The growth factor combination of amplifying candidate stem cell in vitro and its application
阅读说明：本技术 体外扩增造血干细胞的生长因子组合物及其应用 (The growth factor combination of amplifying candidate stem cell in vitro and its application ) 是由 肖业臣 冉秋菊 周佳稔 于 2019-07-29 设计创作，主要内容包括：本发明公开了一种体外扩增造血干细胞的生长因子组合物,包括：干扰素α、肝素以及白细胞介素12。本发明还公开了一种所述的生长因子组合物在体外扩增造血干细胞中的应用,通过将所述生长因子组合物添加到体外培养造血干细胞的培养液中,可实现快速、有效扩增造血干细胞,可提高体外造血干细胞扩增数量达到7689倍,以解决现有技术中培养效率低下、数量不足的问题。(The invention discloses a kind of growth factor combinations of amplifying candidate stem cell in vitro, comprising: interferon-' alpha ', heparin and interleukin 12.The invention also discloses the applications in the growth factor combination described in one kind in vitro amplifying candidate stem cell, by the way that the growth factor combination is added in the culture solution of in vitro culture candidate stem cell, quick, effective amplifying candidate stem cell can be achieved, Hematopoiesis in Vitro expansion of stem cells quantity can be improved and reach 7689 times, to solve the problems, such as that culture efficiency is low in the prior art, lazy weight.)
1. a kind of growth factor combination of amplifying candidate stem cell in vitro characterized by comprising interferon-' alpha ', heparin and Interleukin 12.
2. the growth factor combination of amplifying candidate stem cell in vitro as described in claim 1, which is characterized in that further include: Stem cell factor, interleukin-13, thrombopoietin, Fms sample tyrosine kinase receptor 3 ligand and insulin-like growth The factor.
3. a kind of such as answering in the described in any item growth factor combinations of claim 1-2 in vitro amplifying candidate stem cell It is that will contain the DMEM culture solution of 10% serum and growth factor combination mixing amplification in vitro with, which is characterized in that the application Candidate stem cell.
4. growth factor combination as claimed in claim 3 application in amplifying candidate stem cell in vitro, which is characterized in that The growth factor includes interferon-' alpha ', heparin, interleukin 12, stem cell factor, interleukin-13, thrombocytopoiesis Element, the ligand of Fms sample tyrosine kinase receptor 3 and insulin-like growth factor.
5. growth factor combination as claimed in claim 3 application in amplifying candidate stem cell in vitro, which is characterized in that The interferon-' alpha ', heparin, interleukin 12, stem cell factor, interleukin-13, thrombopoietin, Fms sample tyrosine The ligand of kinases receptors 3 and the addition concentration of insulin-like growth factor are respectively 1-100ng/ml.
6. growth factor combination as claimed in claim 3 application in amplifying candidate stem cell in vitro, which is characterized in that The interferon-' alpha ' addition concentration is 50ng/ml, heparin addition concentration is 10ug/ml, interleukin 12 addition is dense Degree is 10ng/ml, stem cell factor addition concentration is 100ng/mL, interleukin-13 addition concentration is 100ng/ ML, thrombopoietin addition concentration are 100ng/mL, the ligand of the Fms sample tyrosine kinase receptor 3 adds concentration The addition concentration of 100ng/ml and the insulin-like growth factor is 10ng/ml.
7. growth factor combination as claimed in claim 3 application in amplifying candidate stem cell in vitro, which is characterized in that The amplifying candidate stem cell in vitro is obtained from the marrow of people or other lactations such as umbilical cord blood hematopoietic stem cell sample and mouse are moved The hematopoietic stem/progenitor in object source.
8. growth factor combination as claimed in claim 3 application in amplifying candidate stem cell in vitro, which is characterized in that The condition of the amplifying candidate stem cell in vitro are as follows: 37 DEG C, cultivate 14-21 days under 5% carbon dioxide conditions.
Technical field
The present invention relates to stem cell culture technical field, in particular to a kind of growth of amplifying candidate stem cell in vitro because Sub-portfolio object and its application.
Background technique
Bone-marrow transplantation, i.e. candidate stem cell (HSCs) transplanting are current clinical application stem-cell therapy sides the most mature Method is also considered as the treatment means that can uniquely cure the diseases such as leukaemia, alpastic anemia and lymthoma.It is so far Only, HSCs transplanting is all to acquire the procedure for peripheral blood ancestral cells after donor bone marrow is mobilized according to bone marrow transplantation or Cord blood is direct It is transplanted, without in vitro culture, while there is also the problems for finding matching HSCs difficulty；In addition matched donor HSCs Donor is needed to provide a large amount of candidate stem cell, the physiology and psychology to donor all affect, this is also in recent years A lot of hematopoiesis donors that the country occurs, which temporarily go back on one's word, abandons the main reason for contributing.If donor is Cord blood, due to Cord blood HSCs negligible amounts, it is therefore desirable to which the more parts of suitable Cord bloods of distribution type are just able to satisfy adult patients needs, to the collection belt in blood source Carry out very big difficulty, this also results in the viewpoint that useless opinion is much saved about Cord blood.Presence based on above-mentioned many factors The problem of causing HSCs lazy weight, it is difficult to quantity needed for meeting HSCs transplanting.And amplification in vitro is simulated in engine body exterior Intracorporal environment gives certain nutrition and stimulating factor and promotes cell Proliferation.It is thin that Hematopoietic Stem is improved by Amplification Technologies The quantity of born of the same parents, while not reducing its self-renewal capacity, to meet the needs of bone-marrow transplantation, this be also domestic and international scientist always The direction of research.
