CN100554415C - The method of monoclonal antibody ZUB4 immunological magnetic bead sorting primary generation human marrow mesenchyme stem cell - Google Patents
The method of monoclonal antibody ZUB4 immunological magnetic bead sorting primary generation human marrow mesenchyme stem cell Download PDFInfo
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- CN100554415C CN100554415C CNB2006100507628A CN200610050762A CN100554415C CN 100554415 C CN100554415 C CN 100554415C CN B2006100507628 A CNB2006100507628 A CN B2006100507628A CN 200610050762 A CN200610050762 A CN 200610050762A CN 100554415 C CN100554415 C CN 100554415C
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Abstract
The invention provides a kind of method with monoclonal antibody ZUB4 immunological magnetic bead sorting primary generation human marrow mesenchyme stem cell, be from the marrow suspension, to separate to obtain mononuclearcell, with monoclonal antibudy ZUB 4 is mark, obtains the human marrow mesenchymal stem cell of ZUB4 antigen positive by indirect magnetic activated cell (sorting) forward sorting.The human marrow mesenchymal stem cell that the inventive method obtains has growth activity and proliferation potential preferably, and ZUB4 antigen stably express in the passage process, and can be divided into adipocyte, scleroblast, neuron cell at directional induction in vitro.The present invention is to provide a kind of method of comparatively ideal human marrow mesenchymal stem cell sorting purifying, improve the purity of former generation mescenchymal stem cell and the efficient of separation and Culture, helped mesenchymal stem cell biological to learn The Characteristic Study and in clinical detection with the popularization in using.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of separation and purification of human marrow mesenchymal stem cell primary cell and the novel method of vitro culture, this method is used a kind of freshly prepd anti-human marrow mesenchymal stem cell monoclonal antibudy ZUB 4, method by immune sorting is separation and purification mescenchymal stem cell from people's BMNC effectively, and at cultured and amplified in vitro, induce multidirectional differentiation.
Background technology
(Mesenchymal stem cells is to be present in the stem cell that has height self and multidirectional differentiation potential in the marrow MSCs) to human marrow mesenchymal stem cell, is organizational project and regenerative medicine ideal seed cell.The report that a large amount of bone marrow MSCs basis and clinical study are at home and abroad arranged, it holds out broad prospects in the clinical application of stem-cell therapy.
The content of MSCs is very low in normal people's marrow, and about 10
4～10
5Contain a MSCs in the individual mononuclearcell, be applied to basis and clinical study, need separation effectively, purifying, cultured and amplified in vitro MSCs for obtaining sufficient amount MSCs.The scheme of separating MSCs from marrow at present commonly used mainly contains three kinds: 1. stick characteristic according to the substrate of bone marrow MSCs and realize isolating adherent sieve method; 2. according to the density different next isolating density gradient centrifugations of MSCs with other cells; 3. according to some special markings of cell surface, carry out immune sorting, as forward immunity sorting, negative sense immunity sorting and combined sorting with various monoclonal antibodies.The primary generation human marrow MSCs length consuming time that method by traditional adherent culture obtains, cell heterogeneous big, this is restricted the fundamental research of MSCs and the popularization of clinical application.With the cell-specific surface molecular is the method for mark by immune sorting, and the few MSCs of content in the enrichment marrow improves former being commissioned to train and supports gained MSCs purity effectively.
People's bone marrow MSCs phenotypic characteristic of generally acknowledging in the past: do not express the surface antigen of hematopoietic cell, the surface marker of CD34, CD45, CD14, glycophorin A (Glycophorin A), T or bone-marrow-derived lymphocyte is all negative; Special relatively surface antigen has SH2, SH3, SH4, STRO-1, CD166 etc.Immune labeled SH2, STRO-1, low-affinity nerve growth factor receptor (the low-affinity nerve growth factor receptor of mostly being of forward immunity sorting, LNGFR): SH2 is the monoclonal antibody that obtains with people's bone marrow MSCs immune mouse, studies confirm that SH2 identification CD105 molecule, also CD105 is expressed in myelomonocyte, erythroblast, part lymphocyte simultaneously; STRO-1 is the new monoclonal antibody of human bone marrow substrate cell that obtains with marrow CD34+ cellular immunization mouse, but contains a large amount of erythroblasts and sub-fraction bone-marrow-derived lymphocyte in the medullary cell by the STRO-1 sorting; LNGFR is expressed in most of marrow stromal cell, but LNGFR is not the bone marrow MSCs specific marker, and it is expressed in the tunica adventitia vasorum of the multiple organ of human body, and the LNGFR+ cell is about 2.3% in BMNC.MSCs content is extremely low in the BMNC, content by MSCs in the cell after negative sense immune sorting can the raising effectively sorting, it is the CD45-Glycophorin A-cell mass composition complexity that label screening obtains with CD45 and Glycophorin A that the investigator is arranged, and still need be further purified.Therefore, the above-mentioned immune labeled ideal mark that all is difficult to become direct sorting human marrow MSCs.
