CN101215545A - Method for obtaining neural progenitor cells induced by bone marrow mesenchymal stem cells - Google Patents
Method for obtaining neural progenitor cells induced by bone marrow mesenchymal stem cells Download PDFInfo
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Abstract
The invention provides a method for inducing mesenchymal stem cell in the bone marrow to obtain the nerve prosoma cell, which comprises the following steps: doing generation culture for the separated and purified mesenchymal stem cell in the bone marrow, switching stable generation mesenchymal stem cell into induction culture medium, inducing and culturing for 7-20 d, obtaining nerve prosoma cell. The induction culture medium is basic culture medium which is charged with growth factor, wherein the growth factor mainly comprises an epiderm growth factor (EGF), a base fibroblast growth factor (bFGF), an insulin growth factor-1 (IGF-1) and a nerve nourishment factor 3 (NT-3). The invention provides a method for inducing mesenchymal stem cell in the bone marrow to obtain the nerve prosoma cell which can be differentiated into nerve cell and the nerve prosoma cell can be switched into inner ear hair cell and inner ear supporting cells, which provides experiment evidence for cochlear cell transplantation therapeutic sound perception nerve deafness and solves the problem of the transplantation cell resource.
Description
(1) technical field
The present invention relates to a kind of mesenchymal stem cells MSCs that utilizes and induce the method that obtains neural precursor.
(2) background technology
Mesenchymal stem cells MSCs (mesenchymal stem cells, MSCs) except that participating in constituting the microenvironment of hematopoiesis support, also have powerful the of self-replication capacity and multidirectional differentiation potential, different external evoked conditions can determine its differentiation direction, may be the transcription factors that these inductive conditions have started the decision differentiation direction.And when MSCs is transplanted in the body different organizing by intravenous route or local injection, can in corresponding tissue, be differentiated to form such histocyte.The regeneration of neurocyte is transplanted and to be perplexed people behind the nervous system injury for a long time, and embryo neural stem cells is transplanted and can be promoted the neural recovery of decreasing function, but has limited its application because of ethics problem.It is abundant that MSCs has the source, do not relate to advantages such as ethics problem, may provide new way for the treatment of nervous system disorders.
Although currently reported MSCs has the potential of neuralward cytodifferentiation, the system of inducing still needs to improve, and is active bad behind the relatively poor or cell induction of system's repeatability, finally can not obtain functioning cell.
(3) summary of the invention
The object of the invention provides the mesenchymal stem cells MSCs that utilizes of a kind of system good reproducibility and induces the method that obtains neural precursor.
The technical solution used in the present invention is:
A kind of mesenchymal stem cells MSCs that utilizes is induced the method that obtains neural precursor, described method is to separate the mesenchymal stem cells MSCs of purification through the cultivation of going down to posterity, get the stable mesenchymal stem cells MSCs that goes down to posterity and change inducing culture over to, inducing culture 7～20 days obtains neural precursor; Described inducing culture is the basic medium that is added with somatomedin, described somatomedin mainly comprises Urogastron (EGF), Basic Fibroblast Growth Factor (bFGF), insulin-like growth factor-i (IGF-1), neurotrophic factor 3 (NT-3), addition is: EGF 10～30ng/mL basic medium, bFGF 5～20ng/mL basic medium, IGF-140～60ng/mL basic medium, NT-310～30ng/mL basic medium.
Described somatomedin also can be the combination of Urogastron (EGF), Basic Fibroblast Growth Factor (bFGF), insulin-like growth factor-i (IGF-1), Brain Derived Neurotrophic Factor (BDNF) and neurotrophic factor 3 (NT-3), addition is: EGF 10～30ng/mL basic medium, bFGF 5～20ng/mL basic medium, IGF-140～60ng/mL basic medium, BDNF 10～30ng/mL basic medium, NT-310～30ng/mL basic medium.
The mesenchymal stem cells MSCs wide material sources are drawn materials conveniently, are not subjected to the restriction of medical ethics and law; And mesenchymal stem cells MSCs can suppress the ripe and propagation of T lymphocyte, has the characteristics of escaping immunological rejection after the transplanting.Therefore neural precursor further is converted into inner ear hair cells if mesenchymal stem cells MSCs can be induced differentiation, and the cell that well solves the cellular replacement therapy phonosensitive nerve deafness is come source problem.
The present invention adopts adherent method to extract mesenchymal stem cells MSCs, behind the vitro culture purifying, choose Urogastron (EGF), Basic Fibroblast Growth Factor (bFGF), insulin-like growth factor-i (IGF-1), Brain Derived Neurotrophic Factor (BDNF), neurotrophic factor 3 (NT-3) and be combined into different inductive conditions, add in the mesenchymal stem cells MSCs basic culture solution inducing bone mesenchymal differentiation of stem cells.In inducing process, the observation of cell metamorphosis, and detect the whether cell expressing after inducing of neural precursor or neuronal cell specific antigen: nestin, P27 with immunocytochemistry, thereby identify that can mesenchymal stem cells MSCs be induced to differentiate into neural precursor, attempt for the cellular replacement therapy phonosensitive nerve deafness provides experiment basis, and higher advance and important clinical significance are arranged.Through screening, the combination of EGF+bFGF+IGF-1+NT-3 or the combination of EGF+IGF-1+bFGF+BDNF+NT-3 are considered to the best that mesenchymal stem cells MSCs is divided into neural precursor and induce differentiation condition.
