CN1546656A - Method for adult mesenchymal stem cells in vitro directional induction and differentiation to endothelial cell of blood vessel - Google Patents
Method for adult mesenchymal stem cells in vitro directional induction and differentiation to endothelial cell of blood vessel Download PDFInfo
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- CN1546656A CN1546656A CNA2003101184235A CN200310118423A CN1546656A CN 1546656 A CN1546656 A CN 1546656A CN A2003101184235 A CNA2003101184235 A CN A2003101184235A CN 200310118423 A CN200310118423 A CN 200310118423A CN 1546656 A CN1546656 A CN 1546656A
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Abstract
The method provided by the present invention comprises, utilizing Percoll (1.073g/ml) to separate marrow mesenchymal stem cells (MSCs) from ordinary adult bone marrow, using 10% FBS of LG-DMEM for culture medium purification and expansion, using a flow type cell instrument for purity determination, using vascular endothelial growth factor (VEGF) to evoke MSCs for endothelial cells (ECs) differentiation, VIII factor (vWF) and Tie-2 immunity grouping, and using penetrance electric mirror (TEM) to assess the endothelial cell nature.
Description
One, technical field
The outer directional induction of the dried light face type of medulla mesenchyma that the present invention relates to be grown up is divided into a kind of method of vascular endothelial cell.
Reach at present domestic all simple employing separation of C D34+ cell in the world as the precursor cell of inducing the differentiation endotheliocyte, do not see and utilize adult bone bone marrow-drived mesenchymal stem directional induction in vitro to be divided into endotheliocyte.
In recent years, distinctive biological characteristics of stem cell and potential using value are subjected to showing great attention to of various countries scholar.Marrow, blood and other tissue-derived hemopoietic stem cell, mescenchymal stem cell, become blood-vascular cell, blood vessel stem cell and other tissue stem cell to provide possibility for the repopulating cell of organizational project source.The CD34 of peripheral blood, derived from bone marrow
+Hemopoietic stem cell can form " pebbles " sample (cobblestone) endothelial layer after 15～20 days under external evoked condition.At present to CD34
+The extraction of cell, amplification in vitro, and had the complete method of a cover to endotheliocyte directional induction differentiation.
Marrow is made up of NA hemopoietic stem cell and adhesivity stroma cell, contain in these adherent stroma cells abundant mescenchymal stem cell (mesenchymal stem cells, MSCs).MSCs is the early stage cell that mesoderm is grown, and has the tissue stem cell of multidirectional differentiation potential.Can not only be divided into hematopoiesis matrix, hematopoiesis support also can be divided into the multiple hematopoiesis histocyte, particularly mesoderm in addition and the histocyte in neuroderm source.In the presence of suitable environment and inducible factor, adult's bone marrow MSCs can be divided into multiple human tissue cell, comprises bone marrow matrix, bone, cartilage, muscle, tendon, fat, inoblast and myocardial cell etc.; In addition, theoretically, MSCs also can be to the differentiation of internal organs mesoderm, as (endothelial cells ECs) with the smooth muscle cell differentiation, but does not also carry out the in-vitro directed experiment that is divided into vascular endothelial cell with MSCs both at home and abroad at present to vascular endothelial cell.
Two, background technology
The key that decision MSCs is divided into ECs is the condition of inducing differentiation.In vivo, the generation of tissue stem cell and function obtain with tissue in the transcription factor of genes encoding conduct relevant with the extracellular signal.But external, the directed differentiation mechanism of tissue stem cell is still not fully aware of.Basis nutrition, cell density, spatial organization, mechanical force, somatomedin etc. all have very big influence to the differentiation of MSCs.Tremain points out that MSCs clone's mRNA can give expression to the characteristics with endotheliocyte, epidermic cell and neurocyte.Lodie points out that under conditions suitable, MSCs can be divided into endotheliocyte.Studies show that VEGF induces MSCs to be divided into the key point of ECs.VEGF is the aglucon of a kind of tyrosine kinase receptor Flk-1/KDR and Flt-1, and it can specificity urge endothelial cell division, and people's marrow medullary system progenitor cell is had regulating effect.After containing human vascular endothelial growth factor and cultivating about 2 weeks, can differentiate ECs.MSCs is inducing in the atomization to ECs, directly differentiation does not produce ECs, but break up and of short duration amplifying cells (transient amplifying cell) earlier, again through several times after the division that does not wait for tens times directed differentiation be endothelium ancestral/precursor cell (committedprogenitors/precursor), and then can be divided into postmitotic cells (post-mitotic cells) and whole undifferentiated cell (terminally-differentiated cells), i.e. endotheliocyte.
Three, summary of the invention
Content of the present invention is unexposed to be delivered, and those skilled in the art can not infer the method that mesenchymal stem cells MSCs directional induction in vitro of the present invention is divided into endotheliocyte that obtains according to existing technology as not spending creative work at all.