Up to now, the investigator is always by a plurality of surface molecular combined marks both at home and abroad, and come identifier's bone marrow MSCs in conjunction with the self of cell and the characteristic of multidirectional differentiation potential, still in seeking, this has brought certain difficulty for the separation of MSCs, the practical application of purifying to the single specific surfaces molecule of MSCs.The phenotypic characteristic of further investigation people bone marrow MSCs will help separation and purification people bone marrow MSCs and further cultivation amplification effectively, to promote its clinical application in the stem-cell therapy field.Adopt the new anti-human marrow MSCs monoclonal antibudy ZUB 4 of hybridoma technology research preparation, confirm that by immunocytochemistry and immunofluorescence dyeing detection ZUB4 and people's bone marrow MSCs bonded positive rate are about 95%～99%, the MSCs stably express ZUB4 antigen that goes down to posterity and cultivate; The clone no cross reaction of ZUB4 and people's derived from bone marrow; Except that myeloid tissue, ZUB4 and people's mesenchyme tissue and the equal no cross reaction of non-mescenchymal tissue.ZUB4 is the new monoclonal antibody specific at people's bone marrow MSCs, has the specificity and the susceptibility of height with the reaction of people's bone marrow MSCs.
Summary of the invention:
The method that the purpose of this invention is to provide monoclonal antibody ZUB4 immunological magnetic bead sorting primary generation human marrow mesenchyme stem cell (MSCs), be by the separation and purification of people's bone marrow MSCs primary cell and the novel method of vitro culture, ZUB4 antigen stably express is a kind of new ideal mark of people's bone marrow MSCs in the MSCs in early stage, late period that people's marrow MSC primary cell and subculture in vitro separately are cultivated.The present invention is achieved through the following technical solutions: the separation and purification of people's bone marrow MSCs primary cell, promptly from the marrow suspension, separate and obtain mononuclearcell, with number of patent application is that 200510061036.1 disclosed monoclonal antibudy ZUB 4s are mark, obtain people's bone marrow MSCs of ZUB4 antigen positive by indirect magnetic activated cell (sorting) forward sorting, ZUB4 antigen-positive cell adherent growth, be fusiformis, in the vitro culture system, cultivate 5 days left and right sides cells and begin obvious amplification, cell phenotype detected and shows after amplification was gone down to posterity: CD29, CD44, CD166, CD105 (SH2) positive, CD14, CD34, CD45, HLA-DR expresses all negative, is people's bone marrow MSCs of homogeneous.
In the described method, be by gathering healthy donor marrow, anticoagulant heparin, Ficoll-paque density gradient centrifugation is separated the acquisition BMNC, with number of patent application is 200510061036.1 disclosed monoclonal antibudy ZUB 4s, antibody subtype is IgG1, hatch with BMNC, add anti-mouse IgG magnetic bead two anti-hatching behind the centrifuge washing, from BMNC, separate the people's bone marrow MSCs that obtains the ZUB4 antigen positive by magnetic activated cell (sorting) behind the centrifuge washing, respectively with ZUB4 antigen-positive cell and ZUB4 antigen negative cell inoculation in culture dish, with the low sugar DMEM nutrient solution that contains 10% foetal calf serum, place 37 ℃, volume fraction is 5%CO
2Incubator is cultivated, and changes liquid 1 time every 3 days afterwards.
In the described method, ZUB4 antigen-positive cell and negative cells are in vitro culture, cultivate after 5 days that cell attachment is fusiformis in the culture dish of inoculation positive cell, beginning is amplification obviously, cultivating 18～22 days attached cells 80%～90% merges, the cellular form homogeneous with the inoblast plesiomorphism, is arranged in whirlpool shape, netted, radial; The ZUB4 antigen negative cell that sorting obtains is cultivated and is seen a large amount of circular suspension cells after 5 days, wherein has the minute quantity cell attachment to be spindle cell, cultivates and still sees round cell after 20 days, and a small amount of adherent cell is more roomy, and the form heterogeneity is arranged random.