The used substratum of described inducing culture is the basic medium that this area is usually used in the mesenchymal stem cells MSCs inducing culture, and among the present invention, described basic medium is the low sugar DMEM liquid that contains 2% top grade foetal calf serum and 1%N2 assistant agent.The concrete compound method of described basic medium is: every 100mL low sugar DMEM liquid adds 2g foetal calf serum and 1g N2 assistant agent.
The present invention adds somatomedin again and carries out inducing culture after described mesenchymal stem cells MSCs cultivation was passaged to for the 4th generation.Experiment finds that P6 after date, part cell lose typical spindle shape cell sample, and it is big to broaden, and triangular in shape, Polygons, rate of propagation begin to slow down, and the pitch time of going down to posterity is elongated.2004, the same mesenchymal stem cells MSCs of finding such as Oswald broadened greatly for the back cell gradually at P6, experiment confirm this result.In the process of going down to posterity, stem cell may carry out spontaneous differentiation, and passage number is many more, loses stem cell property more.External report mesenchymal stem cells MSCs can be passaged to more than 30 generations, but this moment, the stem cell characteristic was doubtful.Keeping of stem cell characteristic may be closely related with nutritional condition, and note simultaneously to avoid in the culturing process chemical factors, as in the process that goes down to posterity, note the control trysinization time the infringement of mesenchymal stem cells MSCs, and soft when noting the piping and druming cell.The present invention only select P4 for cell as handling cell because this in generation cell, the stem cell characteristic is kept well, and purifying substantially.
The described used substratum of cultivation that goes down to posterity is that this area is usually used in the substratum that mesenchymal stem cells MSCs goes down to posterity and cultivates, go down to posterity described in the present invention to cultivate and carry out in the MSC nutrient solution, described MSC nutrient solution is low sugar DMEM liquid and 250: 44.117 mixing solutions of foetal calf serum liquid volume ratio.
Preferably, described somatomedin is the combination of Urogastron, Basic Fibroblast Growth Factor, insulin-like growth factor-i and neurotrophic factor 3, addition is: EGF 20ng/mL basic medium, bFGF 10ng/mL basic medium, the IGF-150ng/mL basic medium, the NT-320ng/mL basic medium.
Preferably, described somatomedin is the combination of Urogastron, Basic Fibroblast Growth Factor, insulin-like growth factor-i, Brain Derived Neurotrophic Factor and neurotrophic factor 3, addition is: EGF 20ng/mL basic medium, bFGF 10ng/mL basic medium, the IGF-150ng/mL basic medium, BDNF 20ng/mL basic medium, the NT-320ng/mL basic medium.
Concrete, described method is carried out as follows: (1) will separate the mesenchymal stem cells MSCs of purification in the cultivation of going down to posterity in the MSC substratum, mesenchymal stem cells MSCs is gone down to posterity, and obtain the stable mesenchymal stem cells MSCs that goes down to posterity; (2) sterile-processed cover glass through the poly-lysine bag by after as the standby cover glass that is used for cell climbing sheet; (3) mesenchymal stem cells MSCs for the treatment of step (1) goes down to posterity when reaching for the 3rd generation, and with 0.25% tryptic digestion attached cell, centrifugal back is resuspended with the MSC nutrient solution, obtains cell suspension, and the cell concn of adjusting mesenchymal stem cells MSCs in the cell suspension is 1 * 10
5～2 * 10
5/ ml drips cell suspension on described standby cover glass, covers upper surface, and the cover glass periphery drips basic medium and covers, at 37 ℃, 5%CO
2Cultivate under the condition, cell is promptly grown in the fragmentation at lid, be the 4th generation cell; (4) treat that cell growth covered standby cover glass surface at 50%～60% o'clock, the MSC nutrient solution that inclines adds inducing culture, at 37 ℃, 5%CO
2Cultivate under the condition, cultivated 7～20 days, obtain neural precursor.
The separation and Extraction of mesenchymal stem cells MSCs belongs to known technology to those skilled in the art, can adopt this area common method to carry out.Separate the purification mesenchymal stem cells MSCs among the present invention and adopt adherent method.The present invention only experimentizes with rat bone marrow mesenchymal stem cells; but protection domain is not limited in this; in the practical application; can utilize other Mammalss to extract mesenchymal stem cells MSCs; also can utilize the marrow of extracting out from human body, obtain human marrow mesenchymal stem cell, induce differentiation to obtain neural precursor according to the inventive method again by separation and purification; carry out autotransplantation again, can eliminate immunological rejection fully.The rat bone marrow mesenchymal stem cells separating and purifying method is as follows among the present invention: get 4～5 the week age SD rat, taking off neck puts to death, femur is taken out in the sterilization back, femur is cut off, washed medullary space repeatedly, collect washing fluid with low sugar DMEM liquid, centrifugal, get precipitation, resuspended with 15% stem cell nutrient solution, 37 ℃, 5%CO
2Cultivate 10～16h under the condition in culturing bottle, the not attached cell that inclines adds 0.01M PBS, and adherent insecure cell is removed in piping and druming gently, with after the 0.01M PBS washing, adds 15% stem cell nutrient solution, 37 ℃, 5%CO again
2Cultivate the mesenchymal stem cells MSCs that obtains purifying under the condition.