Initially, HSCs in vitro culture is using stroma cell as auxiliary cell, and stroma cell can secrete a large amount of life The long factor or cell factor, and these growth factors are also to promote the key factor of HSCs proliferation.For many years, discovery promotes The cell factor of HSCs proliferation is always the key issues that domestic and international scientist studies HSCs, now also have multiple cell factors and Active material is reported can promote HSCs proliferation or/and self-renewing in vitro.From the point of view of current document both domestic and external, stem cell Growth factor (SCF), interleukin-13 (IL3) and interleukin 6 (IL6) are usually that Cultured Mouse and human hematopoietic stem cell are necessary Addition 3 kinds of cell factors, and different laboratories or researcher may select fibroblast growth factor (FGF1 or FGF2) [de Haan, G., E, 2003；Yeoh JS, R, 2006], insulin-like growth factor (IGF2) [Zhang CC, 2004], thrombopoietin (TPO) [Sauvageau G, 2004；Ko KH, 2011], erythropoietin(EPO) (EPO), Fms sample Tyrosine kinase receptor 3 ligand (Fltl3) [Purton LE, 2000；Ko KH, 2011], granulocyte-macrophage colony thorn Swash one of cell factors such as the factor (GM-CSF) or it is a variety of be added in cell culture fluid, be used for HSCs in-vitro multiplication.Mesh Before, in vitro culture candidate stem cell is carried out using single or cytokine profiles, candidate stem cell can be increased to a certain extent Proliferation, but when cytokine profiles co-incubation candidate stem cell, different ingredients is there may be antagonism or has same The common presence of kind function growth factor and cell factor, leads in vitro culture inefficiency or there are acquired cell defects etc. Problem is difficult to apply to scientific research or industrialization production, such as: human cord blood CD 34+cell use in vitro IL-3, IL-6 and After SCF culture or IL-11, SCF and FL are cultivated 6 days, expression of the cell of amplification due to changing VLA-4 and VLA-5, and Disturbing marrow and going back to the nest causes the HSCS consistent performance of amplification poor [zhai qiongli, 2004], causes to be difficult to apply to face Bed, the problem of not can solve candidate stem cell lazy weight still.Currently, the presence of cytokine profiles is also that respective play is made With amplification efficiency is poor.Therefore, how to design a kind of different cytokines composition can be only achieved optimal expanding effect, The problem of to meet current hematopoietic stem cell expansion low efficiency, lazy weight, is necessary.
Summary of the invention
It is an object of the invention to solve at least the above problems, and provide the advantages of at least will be described later.
It is a still further object of the present invention to provide a kind of compositions of amplifying candidate stem cell in vitro, by using the combination Growth factor and active material can quick in vitro, effective amplifying candidate stem cell, Hematopoiesis in Vitro expansion of stem cells can be improved Quantity simultaneously guarantees its self-renewal capacity.
In order to realize these purposes and other advantages according to the present invention, a kind of amplifying candidate stem cell in vitro is provided Growth factor combination, comprising: interferon-' alpha ', heparin and interleukin 12.
Preferably, further includes: stem cell factor, interleukin-13, thrombopoietin, Fms sample tyrosine kinase by The ligand and insulin-like growth factor of body 3.
It is described the present invention also provides the application in the growth factor combination described in one kind in vitro amplifying candidate stem cell Using for the DMEM culture solution of 10% serum and growth factor combination mixing amplifying candidate stem cell in vitro will be contained.
Preferably, the growth factor include interferon-' alpha ', it is heparin, interleukin 12, stem cell factor, white Interleukin 3, thrombopoietin, the ligand of Fms sample tyrosine kinase receptor 3 and insulin-like growth factor.
Preferably, the interferon-' alpha ', heparin, interleukin 12, stem cell factor, interleukin-13, blood platelet Generate element, the addition concentration of the ligand of Fms sample tyrosine kinase receptor 3 and insulin-like growth factor 2 (IGF2) is respectively 1-100ng/ml。
Preferably, the interferon-' alpha ' addition concentration is 50ng/ml, heparin addition concentration is 10ug/ml, described Interleukin 12 addition concentration is 10ng/ml, stem cell factor addition concentration is 100ng/mL, the interleukin 3 addition concentration be 100ng/mL, the thrombopoietin addition concentration be 100ng/mL, the Fms sample tyrosine kinase by The ligand addition concentration 100ng/ml of body 3 and the addition concentration of the insulin-like growth factor are 10ng/ml.
