CN102533646A - Cell immunofluorescent staining method for differentiation induction process of bone marrow stromal cells - Google Patents
Cell immunofluorescent staining method for differentiation induction process of bone marrow stromal cells Download PDFInfo
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- CN102533646A CN102533646A CN2011104481401A CN201110448140A CN102533646A CN 102533646 A CN102533646 A CN 102533646A CN 2011104481401 A CN2011104481401 A CN 2011104481401A CN 201110448140 A CN201110448140 A CN 201110448140A CN 102533646 A CN102533646 A CN 102533646A
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Abstract
The invention provides a cell immunofluorescent staining method for the differentiation induction process of bone marrow stromal cells, which is applied to an all-trans retinoic acid combined cell factor induction process of differentiating the bone marrow stromal cells into neuron-like cells. An all-trans retinoic acid (ATRA), a basicfibroblast growth factor (bFGF) and an epidermal growth factor (EGF) are applied in the induction process to induce the bone marrow stromal cells (BMSCs) to be differentiated into the neuron-like cells. The neuron-like cells not only have the typical forms of nerve cells but also can be used for expressing neuron-marked antigen neuron specific enolase and asrocyte cell-marked antigen glial fiber acidic protein; the BMSCs still have the capacity of differentiating a cross-germ layer into non-mesenchymal cells, is capable of enabling the cross-germ layer into neuron-like cells and is expected to become seed cells of replacement therapy of nerve cells.
Description
Technical field
The present invention relates to biological technical field; Be particularly related to the cellular immunofluorescence dyeing process in a kind of mesenchymal stem cells MSCs induction process, it is applied to the all-trans-retinoic acid associational cells factor that mesenchymal stem cells MSCs is divided into neuron cell and induces process.
Background technology
Though mesenchymal stem cells MSCs (BMSCs) transplantation treatment ischemic brain injury has no doubt been obtained certain progress; But BMSCs transplant back neurocyte transformation efficiency external can be up to 80%; Be merely 3%～10% in vivo; And the ratio that is divided into neurogliocyte in vivo under the environment is big and to be divided into the ratio of neuron cell less, and this bring unfavorable factor can for undoubtedly the recovery of nervous function.If can be neuron cell in vitro differentiation with marrow stromal cell, the Transplanted cells of going again has the effect of getting twice the result with half the effort.
The BMSCs in mesoderm source is in the starting stage to the research of the neurone differentiation in ectoderm source.All-trans-retinoic acid is the verivate of vitamin A, can obviously increase the quantity of neurocyte and be dose-dependent effect, can regulate the cell differentiation of nerve cord of EGF reaction, increases the synthetic of neuronal cell and astroglia cell.Itself and neurotrophic factor and cytokine united be used for inducing embryo stem cell neuralward stem cell direction differentiation, growth course is similar in the consubstantiality, and can the partial simulation internal milieu, therefore comes into one's own day by day.But; These researchs rest on more induces back BMSC to have the neuron morphology characteristic and express on the neurocyte affinity tag level; And lack evidence with neurocyte physiology characteristic, therefore many scholars to think that this type cell is called as " neuron cell " more appropriate.
Summary of the invention
The present invention proposes the cellular immunofluorescence dyeing process in a kind of mesenchymal stem cells MSCs induction process; It is applied to the all-trans-retinoic acid associational cells factor that mesenchymal stem cells MSCs is divided into neuron cell and induces process; The said process of inducing is used all-trans-retinoic acid (ATRA), Prostatropin (bFGF), Urogastron (EGF) inducing bone mesenchymal stem cell (BMSCs) and is divided into neuron cell; The representative configuration that has not only had neurocyte; And expression neurone sign antigen neuronspecific enolase (neuron specific enolase; NSE), star spongiocyte sign antigen GFAP (glial fibrilament acidic protein, GFAP); Prompting BMSCs has still kept the ability that germinal layer is divided into non-mesenchymal cell of striding, and can make and stride germinal layer and be divided into neuron cell, is expected to become the seed cell of neurocyte replacement therapy.