Described 15% stem cell nutrient solution is low sugar DMEM liquid and 250: 44.117 mixing solutions of foetal calf serum liquid volume ratio.
Nineteen sixty-eight, Friedenstein adopts adherent method to separate the purification mesenchymal stem cells MSCs the earliest, and Majumdar adopts density gradient centrifugation to separate the purification mesenchymal stem cells MSCs.According to the literature, it is high that the adherent method cell purity is owed, and the density gradient centrifugation cell speed of growth is relatively poor.Experiment finds that an adherent method GPRS cultivation skill also can obtain the very high mesenchymal stem cells MSCs of purity, when the mesenchymal stem cells MSCs primary cultured cell rigidly connects kind, cell is circular, adherent gradually within 24 hours, kytoplasm extends, and is the fusiformis sample, growth rapidly, form each clone's colony through mitotic division, become the growth of colony sample, and adherent jail.According to these growth characteristics of mesenchymal stem cells MSCs, think when changing liquid first the removal of not attached cell and adherent insecure cell and when going down to posterity first the removal of the cell of non-clone's colony growth very important to the purifying mesenchymal stem cells MSCs.
Beneficial effect of the present invention is mainly reflected in: provide a kind of mesenchymal stem cells MSCs that utilizes to induce the method that obtains neural precursor, neural precursor further can break up and obtains neurocyte, and neural precursor can be converted into inner ear hair cells, inner ear supporting cell, provide experimental basis to cells,cochlear transplantation treatment phonosensitive nerve deafness, and the solution transplanted cells comes source problem.
(4) description of drawings
Fig. 1 purifies and vitro culture form under the phase microscope (10 * 10) for mesenchymal stem cells MSCs separates; A is a primary cell, B be the 4th generation cell;
Fig. 2 induces the 7th day (A) and the 12nd day (B), form under the phase microscope (10 * 10) for EGF+bFGF+IGF-1+NT-3;
Fig. 3 induces the 7th day (A) and the 12nd day, form under the phase microscope (10 * 10) for EGF+bFGF+IGF-1+BDNF+NT-3;
Fig. 4 is not for inducing the 7th day (A) and the 12nd day (B) of group, form under the phase microscope (10 * 10);
Fig. 5 induces group immunocytochemical stain result (cell gets rid of sheet 10 * 10) for EGF+bFGF+IGF-1+BDNF+NT-3; A:Nestin 1: 150; B:P27
Kip1
Fig. 6 induces group immunocytochemical stain result (cell climbing sheet 10 * 10) for EGF+bFGF+IGF-1+NT-3; A:Nestin 1: 150; B:P27
Kip1
Fig. 7 is not for inducing group immunocytochemical stain result (cell gets rid of sheet 10 * 10); A:Nestin1: 150; B:P27
Kip1
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
1. materials and methods
1.1 experiment material
1.1.1 laboratory animal: the SD rat, in 4～5 ages in week, body weight 80～120g is available from Zhejiang University's Experimental Animal Center.
1.1.2 experiment reagent
1.1.2.1 general reagent
25cm
2Culturing bottle Orange scientific company Belgium
The 6 well culture plate Becton Dickinson company U.S.
The lucky promise biological medicine technology of PBS liquid company limited China
The 0.25% trypsinase GIBCO company U.S.
The Poly-L-Lysine Solution SIGMA company U.S.
Top grade foetal calf serum FBS Hangzhou folium ilicis chinensis biotechnology company limited China
The low sugar DMEM liquid GIBCO company U.S.
The N2 assistant agent GIBCO/invitrogen company U.S.
2.1.2.2 cell induction reagent
The EGF CYTOLAB/PEPROTECH company U.S.
The bFGF CYTOLAB/PEPROTECH company U.S.
The IGF-1 CYTOLAB/PEPROTECH company U.S.
The BDNF CYTOLAB/PEPROTECH company U.S.
The NT-3 CYTOLAB/PEPROTECH company U.S.
2.1.2.3 immunohistochemical methods reagent
(1) one is anti-
The Nestin BD Biosciences company U.S.
P27
Kip1The NEOMARKERS company U.S.
(2) two is anti-
Horseradish peroxidase enzyme connection IgG Foochow steps Newbiotics Inc China
(3) colouring reagents
DAB colouring reagents box Foochow steps Newbiotics Inc China
1.1.3 common agents preparation
1.1.3.1 the packing of cytokine, storage
(1) packing of bFGF, storage:
BFGF reagent is pulvis, and nt wt net weight 10ug uses solubility to be 10ng/ml, stores concentration 0.01ug/ul.