People's bone marrow MSCs of the ZUB4 antigen positive that the inventive method obtains has growth activity and proliferation potential preferably, and ZUB4 antigen stably express in the passage process, and can be divided into adipocyte, scleroblast, neuron cell at directional induction in vitro.
Beneficial effect of the present invention is:
(1) be mark with single anti-human marrow MSCs monoclonal antibody specific ZUB4, by forward immunity sorting can be directly, the former generation MSCs of separation and purification from people's BMNC apace, shortened the time of primary cell culture, improved the efficient of the separation and Culture of people's bone marrow MSCs, improved effectively and adopted traditional adherent sieve method to cultivate former generation MSCs length consuming time, the heterogeneous big defective of cell, and keep the biological characteristics of former generation MSCs, for research with use the former generation MSCs of marrow an effective means is provided.
(2) content of MSCs in marrow is extremely low, be that mark passes through forward immunity sorting and can obtain the higher former generation MSCs of purity with ZUB4, and have higher propagation and multidirectional differentiation potential, can increase at subculture in vitro separately, and orientable adipocyte, scleroblast, the neuron cell of being induced to differentiate into, help people's bone marrow MSCs applying in organizational project, regenerative medicine.
(3) ZUB4 is a kind of anti-human marrow MSCs monoclonal antibody of new development, with single ZUB4 is that mark passes through the MSCs that forward immunity sorting can obtain the phenotype homogeneous, the corresponding film surface antigen of monoclonal antibudy ZUB 4 is stably express in people's bone marrow MSCs vitro culture goes down to posterity amplification procedure, provides platform for studying the biological significance and the further perfect understanding to the MSCs characteristic of this surface molecular in MSCs propagation and atomization.
Description of drawings:
Fig. 1 is that to be with anti-human marrow mesenchyma stem cell (MSCs) monoclonal antibudy ZUB 4 that the positive cell that obtains after the forward immunity sorting of mark and negative cells former is commissioned to train foster.
Fig. 2 is to be the positive cell cultured and amplified in vitro first-generation and the growth curve in the 5th generation that obtains after the forward immunity sorting of mark with ZUB4.
Fig. 3 is to be the flow cytometry figure of positive cell CD14, the CD29, CD34, CD44, CD45, CD105, CD166, HLA-DR and the ZUB4 antigen presentation that obtain after the forward immunity sorting of mark with ZUB4.
Fig. 4 is an induced osteogenesis differentiation back Von Kossa method colored graph.
Fig. 5 is induced lipolysis differentiation back oil red O stain figure.
Fig. 6 is the like cell directional induction differentiation back immunocytochemical stain figure of neuralward unit.
Embodiment:
The present invention is described further with accompanying drawing in conjunction with the embodiments.
Gather healthy donor marrow, anticoagulant heparin, Ficoll-paque (proportion 1.077) density gradient centrifugation is separated the acquisition BMNC, with number of patent application is that 200510061036.1 disclosed monoclonal antibudy ZUB 4s (antibody subtype is IgG1) are hatched with BMNC, adding anti-mouse IgG magnetic bead two anti-(Miltenyi Biotec Inc.) behind the centrifuge washing hatches, from BMNC, separate the people's bone marrow MSCs that obtains the ZUB4 antigen positive by magnetic activated cell (sorting) behind the centrifuge washing, respectively ZUB4 antigen-positive cell and negative cells are inoculated in culture dish, with the low sugar DMEM nutrient solution that contains 10% (V/V) foetal calf serum, place 37 ℃, volume fraction is 5%CO
2Incubator is cultivated, and changes liquid 1 time every 3 days afterwards.
Cultivate after 5 days that cell attachment is fusiformis in the culture dish of inoculation positive cell, beginning is amplification obviously, cultivates about 80% fusion of left and right sides attached cell in 20 days, and the cellular form homogeneous with the inoblast plesiomorphism, is arranged in whirlpool shape, netted, radial; The negative cells that sorting obtains, cultivate and see a large amount of circular suspension cells after 5 days, wherein there is the minute quantity cell attachment to be spindle cell, cultivate and still see round cell after 20 days, a small amount of adherent cell is more roomy, the form heterogeneity, arrange random, referring to Fig. 1, wherein A, B are respectively the morphological feature (* 100) that positive cell was cultivated 8 days, 20 days, and C, D are respectively the morphological feature (* 100) that negative cells was supported 8 days, 20 days.