Concrete, the all-trans-retinoic acid associational cells factor induction method that mesenchymal stem cells MSCs of the present invention is divided into neuron cell comprises the steps:
S1. separation and Culture cell: all operations all carries out under aseptic condition, collects during spongy bone fragment or the Lower limb bone open operation of derived from bone marrow when operation on hip joint.The marrow suspension that anticoagulant heparin is collected, D-Hanks liquid dilution (V: V, 1: 1) slowly is added on the Ficoll parting liquid of half volume, and the centrifugal 15min of 600g collects the interfacial layer cell, and D-Hanks liquid is washed 1 time, and the adjustment cell concn is 1 * 10
5/ ml is inoculated in 25cm
2Culturing bottle, substratum is DMEM-LG+1%Penicillin-Streptomycin+10%FBS, 37 ℃, 5%CO
2, saturated humidity leaves standstill cultivation.Cell density reaches 70% and merges the back with 0.25% tryptic digestion, the ratio that 1 bottle of branch the is 2 bottles cultivation of going down to posterity.
The morphological observation of S2.BMSCs: observe the growing state and the morphological specificity of former generation and passage cell following every day at inverted phase contrast microscope, and take the photograph the sheet record.
S3. flow cytometer detects antigen presentation: culturing cell (2-3 generation) contains 1% bSA PBS adjustment cell to 5 * 10 with behind 0.25% the tryptic digestion
6/ ml.Get the 50ul cell suspension, add respectively following mouse-anti human monoclonal antibodies: CD34-FITC, CD45-PE, CD29-FITC, CD44-PE, CD105-FITC, CD106-FITC,, put 4 ℃ and incubated 30 minutes, PBS washes 2 times, carries out flow cytometry analysis.
S4. induce Analytical Chemical Experiment: get the 2-3 subtituted culturing cell, be inoculated in 6 orifice plates respectively, stand density reaches at 60% o'clock, changes inducing culture (DMEM+10%FBS+1 μ mol/L ATRA+20ng/ml bFGF+20ng/ml EGF) into.37 ℃, 5%CO
2, saturated humidity leaves standstill cultivation.
S5. cellular immunofluorescence dyeing: culturing cell is inducing differentiation to adopt immunofluorescence dyeing after 3 days.
Description of drawings
Through the detailed description below in conjunction with accompanying drawing, aforesaid purpose, the feature and advantage with other of the present invention will become obvious.Wherein:
The steps flow chart synoptic diagram that is divided into the all-trans-retinoic acid associational cells factor induction method of neuron cell for mesenchymal stem cells MSCs of the present invention shown in Figure 1.
Embodiment
Be illustrated in figure 1 as the steps flow chart synoptic diagram that mesenchymal stem cells MSCs of the present invention is divided into the all-trans-retinoic acid associational cells factor induction method of neuron cell, the method for said embodiment comprises the steps:
S1. separation and Culture cell: all operations all carries out under aseptic condition, collects during spongy bone fragment or the Lower limb bone open operation of derived from bone marrow when operation on hip joint.The marrow suspension that anticoagulant heparin is collected, D-Hanks liquid dilution (V: V, 1: 1) slowly is added on the Ficoll parting liquid of half volume, and the centrifugal 15min of 600g collects the interfacial layer cell, and D-Hanks liquid is washed 1 time, and the adjustment cell concn is 1 * 10
5/ ml is inoculated in 25cm
2Culturing bottle, substratum is DMEM-LG+1%Penicillin-Streptomycin+10%FBS, 37 ℃, 5%CO
2, saturated humidity leaves standstill cultivation.Cell density reaches 70% and merges the back with 0.25% tryptic digestion, the ratio that 1 bottle of branch the is 2 bottles cultivation of going down to posterity.
The morphological observation of S2.BMSCs: observe the growing state and the morphological specificity of former generation and passage cell following every day at inverted phase contrast microscope, and take the photograph the sheet record.
S3. flow cytometer detects antigen presentation: culturing cell (2-3 generation) contains 1% bSA PBS adjustment cell to 5 * 10 with behind 0.25% the tryptic digestion
6/ ml.Get the 50ul cell suspension, add respectively following mouse-anti human monoclonal antibodies: CD34-FITC, CD45-PE, CD29-FITC, CD44-PE, CD105-FITC, CD106-FITC,, put 4 ℃ and incubated 30 minutes, PBS washes 2 times, carries out flow cytometry analysis.