Preferably, the amplifying candidate stem cell in vitro is obtained from mouse or the marrow or umbilical cord blood hematopoietic stem cell sample of people Product.
Preferably, the condition of the amplifying candidate stem cell in vitro are as follows: 37 DEG C, cultivate 14- under 5% carbon dioxide conditions 21 days.
The present invention is include at least the following beneficial effects:
The interleukin 12 (IL12) for including in growth factor combination disclosed by the invention is the important tune for promoting TH1 reaction The factor is saved, TH0 cell can be promoted to TH1 cell differentiation, inhibit it to TH2 cell differentiation, and interferon (IFN) is that TH1 class is thin One of intracellular cytokine Major Secretory object acts on the table that can promote the intracellular IL12 receptor mrna of Th0 by IL12 and IFN α simultaneously It reaches, also increases the combination probability of IL12 and IL12 receptor；In addition, the also reversible allergen of IL12 is to IL12 receptor mrna Inhibit, the same combination for increasing IL-12 and IL-12R, therefore, it is thin that IL12 and IFN α adds common in vitro culture Hematopoietic Stem simultaneously Born of the same parents, conducive to the expression of enhancing histocompatibility antigen and peripheral blood mononuclear cells surface FC receptor.Heparin (heparin) is a kind of The mucopolysaccharide of sulfur-bearing acid groups can reinforce antithrombin Ⅲ (AT- III) inactivation serine protease, prevent fibrin ferment to have It is formed, to antithrombase and prevents a variety of effects such as platelet aggregation that from can hindering by the mixing of heparin and IL-12 and IL-12R The only condensation of the candidate stem cell after amplification in vitro, greatly improves amplification efficiency.In addition hematopoietic stem/progenitor itself can divide certainly It secretes or the growth factors such as paracrine FGF, and heparin can promote FGF in conjunction with the FGF receptor of hemopoietic stem cell surface, activate AKT Promote cell Proliferation with signal paths such as ERK.The application is added by growth factor combination IL-12, IFN α and heparin's Add, in conjunction with some other cell factor and amplification in vitro culture medium, amplification in vitro mouse/source of people hematopoietic stem cell expansion can be made 7689 times, compared with the prior art, a large amount of candidate stem cells can be obtained quickly through Amplification Technologies, be advantageously applied to science In research or industrialization production, bone-marrow transplantation is carried out by amplifying candidate stem cell in vitro for modern medicine and is laid the foundation.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this The research and practice of invention and be understood by the person skilled in the art.
Detailed description of the invention
Fig. 1 is the selection result of the different growth factors of the present invention；
CD117+ mouse hematopoietic stem cell expands number when Fig. 2 is present invention addition various concentration growth factor；
CD117+ mouse hematopoietic stem cell amplification efficiency when Fig. 3 is present invention addition various concentration growth factor；
Fig. 4 is that present invention detection CD117+ mouse hematopoietic stem cell generates colony in different growth factor combinations (CFU) potential；
Fig. 5 is the proliferation quantity of the present inventor's cord blood cell candidate stem cell in different growth factor combinations.
Specific embodiment
The present invention is described in further detail below, to enable those skilled in the art's refer to the instruction text being capable of evidence To implement.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein are not precluded one or more The presence or addition of a other elements or combinations thereof.
The present invention provides a kind of growth factor combination of amplifying candidate stem cell in vitro, comprising: interferon-' alpha ', heparin with And interleukin 12.
In one preferred embodiment, further includes: stem cell factor, interleukin-13, thrombopoietin, Fms sample tyrosine The ligand and insulin-like growth factor of kinases receptors 3.
The present invention also provides the applications in the growth factor combination described in one kind in vitro amplifying candidate stem cell, special Sign is, the application be will contain 10% serum DMEM culture solution and growth factor combination mixing amplification in vitro Hematopoietic Stem it is thin Born of the same parents.
In one preferred embodiment, the growth factor include interferon-' alpha ', heparin, interleukin 12, stem cell growth because Son, interleukin-13, thrombopoietin, the ligand of Fms sample tyrosine kinase receptor 3 and insulin-like growth factor.
In one preferred embodiment, the interferon-' alpha ', heparin, interleukin 12, stem cell factor, interleukin-13, The addition concentration of thrombopoietin, the ligand of Fms sample tyrosine kinase receptor 3 and insulin-like growth factor is respectively 1-100ng/ml。
In one preferred embodiment, the interferon-' alpha ' addition concentration is 50ng/ml, heparin addition concentration is 10ug/ Ml, interleukin 12 addition concentration are 10ng/ml, stem cell factor addition concentration is 100ng/mL, institute State interleukin-13 addition concentration be 100ng/mL, the thrombopoietin addition concentration be 100ng/mL, the Fms sample junket ammonia The ligand addition concentration 100ng/ml of acid kinase receptor 3 and the addition concentration of the insulin-like growth factor are 10ng/ ml。
In one preferred embodiment, the amplifying candidate stem cell in vitro is obtained from the marrow or umbilical cord blood hematopoietic stem cell sample of people The candidate stem cell of other mammals such as product and mouse.
