyuan street, Beijing economic and Technological Development Zone Patentee before: Beijing Jia Chen Hong Biological Technologies Company Limited |
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Assignee: Guangzhou Tianhe Nuoya Biological Engineering Co., Ltd. Assignor: Beijing Jia Chen Hong Biological Technologies Company Limited Contract fulfillment period: 2009.6.1 to 2015.5.31 Contract record no.: 2009440001050 Denomination of invention: Method of separating megakaryo archeocyte Granted publication date: 20040317 License type: Exclusive license Record date: 20090727 |
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Free format text: EXCLUSIVE LICENSE; TIME LIMIT OF IMPLEMENTING CONTACT: 2009.6.1 TO 2015.5.31; CHANGE OF CONTRACT Name of requester: GUANGZHOU TIANHE NUOYA BIO-ENGINEERING CO., LTD. Effective date: 20090727 |
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Assignee: Guangzhou Tianhe Nuoya Biological Engineering Co., Ltd. Assignor: Beijing Jia Chen Hong Biological Technologies Company Limited Contract record no.: 2009440001050 Date of cancellation: 20100504 |
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