By the direct former generation MSCs of separation and purification from people's BMNC of forward immunity sorting, improved effectively and adopted traditional adherent sieve method to cultivate former generation MSCs length consuming time, the heterogeneous big defective of cell, provide an effective means for studying and use bone marrow MSCs simultaneously.
With ZUB4 is that mark passes through the positive cell multiplication capacity analysis that forward immunity sorting obtains
Get the ZUB4 male people bone marrow MSCs first-generation that vitro culture goes down to posterity and the 5th generation cell, by 2 * 10
4The density of individual cells/well is inoculated in 24 well culture plates, cultivates respectively 1,2,3,4,5,6,7,8 day, carries out cytokinetic analysis.Cultivation has following common feature: after cell was cultivated through going down to posterity, cell was adherent fully about 10 hours, and cellular form becomes spindle cell again; Go down to posterity to cultivate and be about 24～36 hours latent period; Go down to posterity and cultivate the logarithmic proliferation phase and be about 4～5 days; The logarithmic proliferation phase enters plateau after finishing; The growth of the 5th generation and first-generation cell does not have significant difference, referring to Fig. 2.With ZUB4 is that mark has good proliferation activity by the positive cell that forward immunity sorting obtains, and keeps the cellular form of fusiformis in vitro culture goes down to posterity process.
With ZUB4 is that mark passes through the positive cell phenotypic characteristic that forward immunity sorting obtains
Get the ZUB4 male people bone marrow MSCs first-generation that vitro culture goes down to posterity and the 5th generation cell, with CD 14-FITC, CD29-PE, CD34-PE, CD44-FITC, CD45-FITC, CD105-PE, CD166-PE, HLA-DR-FITC monoclonal antibody is probe, with the expression of flow cytometry analysis MSCs surface molecular.The result shows: CD29, CD44, CD166, CD105 (SH2) positive mark occur unimodal, hematopoietic cell differentiation antigen CD14, CD34, CD45, HLA-DR express all negative, referring to Fig. 3, Fig. 3 is to be the immunophenotype analysis in five generations of positive cell cultured and amplified in vitro to the of obtaining after the forward immunity sorting of mark with ZUB4.Phenotypic characteristic with positive cell phenotypic characteristic behaviour bone marrow MSCs after the ZUB4 forward immunity sorting, the first-generation and the 5th generation MSCs surface markers are expressed no significant difference (referring to table 1) after vitro culture, the amplification of going down to posterity, showing that with positive cell after the ZUB4 forward immunity sorting be MSCs, is the cell mass of a homogeneous.
The sorting of table 1 monoclonal antibudy ZUB 4 immunomagnetic beads method obtains the immunophenotype (%) of human marrow mesenchymal stem cell
With ZUB4 is that mark passes through the multidirectional differentiation potential of positive cell that forward immunity sorting obtains
When people's bone marrow MSCs of the ZUB4 antigen positive of vitro culture passage cell merges near 70%, change nutrient solution, add osteogenic induction nutrient solution (Stem Cell Technologies Inc.), fatty inducing culture liquid (Stem Cell Technologies Inc.), the pre-induced liquid of Neural Differentiation (LG-DMEM, 20% foetal calf serum, 1mM thioglycerin).
Cultivate about 1 week through osteogenic induction, about 50% cellular form changes, become cube type or multiangular by original spindle-type, and engender the calcification spot, prolongation along with induction time, cube type or multiangular cell and calcified plaque obviously increase, and about 15～21 days cells form the mineralising nodal-like structure of extensive homogeneous.Cultivate after 21 days, the dyeing of Von Kossa method, can see the extracellular matrix calcium deposition of dying brownish black, the result is referring to Fig. 4, be to be the positive cell vitro culture that obtains after the forward immunity sorting of mark with ZUB4, to skeletonization directional induction differentiation, figure is induced osteogenesis differentiation back Von Kossa method dyeing (* 100).
Through becoming fatty inducing culture after 1 week, cellular form begins to become circular or polygonal property, and cell is arranged unordered, can be observed the little fat that occurs high refractivity in the endochylema about 2 weeks and drips, and drip along with the prolongation of induction time is gathered into big fat gradually.Cultivate after 21 days, oil red O stain, fat drip for orange red, and the result is to be the positive cell vitro culture that obtains after the forward immunity sorting of mark with ZUB4, to fatty directional induction differentiation back oil red O stain (* 100) referring to Fig. 5.