(2) packing of EGF, storage:
EGF reagent is pulvis, and nt wt net weight 100ug uses solubility to be 50ng/ml, stores concentration 0.02ug/ul.
(3) packing of IGF-1, storage:
IGF-1 reagent is pulvis, and nt wt net weight 20ug uses solubility to be 50ng/ml, stores concentration 0.05ug/ul.
(4) packing of BDNF and NT-3, storage:
BDNF and NT-3 reagent are pulvis, and nt wt net weight 2ug uses solubility to be 20ng/ml, store concentration 0.02ug/ul.
(5) above somatomedin, frozen in-20 ℃ of refrigerators.
1.1.3.2 the preparation of MSC nutrient solution
Low sugar DMEM liquid 250ml+44.117ml foetal calf serum is made into the low sugar DMEM liquid that contains 15% foetal calf serum (FBS), i.e. the MSC nutrient solution
1.1.3.3 the preparation of inducing culture liquid
At first configuration contains the low sugar DMEM liquid of 2%FBS and 1%N2 assistant agent, as basic culture solution, then according to culture condition, adds cytokine when inducing.
1.1.3.4DAB substrate preparation
In 1.5ml eppendorf pipe, add earlier 0.85ml distilled water, add reagent A, each 50 μ l of B, C in the DAB colouring reagents box more successively, mixing the DAB colour developing liquid of 1ml.
1.1.4 major equipment
1.Heraeus cell culture incubator Heraeus company Germany
2.OLMPUS inverted phase contrast microscope OLMPUS company Japan
3.802 whizzer Shanghai Surgical Operation Equipment Factory China
4.Heraeus Biofuge whizzer Heraeus company Germany
5.Heraeus cabfuge 300 whizzer Heraeus company Germany
6.Eppendorf the Centrifuge 5403 whizzer Becton Dickinson company U.S.
7.79-1 magnetic force heating stirrer Changzhou Guohua Electric Appliance Co., Ltd. China
8. Bechtop Heraeus company Germany
9.DK-8D the grand experimental installation of Nereid company limited China on the type electric heating constant temperature tank
1 1. Zeiss company of computer generated image system Germany
14. horizontal sedimentator Heraeus company Germany
2. experimental technique
2.1 the separation of mesenchymal stem cells MSCs and purification
Get 4～5 all SD rats in age (at every turn testing 1～2, shared 20), take off neck and put to death.75% alcohol-pickled 5 minutes, be placed on the foam flat board in the super clean bench, use the pin fixing limbs, cut off skin, separating muscle, taking-up femur, scraping periosteum and residual muscle.The femur center is cut off, washed medullary space with one milliliter of syringe repeatedly with low sugar DMEM liquid again, collect the marrow suspension, 1000rpm, 5 minutes centrifugal removal supernatants use MSC nutrient solution 5ml resuspended again, are inoculated in 25cm
2In the culturing bottle, put into cell culture incubator and cultivate (37 ℃, 5%CO
2).After 12 hours, the not attached cell that inclines, and add 0.01M PBS 3ml and blow and beat gently, remove adherent insecure cell, 0.01MPBS 3ml washing one time adds MSC nutrient solution 5ml again, puts into cell culture incubator and cultivates.The next day change liquid, treat to go down to posterity in about 70～80% o'clock at the bottom of cell is paved with bottle.Process goes down to posterity: the nutrient solution that inclines, add 5mlPBS, and to jiggle at the bottom of the clean bottles, the washings that inclines is added in 0.25% trypsinase 1.5ml of preheating, covers a bottle end; Microscopically observation of cell metamorphosis treats that most cells becomes circle, and breaks away from the bottle end when suspending, add cell culture fluid 3ml, in and pancreatin, a piping and druming bottle end gently, suspension cell, cell suspension is transferred in the 15ml test tube 1000rpm, 5 minutes, centrifugal removal Digestive system, add the 5mlMSC nutrient solution, after the piping and druming evenly, be re-seeded into 25cm
2In the culturing bottle, put into cell culture incubator and continue to cultivate.When reaching for the 4th generation, the beginning cell induction.(generally went down to posterity by 1: 2,1: 3, cell density is 8 * 10
4-2 * 10
5Individual/ml, be calculated as by the culturing bottle floorage: 8 * 10
3-2 * 10
4/ CM
2).
2.2 inducing of mesenchymal stem cells MSCs
2.2.1 cell induction condition
(1) induces group: basic medium+condition inductor
A. basic medium contains the low sugar DMEM liquid of 2%FBS and 1%N2
B. condition inductor grouping EGF+IGF-1
EGF+bFGF
EGF+bFGF+IGF-1
EGF+bFGF+IGF-1+BDNF
EGF+bFGF+IGF-1+NT-3
EGF+IGF-1+bFGF+BDNF+NT-3
C. somatomedin concentration EGF 20ng/ml; IGF-1 50ng/ml; BFGF 10ng/ml;
BDNF20ng/ml；NT-3 20ng/ml；
(2) control group (not inducing group):
Basic medium (the low sugar DMEM liquid that contains 2%FBS and 1%N2)
2.2.2. cell induction step
(1) the lid fragmentation is handled: cover glass is placed on and steeps acid 24 hours in the glass dish, and acid solution is cleaned in the tap water flushing fully, and the aquae destillata rinsing was steep in wine smart 2 hours, and tap water washes, the distilled water rinsing, and autoclave sterilization is standby.