The purpose of this invention is to provide a kind of mesenchymal stem cells MSCs directional induction in vitro and be divided into the thin method of endothelium, for external structure heart tissue engineering lobe provide a kind of economy, convenient, effectively obtain the novel method of repopulating cell, so that be applied to animal experimental model and clinical heart valve replacement surgery.
The present invention utilizes Percoll (1.073g/ml) to isolate MSCs from normal adult marrow, and LG-DMEM substratum purifying and amplification, the flow cytometer of 10%FBS are identified its purity; (VEGF) induces MSCs to break up to ECs with vascular endothelial growth factor, the VIII factor (vWF) and Tie-2 immunohistochemical methods and transmission electron microscope (TEM) identification of cell character.The result shows 5.0 * 10
5Individual MSCs after 15 generations, obtains 8.0 * 10 at amplification in vitro
12Individual MSCs has increased about 1.6 * 10
7Doubly; MSCs was adding the VEGF inducing culture about 14～21 days, and 90% inducing cell is positive to the VIII factor and Tie-2 related antigen; TEM can be observed the Weible-palade corpusculum in the endochylema, turn out to be ECs.Result of study explanation adult bone marrow MSCs has the potential that directional induction is divided into ECs external, and this is a heart tissue engineering lobe external structure, and especially the source of repopulating cell provides possibility in baby's congenital heart trouble Tissue Engineering Study.
With embodiment the present invention is further elaborated below.
Four, description of drawings (seeing the Figure of description page or leaf)
Five, embodiment
Embodiment 1
The in-vitro separation of adult bone bone marrow-drived mesenchymal stem, purifying and amplification
One method
1. the heparin liquid penicillin bottle is prepared: in disinfectant penicillin bottle, add 1ml heparin liquid (250U/ml);
Vessel when getting marrow.Bone marrow prepare is got 5ml for every bottle and is got final product.
The preparation of heparin liquid: 2 heparin (1.25 ten thousand
U/)+100ml physiological saline → 250U/ml/ bottle.
2.Percoll use the preparation of liquid:
Percoll stoste (density is 1.13) → stock solution → application liquid (density is 1.073)
That is: Percoll stoste 5.04ml+0.56ml 1.5M NaCl+4.4ml physiological saline=10ml uses liquid
Or: Percoll stoste: 1.5M NaCl=9: 1 (V/V) → stock solution
Stock solution: physiological saline=5.6ml: 4.4ml → application liquid
3. the final concentration of getting bone marrow prepare 4～5ml/ bottle+heparin liquid 1ml/ bottle → 5～6ml mixed solution → heparin liquid is 40～50U/ml.
Add marrow 4.Percoll use in the liquid bottle, promptly marrow uses on the liquid two at Percoll.General by 2: 1 (pressing 1: 1 during practical application), its objective is in order to save Percoll and use liquid.That is: Percoll uses liquid: marrow=1: 1 (v/v).
5. gradient centrifugation.1800rpm * 20min gets mononuclearcell (mononuclear cells, MNCs) aspect, the preparation MNCs suspension of intermediate interface.
6. cell washs twice through PBS, or counts with twice back of LG-DMEM washing.That is: MNCs+LG-DMEM 10ml → piping and druming gently, mixing → centrifugal, 1500rpm, 5min washing MNCs → repeat.
7. behind the removal supernatant, with LG-DMEM 2ml suspension MNCs (promptly having diluted 2 times).
8. inoculation culture.Inoculum density is: 2 * 10
5/ cm
2(as using 25cm
2Culturing bottle, then multiply by 25).
9. the former foster MSCs that is commissioned to train, its nutrient solution consists of: LG-DMEM+10%FBS (import).
10. under mirror behind the 48h, observe,, then can change nutrient solution entirely if any attached cell; As do not see attached cell, half amount is changed liquid.11. after former foster the going down to posterity for the first time of being commissioned to train, (the 25cm composed as follows of its nutrient solution
2Culturing bottle): LG-DMEM 4.5ml
FBS (homemade) 0.5ml (10%)
L-glutaminate 50 μ l (1mmol/l)
12. adherent confluent monolayer cells presses 6 * 10 through 0.125% tryptic digestion after reaching and merging more than 90%
3Cells/cm
2Density be inoculated in the culturing bottle that goes down to posterity (T-75) and carry out amplification cultivation.
13. former be commissioned to train to support when typical inoblast sample form and cell density occurring merge when reaching 80%～90%, carry out cell make a video recording (seeing Figure of description 1).