S4. induce Analytical Chemical Experiment: get the 2-3 subtituted culturing cell, be inoculated in 6 orifice plates respectively, stand density reaches at 60% o'clock, changes inducing culture (DMEM+10%FBS+1 μ mol/L ATRA+20ng/ml bFGF+20ng/ml EGF) into.37 ℃, 5%CO
2, saturated humidity leaves standstill cultivation.
S5. cellular immunofluorescence dyeing: culturing cell is inducing differentiation to adopt immunofluorescence dyeing after 3 days.Attached cell removes substratum, washes 3 times with PBS, and the Paraformaldehyde 96 through 4% is 30min fixedly; PBS washing 3 times, 0.3%TritonX-100 handles 20min, PBS rinsing 3 times; 10% sheep blood serum room temperature sealing 20min, sucking-off serum adds the anti-people NSE of rabbit (1: 500), GFAP (1: 1000) respectively; Hatch 2h for 37 ℃, PBS washes 3 times then, adds sheep anti mouse-FITC (1: 500) and incubates 30min.The colour developing back is observed under fluorescent microscope.
Experimental result and analysis:
1.BMSCs former be commissioned to train foster: former be commissioned to train support 4h after visible cell adherent, the beginning attached cell is single dispersion, is mostly long shuttle shape.Begin propagation behind the 3d, form is various, and one is fusiformis, has 2～3 long projections, and its nucleolus or ellipse have 1～3 kernel; Other has a kind of be broad flat-section or feather shape, out-of-shape, and nucleus also is circular or oval.Other have some to be tiny fusiformis, circle etc., measure less.The back is with form cell aggregation and form colony, and attached cell begins to merge behind 5～7d, forms individual layer, and wherein spindle cell is arranged and had certain directivity.Visible behind 7～9d have 80% above cytogamy, is growth of whirlpool shape or cluster growth, and along with the increase of cell density, it is elongated that cell space becomes, the similar inoblast of form.Primary cell growth is slower, needs 2-4 week at the bottom of covering with bottle, and under the condition that foetal calf serum exists, passage cell can be bred one times about 3-5d.Cultured cells can go down to posterity by stable growth, and vitro culture is after 10 generations, and the rate of propagation of cell does not have obviously and slows down.
2.BMSCs the surface antigen characteristic
The flow cytometer detected result shows that BMSCs expresses CD29, CD44 and CD105, does not express CD34, CD45 and CD106.
3. induce differentiation and immunofluorescence dyeing result
Cellular form is considerable change behind the adding induced liquid 2h, and the tenuigenin of BMSCs shrinks to nuclear under the light microscopic, is typical perikaryon form.Most cells can form the neuron cell form behind 3～5h, and cell space is rounded, and projection is longer, branch occurs in lug tips, and the projection of part flanking cell connects into net, but cell number does not have obvious increase.Most cells changes into bipolar or multipolar neuron cell appearance form behind the 3d, stretches out cynapse (similar aixs cylinder or dendron), pulls into nettedly between the part cell, and the visible NSE of dyeing, GFAP are positive.
The induction method application all-trans-retinoic acid (ATRA) of present embodiment, Prostatropin (bFGF), Urogastron (EGF) inducing bone mesenchymal stem cell (BMSCs) are divided into neuron cell; The representative configuration that has not only had neurocyte; And expression neurone sign antigen neuronspecific enolase (neuron specific enolase; NSE), star spongiocyte sign antigen GFAP (glial fibrilament acidic protein, GFAP); Prompting BMSCs has still kept the ability that germinal layer is divided into non-mesenchymal cell of striding, and can stride germinal layer and be divided into neuron cell, is expected to become the seed cell of neurocyte replacement therapy.
The present invention is not limited to described embodiment, and those skilled in the art still can do some corrections or change, so rights protection scope of the present invention is as the criterion with claims restricted portion not breaking away from spirit of the present invention promptly openly in the scope.