(2) poly-lysine is prepared, is wrapped by cover glass:
A. Mie Jun distilled water dilutes Poly-L-Lysine Solution at 1: 10;
B. the Poly-L-Lysine Solution with dilution is placed on indoorly, is cooled to 18-26 ℃;
C. slide is immersed in the Poly-L-Lysine Solution 5 minutes of dilution;
D. after taking out, in 1 hour drying of 60 ℃ of baking ovens, or 18～26 ℃ of dried overnight of room temperature are stand-by.
(3) cover glass with poly-lysine bag quilt is positioned over six orifice plates, puts the super clean bench disinfection by ultraviolet light and spends the night standby.
(4) cell climbing sheet: treat passage to the during 3 generations, with 0.25% tryptic digestion attached cell, centrifugal back is resuspended with the MSC nutrient solution, the cell counting count board counting, and adjusting cell concn is 1 * 10
5～2 * 10
5/ ml drips on the cover glass of cell suspension in six orifice plates, covers upper surface.The cover glass periphery drips basic medium and covers, and carefully moves in the incubator and cultivates.Cell is promptly grown in the fragmentation at lid, be the 4th generation cell.
(5) cell induction: respectively at 6 well culture plates and 25cm
2Inducing cell in the culturing bottle.Treat that cell covered the cover glass surface at 50%～60% o'clock, begin to induce that the common nutrient solution of the MSC that inclines adds 2ml (6 well culture plate) and 5ml (25cm
2Culturing bottle) basic medium: contain the low sugar DMEM liquid of 2% foetal calf serum and 1%N2 assistant agent, according to the grouping of condition inductor, add somatomedin successively again, move in the incubator and cultivate, change induced liquid every other day.
2.2.3 cellular form is observed
Every day is the dynamic change of observation of cell form under phase microscope, and totally 15～20 days, with digital camera Taking Pictures recording cellular form, picture went to the computer arrangement, preserves.Because cell has obvious characteristic and representativeness the 7th day～the 15th day the time, so, choose the 7th day and 14 days cells carry out immunocytochemical stain.
2.2.4 immunocytochemical stain detects cell antigen
2.2.4.1 immunocytochemistry grouping
Induce group: the mesenchymal stem cells MSCs of under various inductive conditions, inducing the 12nd day.
Do not induce group: under basic medium, cultivate the 12nd day mesenchymal stem cells MSCs.
2.2.4.2 immunocytochemical stain step
The immunocytochemical stain two step method is adopted in experiment.
(1) sample preparations: adopted cell to get rid of sheet and two kinds of methods of cell climbing sheet: a. cell gets rid of sheet:
With 0.25% tryptic digestion 25cm
2The culturing bottle attached cell, centrifugal back is resuspended with PBS liquid, and adjusting cell density is 2 * 10
5-2 * 10
6/ ml.Draw 100 μ l cell suspensions and add in the smearing machine, centrifugal 4 minutes of 1000rpm, cell is coated on the slide glass uniformly.
B. cell climbing sheet (seeing the process of inducing).After sample preparations is good, shakes with 0.01M PBS and to wash 2 minutes * 3 times.
(2) cell fixation: every Paraformaldehyde 96 100 μ l that get rid of sheet or creep plate cell dropping 4%, at room temperature placed 15 minutes, shake with 0.01M PBS and wash, 5 minutes * 3 times (in the wet box).
(3) saturating film: drip 0.3%triton x-100 50 μ l (getting rid of sheet) or 100 μ l (creep plate), incubated at room 10 minutes is shaken with 0.01M PBS and to be washed 2 minutes * 3 times (wet box is interior).
(4) heterogenetic antigen sealing: the lowlenthal serum 50 μ l (getting rid of sheet) or the 100 μ l (creep plate) of dropping 10%, incubated at room is (in the wet box) after 30 minutes, gets rid of serum deprivation.
(5) one is anti-: drip anti-50 μ l (getting rid of sheet) or 100 μ l (creep plate).One anti-concentration: Nestin1: 150; P27
Kip11: 100.Hatch 2 hours (in the wet box) under the room temperature, shake with 0.01MPBS and wash, 5 minutes * 3 times (in the wet box).
(6) two is anti-: drip two anti-50 μ l (getting rid of sheet) or 100 μ l (creep plate) of horseradish peroxidase mark, hatched under the room temperature 30 minutes, 5 minutes * 3 times (in the wet box).