Cellular form is not seen considerable change after Neural Differentiation is induced 24 hours in advance, replacing contains the serum-free LG-DMEM of 5mM thioglycerin, inducing in back 3～5 hours, most cells can be changed into typical neuron cell, simple twin-stage cell and complicated multilevel cell occur, and a plurality of neuron cell projections can extend and form netted mutually.Immunohistochemical staining is analyzed, the cell space of neuron cell and part projection neural stem cell mark Nestin, neurone mark NSE, neurone mark NF-M expresses positive, astroglia cell mark GFAP expresses negative, the result is referring to Fig. 6, be to be the positive cell vitro culture that obtains after the forward immunity sorting of mark with ZUB4, the like cell directional induction differentiation of neuralward unit, wherein A, B, C, D detects neural stem cell mark Nestin with the immunocytochemical stain method after being respectively and inducing Neural Differentiation, neurone mark NSE, neurone mark NF-M, the expression (* 100) of astroglia cell mark GFAP.
To sum up, be mark with single anti-human marrow MSCs monoclonal antibody specific ZUB4, people's bone marrow MSCs of the ZUB4 antigen positive that obtains by forward immunity sorting can be divided into adipocyte, scleroblast, neuron cell at directional induction in vitro.
Claims (3)
1, the method for monoclonal antibody ZUB4 immunological magnetic bead sorting primary generation human marrow mesenchyme stem cell, it is characterized in that, described method realizes by following steps: (1) is separated from the marrow suspension and is obtained mononuclearcell, (2) be that the hybridoma excretory monoclonal antibudy ZUB 4 of CCTCC No.C200511 is a mark with preserving number, obtain the human marrow mesenchymal stem cell of ZUB4 antigen positive by indirect magnetic activated cell (sorting) forward sorting; (3) with the human marrow mesenchymal stem cell of ZUB4 antigen positive of (2) step gained in the vitro culture system adherent growth, be fusiformis, cultivate 5 days left and right sides cells and begin obvious amplification, cell phenotype detected and shows after amplification was gone down to posterity: CD29, CD44, CD166, the CD105 positive, CD14, CD34, CD45, HLA-DR express all negative, are the human marrow mesenchymal stem cells of homogeneous.
2, method according to claim 1, it is characterized in that, step (2) is to be the hybridoma excretory monoclonal antibudy ZUB 4 of CCTCC No.C200511 with preserving number, antibody subtype is IgG1, hatch with BMNC, add anti-mouse IgG magnetic bead two anti-hatching behind the centrifuge washing, the human marrow mesenchymal stem cell that from BMNC, separates the ZUB4 antigen positive behind the centrifuge washing by magnetic activated cell (sorting), respectively with ZUB4 antigen-positive cell and ZUB4 antigen negative cell inoculation in culture dish, with the low sugar DMEM nutrient solution that contains 10% foetal calf serum, place 37 ℃, volume fraction is 5%CO
2Incubator is cultivated, and changes liquid 1 time every 3 days afterwards.
3, method according to claim 1, it is characterized in that, the ZUB4 antigen-positive cell that step (3) sorting obtains was cultivated after 5 days, cell attachment is fusiformis in the culture dish, beginning is amplification obviously, cultivates 18～22 days attached cells 80%～90% and merges the cellular form homogeneous, with the inoblast plesiomorphism, be arranged in whirlpool shape, netted, radial; The ZUB4 antigen negative cell that sorting obtains is cultivated and is seen a large amount of circular suspension cells after 5 days, wherein has the minute quantity cell attachment to be spindle cell, cultivates and still sees round cell after 20 days, and a small amount of adherent cell is more roomy, and the form heterogeneity is arranged random.
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| New Monoclonal Antibodies Reactive withHumanBoneMarrow-Derived Mesenchymal Stem Cells. Xiaoyu Lai等.Blood(ASH Annual Meeting Abstracts),Vol.106 . 2005 |
| New Monoclonal Antibodies Reactive withHumanBoneMarrow-Derived Mesenchymal Stem Cells. Xiaoyu Lai等.Blood(ASH Annual Meeting Abstracts),Vol.106 . 2005 * |
| 人骨髓间充质干细胞的鉴定：抗SH2和SH3单克隆抗体的制备与应用. 刘培光等.中国实验血液学杂志，第13卷第4期. 2005 |
| 人骨髓间充质干细胞的鉴定：抗SH2和SH3单克隆抗体的制备与应用. 刘培光等.中国实验血液学杂志，第13卷第4期. 2005 * |
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