In one preferred embodiment, the condition of the amplifying candidate stem cell in vitro are as follows: 37 DEG C, under 5% carbon dioxide conditions Culture 14-21 days.
Interferon-' alpha ' (IFN α), it is a kind of substance of solubility, can enhance histocompatibility antigen and peripheral blood mononuclear cells The expression of surface FC receptor, inhibits the proliferation of lymphocyte, enhances the cytotoxic activity of macrophage, adjusts immunologic homeostasis Function etc..
Heparin (heparin) is a kind of mucopolysaccharide of sulfur-bearing acid groups, can promote the combination of FGF and FGFR, moreover it is possible to reinforce Antithrombin Ⅲ (AT- III) inactivates serine protease, prevents fibrin ferment from being formed to have, and to antithrombase and prevents blood small A variety of effects such as plate aggregation.
Interleukin 12 (IL12) is a kind of heterodimeric cytokine, it is considered to be during cellullar immunologic response Key regulator.
Stem cell factor (SCF) is a kind of group with the protein and enzyme that stimulate self endogenous retinal stem cells growth Conjunction, is early stage candidate stem cell/progenitor cells stimulating factor, is maintaining hematopoietic cell survival, promote hematopoietic cell proliferation and is dividing Change, regulates and controls in the growth and development of hematopoietic cell and play an important role.
Interleukin-13 (IL3) is one of interleukins family important member, is a kind of by the produced sugared egg of T lymphocyte It is white, the cell Proliferation for participating in being immunoreacted, differentiation can be stimulated and improve its function.
Thrombopoietin element (TPO) is the glycosylated polypeptide chain comprising 834 amino acid, and Physiological effect hematopoiesis ancestral is thin Born of the same parents are evolved into mature megakaryocyte proliferation and differentiation.
The ligand (Fltl3) of Fms sample tyrosine kinase receptor 3) it is a kind of early stage hematopoietic growth related with hematopoietic regulation The factor can act synergistically with cytokine profiles.The ligand of Flt3 in conjunction with Flt3 after can adjust primitive hematopoietic stem cell/ancestral Medullary system and lymphoid progenitor cell, Dendritic Cells, natural killer cells and natural kill tree are mobilized and stimulate in cell Proliferation and differentiation Prominent shape cell Proliferation and differentiation, are the mostly important growth factors of Dendritic Cells.
A kind of multi-functional regulation of cell proliferation factor of insulin-like growth factor (IGFs), it has now been found that include IGF-1 With two kinds of IGF-2, the IGF2 of the application can promote the proliferation rate of candidate stem cell.
1, object
Animal source cell: mouse CD117+ ancestral cells are obtained from；
Human archeocyte: obtained from be not suffering from hemopathic marrow, umbilical cord blood hematopoietic stem cell sample (from human umbilical cord blood, has Puerpera knows book).
2, test method
2.1 obtain mouse CD117+ ancestral cells, include the following steps:
1) CO2 smother play is taken to put to death mouse；
2) mouse is fixed in dissection plate, 75% ethyl alcohol is directly sprayed on mouse surface sterilization；
3) mouse disinfected is transferred in Biohazard Safety Equipment；
4) leg skin after mouse is subtracted, exposes mouse leg, mouse foot is separated at mouse ankle；
5) fascia and muscle after mouse knee joint are cut, fixes knee joint with tweezers, the direction Xiang Guanjie fractures femur Nearly knee joint.It pushes down at musculature to pelvis, by whole femur exposure and cuts；
6) knee joint is fixed with tweezers, opposite direction fractures at the nearly knee joint of shin bone, pushes down on musculature and remove shin bone；
7) mouse femur and shin bone are placed in the 6 orifice plates of the sterile PBS of 3ml；
8) marrow is gone out into the bone of both ends open with the PBS that 1ml syringe draws immersion femur and shin bone, be repeated several times Until bone flushing is whitened, the flushing liquor gone out then is dispelled into cell with syringe；
9) 70 μm of filters are placed on 50ml centrifuge tube, the resulting cell suspension of step 8,1200RPM is added into filter It is centrifuged 10min；
10) bone marrow cell is transferred in the sterile fluidic cell pipe of sterile 5mL, passes through centrifugal concentrating volume most 200 μ L, is then added the anti-CD117 antibody of PE label, and subsequent operation program is detailed in kit specification (EasySepTM Mouse CD117 (cKIT) Positive Selection kit, STEMCELL), it is dry that CD117+ mouse hemopoietic is obtained with magnetic bead sorting method Cell.
2.2 CD117+ mouse hematopoietic stem cell in-vitro multiplications
2.2.1 different growth factor optimal combination objects are probed into
The 7000 above-mentioned 2.1 CD117+ mouse hematopoietic stem cells obtained are taken to be planted in the DMEM culture containing 10% serum In 96 orifice plates (round bottom) of liquid (HyClone-DMEM/HIGH GLUCOSE), DMEM culture solution (containing 10% serum) is used in every hole Amount is 200 μ L, according to experiment needs, adds the growth factor of various combination, every kind of combination sets 5 repeating holes, in 37 DEG C, 5% CO2Condition in vitro culture 14-21d after, collect cultured products in cell, calculate total number of cells.