(8) colour developing: drip freshly prepared DAB substrate solution 100 μ l (getting rid of sheet) or 200 μ l (creep plate), put under the light microscopic and observe, after 5 minutes, immerse the tap water stopped reaction, with tap water flushing 5 minutes * 3 times.
(9) redye: soaked the Hematorylin dye liquor 10 minutes, the nucleus lining dyes, with tap water flushing, 5 minutes * 3 times.The persalt spirituous solution dedifferentes rapidly, with tap water flushing, 5 minutes * 3 times.60 ℃ of tap water returned indigo plant in 1 minute
(10) dehydration: 70% alcohol 3 minutes, 95% alcohol 3 minutes, raw spirit 3 minutes.
(11) mounting: dry up slide with blower, the resinene mounting behind the mark, is put into slide box and is preserved.
2.2.5 the immunocytochemistry slide is observed
Under light microscopic, observe the immunohistochemical methods slide, and Taking Pictures recording, picture deposits computer in.
2.2.6 contrast is provided with
Negative control: select the blank method, replace one to resist as negative with PBS.
2.2.7 immunocytochemical stain is judging criterion as a result
Criterion is with reference to Fromowitz sxemiquantitative staging:
1) according to the score of the dyeing depth: non-coloring is 0 minute, and faint yellow is 1 minute, and pale brown look is 2 minutes, and brown is 3 minutes;
2) under 100 times of light microscopics, count 10 not visuals field, iteron, the pale brown cytochrome that dyes accounts for the ratio of total cell, and＜5% is 0 minute, and 5%～25% is 1 minute, and 26%～50% is 2 minutes, and 51%～75% is 3 minutes, and＞75% is 4 minutes.
3) two results added:＜2 minutes negative (-), 2～3 are divided into the weak positive (+), and 4～5 are divided into moderate positive (++), and 6～7 are divided into strong positive (+++).
3. experimental result
3.1 growth of marrow mesenchyme stem cell characteristic
When primary cultured cell rigidly connected kind, cell was circular, and half an hour to 24 is hour adherent gradually, and kytoplasm extends, and is the fusiformis sample, clear-cut, and nucleus is big, and visible more nuclear fission phase forms each clone's colony through mitotic division, becomes the growth of colony sample.Propagation just can cover with one bottle (Figure 1A be a primary cell, inoculates afterwards the 5th day, has formed clone's colony, and based on the spindle shape cell, but misaligned, the form heterogeneity has more impurity cell) rapidly in general 7～10 days.
Cell after going down to posterity, cell are arranged and are become neat, and based on paliform, a small amount of colony is arranged in the whirlpool shape.In five generations of the first-generation to the,, (P1～P5) cellular form still became typical spindle shape, clear-cut, growth is rapid, and cell can increase two to three times in general 4 days, and the order of magnitude can reach 106, need go down to posterity once more (Figure 1B be the 4th generation cell, cell is typical spindle shape, form homogeneous, clear-cut, marshalling, heteroproteose cell is few); Behind the P6, the part cell loses typical spindle shape cell sample, and it is big to broaden, and triangular in shape, Polygons, rate of propagation begin to slow down, and the pitch time of going down to posterity is elongated.
Mesenchymal stem cells MSCs surface lacks absolute specificity antigen, does not therefore come identification of cell by the method that detects cell antigen, but according to cellular form (spindle shape), growth pattern (adherent growth) with can the branch voltinism identify.The hemopoietic stem cell suspension growth, rounded.
Each organizes growth factor-induced the 7th day and the 12nd day cellular form is as follows:
EGF+IGF-1 induces group:
A is for inducing the 7th day, and cell still is fusiformis; B is for inducing the 12nd day, and most cell cytoplasm retractions are short fusiformis, rectangle sample, a few cell spindle, but do not form cynapse, network-like, big ring texture does not appear.
EGF-1+bFGF induces group:
Induced the 7th day, cell has formed network-like, big ring texture.Induced the 12nd day, and formed cytosis network-like, big ring texture.In same bottle cell, the cell compact district: cell two ends retraction, rounded or stretch out 2-6 short and small cynapse, join with contiguous cell space or cynapse, reticulate.Cell is the compact district not: the elongation of cell two ends, be elongated shape, and long cynapse is arranged, to join with apart from each other cell space or cynapse, part forms big annular through a plurality of cell transmission.
EGF+bFGF+IGF-1 induces group:
Induced the 7th day, the part cell rounding, but stretch out lacking of cynapse, the part cell is elongated shape, and cynapse is arranged, and links to each other; B is for inducing the 12nd day, and the part cell is still rounded, and as the paving stone sample, the part cell is elongated shape and stretches out cynapse but form netted lacking.
EGF+bFGF+IGF-1+BDNF induces group:
Induced the 7th day, the part cell is elongated shape, and two ends form cynapse.Part cell two ends retraction, rounded or rectangle sample; Induced the 12nd day, elongated and round cell increases, and round cell begins to stretch out cynapse.
EGF+bFGF+IGF-1+NT-3 induces the group (see figure 2):
Induced the 7th day, cell is elongated shape in not intensive zone, and long cynapse is arranged, as star and bipolar; Cell is intensive regional rounded.Induced the 12nd day, cell forms big ring texture in not intensive zone.Still keep circle at intensive regional cell.