Annotate: above operation is all in strict accordance with the aseptic technique rules
Two. the result
Can be through aforesaid method from 5.0 * 10
5Individual former generation MSCs after 15 generations, obtains 8.0 * 10 at amplification in vitro
12Individual MSCs has increased about 1.6 * 10
7Doubly.
Embodiment 2
MSCs surface antigen Molecular Detection
One, method
During the s-generation, use the tryptic digestion harvested cell 1.MSCs cell cultures increases, get 2 * 10 respectively
5React 30min in individual cell and mouse-anti people CD13, CD29, CD34, CD45, CD105 and the HLA-DR antibody room temperature, remove behind the unreacted antibody two anti-lucifuges reaction 15min with the FITC mark with the PBS washed twice.
2. get 2 * 10
5The straight labeling antibody room temperature of individual cell and CD166 lucifuge is hatched 20min, and 1%BSA-PBS cleans 2 times, and it is resuspended to add 500 μ l PBS, and establish IgG1-FITC, IgGl-PE, IgG1-PC5 is contrast.
3. use flow cytometer (COULTER Epics XL-AD 2517, BECKMAN COULTER company, the U.S.) and detect at least 10,000 cell, with Cofit software analysis result.
Two results
Flow cytometer detects the surface antigen characteristic of MSCs.CD34 (hematopoietic stem and endothelial cell specific sign), CD45 (leukocyte common antigen (LCA)) and HLA-DR expression such as (HLA II quasi-molecule, antigen presenting cell and activated T cells surface markers) presents feminine gender; And CD105 (mainly being expressed in endotheliocyte and activated monocyte), CD13 (mainly being expressed in monocyte and granulocyte), the CD29 (acceptor of fibronectin and transparency grease hydrochlorate, the stroma cell surface marker) and CD166 (mesenchymal cell surface marker) express and to present the positive, all more than 98%.Through the back (cell approximately divides 14 times) of going down to posterity more than 2 generations, the MSC of amplification in vitro has reached the homogeneous more than 95% (seeing Figure of description 2) on form and cell phenotype.
Embodiment 3
MSCs is to the directional induction differentiation of endotheliocyte
One. method
1. the MSCs that selected for the 3rd～10 generation is as experiment material.
2. consisting of of its inducing culture system: DMEM-HG, 20%FBS (river, Tianjin page or leaf biochemical product company limited, 030509), VEGF (10ng/ml lot number:, Pepro Tech EC LTD company, Britain), bFGF (5ng/ml, Pepro Tech EC LTD company, Britain), L-glutaminate (2mmol/L), penicillin liquid (100U/ml), streptomycin solution (100 μ g/ml).
3. per 2～3d changes nutrient solution, when adherent confluent monolayer cells reaches 90% or more after the fusion, goes down to posterity through 0.125% tryptic digestion; By 6 * 10
3Cells/cm
2Density be inoculated in 25cm
2In the culturing bottle.The operation that repeats above-mentioned passage can realize effective amplification of cell.
Two. the result
MSCs is after the about 14～21d of differentiation culture system is induced in adding, and how unfixing form is, and adherent cell shows as bigger, is flat, big polygon, spindle shape, garden shape, ellipse original shape or tabular, and cell has abundant endochylema and irregular projection.The born of the same parents that grow up to individual layer are typical " pebbles " sample, are endothelial cell morphology feature (seeing Figure of description 3).
Embodiment 4
The endotheliocyte of MSCs directional induction differentiation is identified
One. method
1. the inductive cell grows up to individual layer under the light microscopic, is " pebbles " and (cobblestone) prepares cell suspension during sample, fixes with 5% glutaraldehyde and 1% perosmic anhydride respectively, makes the capable transmission electron microscope of electron microscope specimen (TEM, JEM-1010, Japan) observation.
2.VIII the immunohistochemical methods of the factor (vWF) and Tie-2 related antigen detects (ABC enzyme labelling method): select MSCs through inducing 14～21 days cell of differentiation culture system, the digestion back with centrifugal smearing machine smear on slide glass, the acetone fixed cell.Adopt ABC enzyme labelling method to detect, and establish negative control group; Drip 1: 100 the anti-people VIII of rabbit factor antibody (is anti-) and 1: 200 Tie-2 mouse-anti human monoclonal antibodies.Nikon inverted phase contrast microscope (Olympus, Japan) is observed down, takes pictures.The positive reaction of brown granular appears in the cytoplasm.
Two. the result
Visible Weible-palade corpusculum (being called for short the W-P corpusculum) in the endochylema is the distinctive particle of ECs (seeing Figure of description 5) under the TEM.The Showed by immune group result cell of VIII (vWF) factor and Tie-2 related antigen is multiangular or round, be covered with fine and closely woven brown granular in the endochylema, do not see paintedly in the nucleus, be a slice negative control area, prove that what cultivate is endotheliocyte (seeing Figure of description 4).