Each cell mass percentage of streaming instrument cell analysis, the specific steps are as follows: above-mentioned each sample addition 4uL contains as follows Then the antibody mixed liquor of streaming antibody, room temperature or 4 degree of dyeing 30min are added PBS buffer solution and clean 1-2 times, add 200ul PBS buffer solution, flow cytomery (BD AccuriTM C6 Flow Cytometer,BD)。
Wherein, streaming antibody is purchased from eBioscience company without specified otherwise, and wherein Lin includes FITC label CD11b, Gr-1, TER-119, CD3, CD4, CD8, B220 and CD41；The Sca- of c-Kit and the PE-cy5.5 label of APC label 1。
The preparation of PBS buffer solution: NaCl 8g, KCL0.2g, Na are weighed2HPO4·12H2PO4It is bis- that 0.24g is dissolved in 900mL It steams in water, with hydrochloric acid tune pH value to 7.4, water is added to be settled to 1L, be stored at room temperature spare.Above-mentioned sterile PBS is obtained from prepared PBS Buffer is in 121 DEG C, high pressure sterilization 20-30min.
The method and step of the growth factor of above-mentioned addition various combination: (INF α is 10ng/ml, and heparin concentration is 20ug/ml, the addition concentration of other various factors are 50ng/mL)
DMEM culture solution+SCF to contain 10% serum is added respectively as the control of culture monokaryon CD117+ cell IL3, IL6, INF α, EPO, TPO, heparin, desmocyte growth factor-21 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9) and fibroblast growth factor 18 (FGF18) obtain amplification efficiency Highest growth factor, is named as A.
1, on the basis of above-mentioned the selection result, to contain the DMEM culture solution+SCF+A of 10% serum as culture monokaryon Fltl3, IGF2, hepatocyte growth factor (HGF), TPO, IL12, stroma cell derivative are added in the control of CD117+ cell respectively The factor 1 (SDF1), epidermal growth factor (EGF), growth inhibition specific gene product 6 (Gas6), EPO and M-CSF, are obtained The highest growth factor of amplification efficiency is taken, B is named as.
2, on the basis of 1 the selection result, to contain the DMEM culture solution+SCF+A+B of 10% serum as culture monokaryon The control of CD117+ cell adds INF α, IGF2, EGF, IL12, FGF1, FGF2, FGF9, FGF18 and blood platelet respectively and spreads out Raw the factor (PDGF) obtains the highest growth factor of amplification efficiency, is named as C.
3, single as culture to contain the DMEM culture solution+SCF+A+B+C of 10% serum on the basis of 2 the selection result IL7, heparin, SDF1 and fibroblast growth factor 21 (FGF21) are added in the control of core CD117+ cell respectively, The highest growth factor of amplification efficiency is obtained, D is named as.
4, on the basis of 3 the selection result, to contain the DMEM culture solution+SCF+A+B+C+D of 10% serum as culture IL6, IGF2, IL12, EPO, HGF and Fltl3 are added in the control of monokaryon CD117+ cell respectively, obtain amplification efficiency highest Growth factor, be named as E.
5, on the basis of 4 the selection result, to contain the DMEM culture solution+SCF+A+B+C+D+E of 10% serum as training Support monokaryon CD117+ cell control, respectively add bon e formation occur albumen 2 (BMP2), IGF2, Fltl3, Wnt3a, SDF1 with And EPO, the highest growth factor of amplification efficiency is obtained, F is named as.
6, on the basis of 5 the selection result, using contain the DMEM culture solution+SCF+A+B+C+D+E+F of 10% serum as The control of monokaryon CD117+ cell is cultivated, adds IGF2, EPO, Wnt3a and SDF1 respectively, obtains the highest life of amplification efficiency The long factor, is named as G.
7, on the basis of 6 the selection result, made with the DMEM culture solution+SCF+A+B+C+D+E+F+G containing 10% serum For the control for cultivating monokaryon CD117+ cell, EPO, Wnt3a and SDF1 are added respectively, are obtained and are promoted monokaryon CD117+ cell The growth factor of amplification composition (inhibited with added every kind of growth factor or with compare play identical effect when The standard of composition is obtained for judgement).