EGF+bFGF+IGF-1+BDNF+NT-3 induces the group (see figure 3):
A is for inducing the 7th day, and cell is bipolar, star, circle; B is for inducing netted, big ring texture that cell forms the 12nd day.
Do not induce the group (see figure 4):
A is for cultivating the 7th day, and B is for cultivating the 12nd day, and most of cell is fusiformis, and obscurity boundary, silk are cotton-shaped, and profile is unclear, and cell debris increases in the nutrient solution.
3.2 each differentiation group cellular immunization cytochemical staining result (seeing Table 1)
3.2.1 whole groups of cells Nestin, P27
Kip1All positive, wherein EGF+bFGF+IGF-1+BDNF+NT-3 group and EGF+bFGF+IGF-1+NT-3 organize equal moderate positive.
Each groups of cells immunocytochemical stain result:
The EGF+IGF-1 group;
The Nestin moderate positive, dyeing part is positioned at endochylema; P27
Kip1Weak positive, dyeing part mainly is positioned at endochylema, and part is in karyon, and all cell all has dyeing.
The EGF+bFGF group:
The Nestin moderate positive, dyeing part is positioned at endochylema; P27
Kip1Weak positive, dyeing part mainly is positioned at endochylema, and part is in karyon, and all cell all has dyeing.
The EGF+bFGF+IGF-1 group is thin:
The Nestin moderate positive, dyeing part is positioned at endochylema; P27
Kip1Weak positive, dyeing part mainly is positioned at endochylema, and part is in karyon, and all cell all has dyeing.
The EGF+bFGF+IGF-1+BDNF group:
The Nestin moderate positive, dyeing part is positioned at endochylema, and cynapse is arranged, and is dark as the cell dyeing of neurocyte sample, the cell dyeing of other gradual changes shallow or dye-free; P27
Kip1Moderate positive, dyeing part mainly are positioned at endochylema, and part is in karyon, and all cell all has dyeing.
EGF+bFGF+IGF-1+BDNF+NT-3 organizes (see figure 5):
Nestin (Fig. 5 A) moderate positive, dyeing part is positioned at endochylema, and cynapse is arranged, and is dark as the cell dyeing of neurocyte sample, the shallow or dye-free of the cell dyeing of other gradual changes; P27
Kip1(Fig. 5 B) moderate positive, dyeing part mainly is positioned at endochylema, and part is in karyon, and all cell all has dyeing.EGF+bFGF+IGF-1+NT-3 organizes (see figure 6):
Nestin (Fig. 6 A) moderate positive, dyeing part is positioned at endochylema, and cynapse is arranged, and is dark as the cell dyeing of neurocyte sample, the shallow or dye-free of the cell dyeing of other gradual changes; P27
Kip1(Fig. 6 B) moderate positive, dyeing part mainly is positioned at endochylema, and part is in karyon, and all cell all has dyeing.Do not induce the group (see figure 7);
Nestin (Fig. 7 A) is weak positive, and dyeing part is positioned at endochylema, and positive cell quantity is few; P27
Kip1(Fig. 7 B) is weak positive, and dyeing part mainly is positioned at endochylema, and part is in karyon, and all cell all has dyeing.
Table 1: induce group and do not induce group cellular immunization cytochemical staining result
| Group | Nestin | P27 kip1 |
| EGF+IGF-1 EGF+bFGF EGF+bFGF+IGF-1 EGF+bFGF+IGF-1+BDNF EGF+bFGF+IGF-1+NT-3 EGF+IGF-1+bFGF+BDNF+NT-3 does not induce group | ++ ++ ++ ++ ++ ++ + | + + + + ++ ++ + |
Annotate: Nestin is the neural precursor sign, expresses the cell of Nestin, has the ability that is divided into ripe neurocyte; And can be divided into inner ear hair cells.P27
Kip1Be inner ear sensation precursor cell and sustenticular cell sign.
But 3.3 bFGF, NT-3, BDNF inducing bone mesenchymal cell neuralward precursor cell differentiation
Under the phase microscope, adding the group of inducing of bFGF, NT-3, BDNF, cell becomes circle or elongated shape gradually, stretch out 2～6 cynapses, be bipolar, star, as neurone, oligodendrocyte, astroglia cell sample, can form netted or big ring texture between cell and the cell, just as neural network.Immunocytochemical stain is as seen: the neural precursor sign Nestin moderate positive that dyes on the cell in this section, and the shallow or dye-free of the cell dyeing of other gradual changes.
EGF+bFGF+IGF-1+NT-3 and EGF+IGF-1+FGF-2+BDNF+NT-3 induce group, and cellularstructure is clear, and the immunocytochemical stain support can be thought the top condition that inducing bone mesenchymal cell neuralward precursor cell breaks up.