Claims (1)
1. the outer directional induction of the dried light face type of adult's medulla mesenchyma is divided into vascular endothelial cell (endothelial cells, a kind of method ECs).It is characterized in that utilizing Percoll (1.073g/ml) from normal adult marrow, isolate mesenchymal stem cells MSCs (mesenchymal stem cells, MSCs), LG-DMEM substratum purifying and the amplification of 10%FBS, flow cytometer is identified its purity; (vascularendothelial growth factor VEGF) induces MSCs to break up to ECs, the VIII factor (vWF) and Tie-2 immunohistochemical methods, and transmission electron microscope (TEM) evaluation vascular endothelial cell character with vascular endothelial growth factor.The result shows that mesenchymal stem cells MSCs (MSCs) is external and can differentiate vascular endothelial cell after about 14～21 days adding the VEGF inducing culture.Confirmed that the adult bone bone marrow-drived mesenchymal stem has directional induction and is divided into the thin potential of blood vessel endothelium external.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101185651B (en) * | 2007-10-24 | 2010-08-11 | 山东大学 | Application of 2,3-dihydrogen-3-hydroxymethyl-6-amino-[1,4]-benzoxazin in preparing medicine |
| CN102776150A (en) * | 2011-05-13 | 2012-11-14 | 上海交通大学医学院附属第九人民医院 | Method for inducing umbilical cord mesenchymal stem cells to differentiate into endothelial cells |
| CN103041453A (en) * | 2013-01-18 | 2013-04-17 | 新乡医学院 | Double-layer skin covering material compositely built by collagen/fibrin glue-VEGF (vascular endothelial growth factor) and mesenchymal stem cell as well as preparation method and application of double-layer skin covering material |
| CN105748523A (en) * | 2016-02-28 | 2016-07-13 | 深圳爱生再生医学科技有限公司 | Stem cell preparation for treating hepatic failure as well as preparation method and application thereof |
| WO2016175618A1 (en) * | 2015-04-29 | 2016-11-03 | 가톨릭대학교 산학협력단 | Method for converting mesenchymal stem cells into endothelial cells by using specific transcription factors |
| CN108410796A (en) * | 2018-03-13 | 2018-08-17 | 太原市中心医院 | A method of induction human mesenchymal stem cell breaks up to vascular endothelial cell |
| CN109666628A (en) * | 2019-02-19 | 2019-04-23 | 北京大学第医院 | The induced medium and abductive approach of a kind of human marrow mesenchymal stem cell to endothelial cell directed differentiation |
| US10428307B2 (en) | 2015-04-29 | 2019-10-01 | The Catholic University Of Korea Industry-Academic Cooperation Foundation | Method for converting mesenchymal stem cells into endothelial cells by using specific transcription factors |
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2003
- 2003-12-16 CN CNA2003101184235A patent/CN1546656A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101185651B (en) * | 2007-10-24 | 2010-08-11 | 山东大学 | Application of 2,3-dihydrogen-3-hydroxymethyl-6-amino-[1,4]-benzoxazin in preparing medicine |
| CN102776150A (en) * | 2011-05-13 | 2012-11-14 | 上海交通大学医学院附属第九人民医院 | Method for inducing umbilical cord mesenchymal stem cells to differentiate into endothelial cells |
| CN103041453A (en) * | 2013-01-18 | 2013-04-17 | 新乡医学院 | Double-layer skin covering material compositely built by collagen/fibrin glue-VEGF (vascular endothelial growth factor) and mesenchymal stem cell as well as preparation method and application of double-layer skin covering material |
| WO2016175618A1 (en) * | 2015-04-29 | 2016-11-03 | 가톨릭대학교 산학협력단 | Method for converting mesenchymal stem cells into endothelial cells by using specific transcription factors |
| US10428307B2 (en) | 2015-04-29 | 2019-10-01 | The Catholic University Of Korea Industry-Academic Cooperation Foundation | Method for converting mesenchymal stem cells into endothelial cells by using specific transcription factors |
| CN105748523A (en) * | 2016-02-28 | 2016-07-13 | 深圳爱生再生医学科技有限公司 | Stem cell preparation for treating hepatic failure as well as preparation method and application thereof |
| CN108410796A (en) * | 2018-03-13 | 2018-08-17 | 太原市中心医院 | A method of induction human mesenchymal stem cell breaks up to vascular endothelial cell |
| CN108410796B (en) * | 2018-03-13 | 2020-12-22 | 太原市中心医院 | Method for inducing differentiation of human mesenchymal stem cells into vascular endothelial cells |
| CN109666628A (en) * | 2019-02-19 | 2019-04-23 | 北京大学第医院 | The induced medium and abductive approach of a kind of human marrow mesenchymal stem cell to endothelial cell directed differentiation |
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