2.2.2 the optium concentration of different growth factors in optimal combination object is probed into
On the basis of the optimal combinations of growth factors that 2.2.1 is probed into, 7000 above-mentioned 2.1 are taken to obtain CD117+ Mouse hematopoietic stem cell, in 96 orifice plates (round bottom) of the DMEM culture solution containing 10% serum, DMEM culture solution in every hole (contain 10% serum) dosage is 200 μ L, according to experiment needs, add growth factor in the optimal combination object of various concentration and Growth factor combination, wherein the addition concentration of single growth factor are as follows: SCF, IL3, TPO, Fltl3, IGF2, INF α and 1ng/mL, 10ng/mL, 50ng/mL, 100ng/mL is respectively adopted in IL12 addition concentration, and heparin additive amount is 1 μ g/mL, 10 μg/mL、50μg/mL、100μg/mL。
The concentration of the combination of growth factor combination and its each growth factor is as follows: composition Z1 includes SCF 100ng/mL, IL3 100ng/mL and TPO 100ng/mL；Composition Z2 include SCF 100ng/mL, IL3 100ng/mL, TPO 100ng/mL and INF α 50ng/mL；Composition Z3 include SCF 100ng/mL, IL3 100ng/mL, TPO 100ng/mL, INF α 50ng/mL, heparin10ug/ml and Fltl3 100ng/mL；Composition Z4 includes SCF 100ng/mL, IL3 100ng/mL, F1t3L 100ng/ml, heparin 10ug/ml and IGF2 10ng/ml；Composition Z5 includes SCF 100ng/ ML, IL3 100ng/mL, F1t3L 100ng/ml, heparin 10ug/ml, IGF2 10ng/ml and INF α 50ng/ml；Group Close object Z6 include SCF 100ng/mL, IL3 100ng/mL, TPO 100ng/mL, INF α 50ng/mL, F1t3L 100ng/ml, Heparin 10ug/ml, IGF2 10ng/ml and IL12 10ng/ml), every kind of combination sets 5 repeating holes, in vitro culture 14- After 21d, cell is collected, calculates total number of cells, while to add the composition culture of SCF100ng/mL and IL3100ng/mL CD117+ mouse hematopoietic stem cell is control.Each cell mass percentage of flow cytometry analysis, specific steps are referring to 2.2.1 institute Show.
2.2.3 detection CD117+ mouse hematopoietic stem cell generates the potential of colony (CFU)
Using Methyl cellulose Semi-solid cell culture method culture CD117+ mouse hematopoietic stem cell.10000 above-mentioned 2.1 are taken to obtain CD117+ mouse hematopoietic stem cell is planted in Methyl cellulose semisolid culturemedium, according to requirement of experiment, add different growths because Sub-portfolio, every kind of combination set 3 repetitions, carry out colony-forming test.
Specific step is as follows: obtained cell being resuspended, Trypan Blue counts, and takes 10000 cells and Methyl cellulose Semisolid culturemedium mixing, control group are Methyl cellulose semisolid culturemedium, are separately added into Methyl cellulose semisolid culturemedium Following composition: composition F1 includes SCF 100ng/mL, IL3 100ng/mL and TPO 100ng/mL；Composition F2 includes SCF 100ng/mL, IL3 100ng/mL, TPO 100ng/mL and INF α 50ng/mL；Composition F3 includes SCF 100ng/ mL,IL3 100ng/mL,TPO 100ng/mL,INFα50ng/mL,heparin10ug/ml；Composition F4 includes SCF 100ng/mL, IL3 100ng/mL, TPO 100ng/mL, F1t3L 100ng/ml, heparin 10ug/ml and IGF2 10ng/ml；Composition F5 include SCF 100ng/mL, IL3 100ng/mL, TPO 100ng/mL, F1t3L 100ng/ml, Heparin 10ug/ml, IGF2 10ng/ml and INF α 50ng/ml；Composition F6 includes SCF 100ng/mL, IL3 100ng/mL、TPO 100ng/mL、F1t3L 100ng/ml、heparin 10ug/ml、IGF2 10ng/ml、INFα 50ng/ Ml and IL12 10ng/ml；Then 37 DEG C, 5%CO are respectively placed in2Under the conditions of cultivate 14-21 days after, detection CD117+ mouse make The quantity of hemocytoblast colony in Methyl cellulose semisolid culturemedium.
2.3 source of people candidate stem cells amplification in vitro in different combinations of growth factors
2.3.1 human archeocyte is obtained, includes following methods step:
(1) it takes without after hemopathic marrow or umbilical cord blood hematopoietic stem cell sample, is drawn onto sample with 3ml plastic suction pipe In 50ml centrifuge tube, add PBS buffer solution to 50ml, 1200RPM centrifugation 5min.
(2) abandon supernatant, according to erythrocyte cracked liquid (Solarbio) specification be added respective volume erythrocyte cracked liquid, 4 DEG C cracking 10min, 1200RPM be centrifuged 5min.
(3) supernatant is abandoned, step 2 is repeated, until erythrocyte splitting is complete.
2.3.2 source of people candidate stem cell in-vitro multiplication
Human archeocyte is obtained according to the above method, takes 50000 cell seedings in the DMEM culture solution containing 10% serum 24 orifice plates in, according to requirement of experiment, the optimum growh factor and combinations thereof that 2.2.1 is obtained is added, in 37 DEG C, 5%CO2Item In vitro culture 14-21 days under part, every 2d flow cytometry analysis CD34+ cell rate.Using flow cytomery method, specifically Step is referring to shown in 2.2.1.
3, statistical analysis
The Student t that all experimental datas are all made of GraphPad Prism Version 5.04 is examined, and data will With Means ± SEM, P < 0.05 is considered as statistically significant.