Claims (1)
1. the cellular immunofluorescence dyeing process in the mesenchymal stem cells MSCs induction process; It is used for the all-trans-retinoic acid associational cells factor that mesenchymal stem cells MSCs is divided into neuron cell and induces process; The said process of inducing comprises that the morphological observation of separation and Culture cell, BMSCs, flow cytometer detect antigen presentation, induce Analytical Chemical Experiment and cellular immunofluorescence dyeing, and said cellular immunofluorescence dyeing process is:
Inducing differentiation to adopt immunofluorescence dyeing after 3 days culturing cell;
Attached cell removes substratum, washes 3 times with PBS, and the Paraformaldehyde 96 through 4% is 30min fixedly; PBS washing 3 times, 0.3%TritonX-100 handles 20min, PBS rinsing 3 times; 10% sheep blood serum room temperature sealing 20min, sucking-off serum adds the anti-people NSE of rabbit (1: 500), GFAP (1: 1000) respectively; Hatch 2h for 37 ℃, PBS washes 3 times then, adds sheep anti mouse-FITC (1: 500) and incubates 30min; And
The colour developing back is observed under fluorescent microscope.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103245648A (en) * | 2013-04-28 | 2013-08-14 | 西安交通大学 | Method for observing attached wall of vascular stem cell |
| CN104215773A (en) * | 2014-08-22 | 2014-12-17 | 武汉高华细胞技术有限公司 | Method for authenticating activity of bone marrow mesenchymal stem cells |
| CN106950203A (en) * | 2015-10-15 | 2017-07-14 | 浜松光子学株式会社 | Phagocytic activity evaluation method and fluorescence analysis |
| CN111471654A (en) * | 2020-04-22 | 2020-07-31 | 山东省立医院 | Method for differentiating mesenchymal stem cells into DAergic neurons and molecular mechanism thereof |
-
2011
- 2011-12-27 CN CN2011104481401A patent/CN102533646A/en active Pending
Non-Patent Citations (3)
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| ZHAO HAN-NING ET AL: "All-trans retinoic acid-induced nerve cell differentiation of rat bone marrow mesenchymal stem cells inhibits T lymphocyte proliferation", 《JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH》 * |
| 施雪英 等: "全反式维甲酸诱导大鼠骨髓间充质干细胞分化为神经元样细胞的研究", 《立体定向和功能性神经外科杂志》 * |
| 陆华 等: "全反式维甲酸联合细胞因子诱导骨髓间充质干细胞分化为神经元样细胞的作用", 《江苏医药》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103245648A (en) * | 2013-04-28 | 2013-08-14 | 西安交通大学 | Method for observing attached wall of vascular stem cell |
| CN103245648B (en) * | 2013-04-28 | 2015-05-27 | 西安交通大学 | Method for observing attached wall of vascular stem cell |
| CN104215773A (en) * | 2014-08-22 | 2014-12-17 | 武汉高华细胞技术有限公司 | Method for authenticating activity of bone marrow mesenchymal stem cells |
| CN104215773B (en) * | 2014-08-22 | 2016-06-29 | 武汉高华细胞技术有限公司 | A kind of method identifying mesenchymal stem cells MSCs activity |
| CN106950203A (en) * | 2015-10-15 | 2017-07-14 | 浜松光子学株式会社 | Phagocytic activity evaluation method and fluorescence analysis |
| US10724953B2 (en) | 2015-10-15 | 2020-07-28 | Hamamatsu Photonics K.K. | Method for evaluating phagocytic capacity and fluorescence measurement method |
| CN106950203B (en) * | 2015-10-15 | 2021-04-06 | 浜松光子学株式会社 | Phagocytosis ability evaluation method and fluorescence measurement method |
| CN111471654A (en) * | 2020-04-22 | 2020-07-31 | 山东省立医院 | Method for differentiating mesenchymal stem cells into DAergic neurons and molecular mechanism thereof |
| CN111471654B (en) * | 2020-04-22 | 2024-03-22 | 广东横琴粤澳深度合作区齐美国际干细胞医院有限公司 | Method for differentiating mesenchymal stem cells into DAergic neurons and molecular mechanism thereof |
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Application publication date: 20120704 |