As a result
1, different growth factor optimal combination objects
The CD117+ candidate stem cell cultivation results of the growth factor of 1.1 addition various combinations
1, it to contain the control of the DMEM culture solution+SCF of 10% serum as culture monokaryon CD117+ cell, as a result shows Show: compared to control, after adding growth factor I L3, IL6, INF α, TPO, heparin, total number of cells expand 17.5 times, 9 respectively Again, 8.5 times, 7.5 times and 10.5 times, remaining growth factor is as control effect, as shown in Figure 1a.
2, to contain the DMEM culture solution+SCF+IL3 of 10% serum as the control of culture monokaryon CD117+ cell, as a result Display: compared to control, after adding Fltl3, IGF2, HGF, TPO, IL12, SDF1 and EPO, total number of cells expand 10.5 respectively Again, 11.5 times, 13.5 times, 17 times, 16 times, 11 times and 9.5 times, remaining growth factor is as control effect, such as Fig. 1 b It is shown.
3, to contain the control of the DMEM culture solution+SCF+IL3+TPO of 10% serum as culture monokaryon CD117+ cell, INF α, IGF2, EGF, FGF1, FGF2, FGF9, FGF18 and PDGF are added respectively, as the result is shown: compared to control, adding INF α, EGF, FGF2, FGF9 and PDGF, total number of cells expand 6.5 times, 5.25 times, 5.75 times, 6 times and 5.35 times respectively, Remaining growth factor suppression of amplification or with control effect as, as illustrated in figure 1 c.
4, to contain the DMEM culture solution+SCF+IL3+TPO+INF α of 10% serum as culture monokaryon CD117+ cell IL7, heparin, SDF1 and FGF21 are added in control respectively, the results show that addition heparin and SDF1 is thin compared to control Born of the same parents' sum expands 6 times and 5.4 times respectively, remaining growth factor is as control effect, as shown in Figure 1 d.
5, to contain the DMEM culture solution+SCF+IL3+TPO+INF α+heparin of 10% serum as culture monokaryon IL6, IGF2, IL12, EPO, HGF and Fltl3 are added in the control of CD117+ cell respectively, the results show that adding compared to control IL12 and Fltl3 total number of cells are added to expand 14.5 times and 9 times respectively, remaining growth factor is as control effect, such as Fig. 1 e institute Show.
6, single as culture to contain the DMEM culture solution+SCF+IL3+TPO+INF α+heparin+IL12 of 10% serum The control of core CD117+ cell, respectively add BMP2, IGF2, Fltl3, Wnt3a, SDF1 and EPO, the results show that compared to pair According to addition IGF2, Fltl3, Wnt3a, SDF1 and EPO total number of cells expand 6.5 times, 10.1 times, 7.5 times, 6.1 times respectively With 6 times, addition BMP2 effect it is similar with control, as shown in Figure 1 f.
7, using contain the DMEM culture solution+SCF+IL3+TPO+INF α+heparin+IL12+Fltl3 of 10% serum as The control of monokaryon CD117+ cell is cultivated, adds IGF2, EPO, Wnt3a and SDF1 respectively, the results show that adding compared to control IGF2, Wnt3a and SDF1 total number of cells are added to distinguish 7 times, 5.9 times and 5.9 times, addition EPO is similar with control, such as Fig. 1 g It is shown.
8, with the DMEM culture solution+SCF+IL3+TPO+INF α+heparin+IL12+Fltl3+IGF2 containing 10% serum As the control of culture monokaryon CD117+ cell, EPO, Wnt3a and SDF1 are added respectively, the results show that adding compared to control Add SDF1 effect similar, other two kinds of suppression of amplification, as shown in figure 1h.
It is analyzed by the screening process of more than 20 kinds of cell factors, when growth factor cultivates monokaryon CD117+ cell in vitro It is interaction, such as: in above-mentioned 2 or 6, when culture medium is the DMEM culture solution+SCF+IL3 containing 10% serum Or when DMEM culture solution+SCF+IL3+INF α+heparin+IL12+Fltl3 containing 10% serum, addition EPO can be improved The amplification efficiency of total number of cells, and add EPO in above-mentioned 4,5,7,8 and cell is inhibited to expand, illustrate that IL12 and EPO has There is collaboration to promote amplification effect, and Fltl3 or Fltl3+IGF2 combination can inhibit the activity of EPO regulation RBC acceptor garland rate. For another example: suppression of amplification after IL12 is added on the basis of above-mentioned 3 used medium, and 2 or 5 add on used medium base IL12 can improve the amplification of total number of cells respectively, illustrate on the medium base used 1, and individually adding IL3 can promote IL12 Activity improve amplification efficiency, but IL3+TPO simultaneously be added 1 used in culture medium when, then can inhibit IL12 activity to Inhibit cell amplification；In addition, adding INF α+heparin on the basis of 1 used culture medium after IL3+TPO and can pierce Swash the activity increases cell amplification efficiency of IL12, therefore every kind of growth factor is not that individual play is made in ex vivo expansion process With, but by the common amplification efficiency for improving amplifying candidate stem cell in vitro of the synergistic effect of a variety of growth factors, it is, The type that growth factor is added in amplification in vitro culture solution is very crucial.Present inventor by the screenings of a variety of growth factors, Can promote CD117+ mouse hematopoietic stem cell expand growth factor include: SCF, IL3, TPO, FLTL3, IGF2, Heparin, INF α and IL12.
When adding 8 kinds of growth factors simultaneously, CD117+ mouse hematopoietic stem cell amplification efficiency highest (as shown in Figure 2), Through flow cytomery, total monocyte can reach 21358300, ratio 1.26% shared by candidate stem cell, total hematopoiesis The quantity of stem cell reaches 269115, is obtained with this and has expanded 7689 times.In the prior art, different growth factor groups is added After closing object, can be improved for 14 days by cultivating by 3139 times, and candidate stem cell reaches 109881, and can expand after the application culture 14-21 days Increase 7689 times.
1, the optium concentration of different growth factor optimal combination objects
When SCF, IL3, TPO, Fltl3, IGF2, INF α and IL12 addition concentration be respectively adopted 1ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, heparin additive amount be 1 μ g/mL, 10 μ g/mL, 50 μ g/mL, 100 μ g/mL, as the result is shown: only adding When adding SCF, four addition concentration amplification in vitros are less obvious, but total number of cells are more when opposite addition 100ng/mL；Addition When SCF100ng/mL, IL3 addition concentration is that 100ng/mL expands ratio and reaches 1 times, for referring to more accurate and conveniently, therefore, Amplification ratio when selection SCF+IL3 is respectively 100ng/mL is control.
It is learnt by Fig. 3 analysis, it is small for culture CD117+ after addition SCF and IL3 in the DMEM culture solution containing 10% serum The control of mouse candidate stem cell, it is 100ng/mL that TPO, which most preferably adds concentration, and it is 100ng/mL that Fltl3, which most preferably adds concentration, It is 10ng/mL that IGF2, which most preferably adds concentration, and heparin most preferably add concentration and most preferably adds concentration for 10 μ g/mL, IL12 and be It is 50ng/mL that 10ng/mL, INF α, which most preferably add concentration,.
On the basis of the optium concentration preferably gone out in different growth factors in individually addition optimal combination object, carry out not I.e. with combination: composition Z1- composition Z6 has found that the addition of composition is any raw compared in individually addition optimal combination object Length is improved because of the amplification efficiency of the period of the day from 11 p.m. to 1 a.m；It is analyzed from table 1, composition Z1- composition Z6 different combining forms expands ratio Difference, Z6 show optimal amplification ratio, are 7689 times of control.That is: SCF 100ng/mL, IL3 are added when simultaneously 100ng/mL、TPO 100ng/mL、INFα50ng/mL、F1t3L 100ng/ml、heparin 10ug/ml、IGF2 10ng/ml When with IL12 10ng/ml, highest amplification ratio can get, it is seen then that the present invention is with optimal growth factor combination and its phase It answers under concentration, the in-vitro multiplication rate of candidate stem cell can be effectively improved.
The amplifying candidate stem cell in vitro of the different growth factor combinations of table 1
2, detection CD117+ mouse hematopoietic stem cell generates the potential of colony (CFU)
By Fig. 4 analysis, it can be seen that, different growth factor combinations presents compared to control and increases hematopoiesis in various degree Cell colony, and in the optimum growh combinations of factors and concentration containing Fig. 2, Fig. 3 as the result is shown, i.e. SCF 100ng/mL, IL3 100ng/mL、TPO 100ng/mL、F1t3L 100ng/ml、heparin 10ug/ml、IGF2 10ng/ml、INFα50ng/ml The quantity formed with hematopoietic colonies in the Methyl cellulose semisolid culturemedium of IL12 10ng/ml) is most, demonstrates again that selected life The long factor can improve the amplification efficiency of Hematopoiesis in Vitro stem cell, but only in the addition simultaneously of 8 kinds of growth factors and optimal Candidate stem cell is cultivated under concentration, could be cooperateed with and be shown optimal colony potential；The quantity that F6 hematopoietic colonies are formed is most, is 6.5 times of control.
3, the proliferation quantity of human archeocyte candidate stem cell in different combinations of growth factors
By Fig. 5 analysis, it can be seen that, different growth factor combinations presents compared to control and increases hematopoiesis in various degree Cell quantity, and containing Fig. 2, Fig. 3 optimum growh combinations of factors as the result is shown and concentration (that is: SCF 100ng/mL, IL3 100ng/mL、TPO 100ng/mL、INFα50ng/mL、F1t3L 100ng/ml、heparin 10ug/ml、IGF2 10ng/ml and IL12 10ng/ml) condition of culture under candidate stem cell quantity it is most, demonstrate again that selected growth factor is equal The amplification efficiency of Hematopoiesis in Vitro stem cell can be improved, but only adds in 8 kinds of growth factors and is trained under optimal concentration simultaneously Candidate stem cell is supported, could cooperate with and show optimal proliferation efficiency.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited In specific details and shown here as with attached drawing.