3.4 bFGF and NT-3 have very strong nerve-inducing
The effect of bFGF, NT-3 inducing bone mesenchymal cell neuralward cell direction differentiation may be stronger than BDNF: add bFGF, NT-3 group, just occur bipolar, star like cell in early days inducing, and form netted or ring texture; And the cell that adds BDNF group formation cynapse is few.
3.5 the density of cell is to the influence of mesenchymal stem cells MSCs differentiation
In same culturing bottle, in the cell compact district, cell two ends retractions, rounded or stretch out 2～6 short and small cynapses, form with contiguous cell space or cynapse and join, reticulate.In cell compact district not, the elongation of cell two ends is elongated shape, forms long cynapse gradually, joins with apart from each other cell space or cynapse, and part forms big annular through a plurality of cell transmission.
Claims (10)
1. one kind is utilized mesenchymal stem cells MSCs to induce the method that obtains neural precursor, described method is to separate the mesenchymal stem cells MSCs of purification through the cultivation of going down to posterity, get the stable mesenchymal stem cells MSCs that goes down to posterity and change inducing culture over to, inducing culture 7～20 days obtains neuron cell; Described inducing culture is the basic medium that is added with somatomedin, and described somatomedin mainly comprises Urogastron, Basic Fibroblast Growth Factor, insulin-like growth factor-i and neurotrophic factor 3; Addition is: EGF 10-30ng/mL, bFGF 5-20ng/mL, IGF-140-60ng/mL, NT-310-30ng/mL.
2. the method for claim 1, it is characterized in that: described somatomedin is the combination of Urogastron, Basic Fibroblast Growth Factor, insulin-like growth factor-i, Brain Derived Neurotrophic Factor and neurotrophic factor 3; Addition is: EGF 10-30ng/mL, bFGF5-20ng/mL, IGF-140-60ng/mL, BDNF 10-30ng/ml, NT-310-30ng/mL.
3. method as claimed in claim 1 or 2, described method is carried out as follows: (1) will separate the rat bone marrow mesenchymal stem cells of purification in the cultivation of going down to posterity in the MSC substratum, mesenchymal stem cells MSCs is gone down to posterity, obtain the stable mesenchymal stem cells MSCs that goes down to posterity; (2) sterile-processed cover glass through the poly-lysine bag by after as the standby cover glass that is used for cell climbing sheet;
(3) mesenchymal stem cells MSCs for the treatment of step (1) goes down to posterity when reaching for the 3rd generation, and with 0.25% tryptic digestion attached cell, centrifugal back is resuspended with the MSC nutrient solution, obtains cell suspension, and the cell concn of adjusting mesenchymal stem cells MSCs in the cell suspension is 1 * 10
5～2 * 10
5/ ml drips cell suspension on described standby cover glass, covers upper surface, and the cover glass periphery drips basic medium and covers, at 37 ℃, 5%CO
2Cultivate under the condition, cell is promptly grown in the fragmentation at lid, be the 4th generation cell; (4) treat that cell growth covered standby cover glass surface at 50%～60% o'clock, the MSC nutrient solution that inclines adds inducing culture, at 37 ℃, 5%CO
2Cultivate under the condition, cultivated 7～20 days, obtain neural precursor.
4. method as claimed in claim 1 or 2 is characterized in that: described basic medium is the low sugar DMEM liquid that contains 2% foetal calf serum and 1%N2 assistant agent.
5. method as claimed in claim 1 or 2 is characterized in that: after described mesenchymal stem cells MSCs cultivation was passaged to for the 4th generation, changes over to and induce differentiation culture in the inducing culture.
6. method as claimed in claim 1 or 2 is characterized in that: the described cultivation of going down to posterity is carried out in low sugar DMEM liquid and 250: 44.117 mixing solutions of foetal calf serum liquid volume ratio.
7. the method for claim 1, it is characterized in that: described somatomedin is the combination of Urogastron, Basic Fibroblast Growth Factor, insulin-like growth factor-i and neurotrophic factor 3, addition is: EGF 20ng/mL, bFGF 10ng/mL, IGF-150ng/mL, NT-320ng/mL.
8. the method for claim 1, it is characterized in that: described somatomedin is the combination of Urogastron, Basic Fibroblast Growth Factor, insulin-like growth factor-i, Brain Derived Neurotrophic Factor and neurotrophic factor 3, addition is: EGF 20ng/mL, bFGF 10ng/mL, IGF-150ng/mL, BDNF 20ng/mL, NT-320ng/mL.
9. the method for claim 1, it is characterized in that described mesenchymal stem cells MSCs from the SD rat, separating and purifying method is as follows: get 4～5 age in week the SD rat, take off neck and put to death, femur is taken out in the sterilization back, femur is cut off, washed medullary space repeatedly, collect washing fluid with low sugar DMEM liquid, centrifugal, get precipitation, resuspended with 15% stem cell nutrient solution, 37 ℃, 5%CO
2Cultivate 10～16h under the condition in culturing bottle, the not attached cell that inclines adds 0.01M PBS and removes adherent insecure cell, with after the 0.01M PBS washing, adds 15% stem cell nutrient solution, 37 ℃, 5%CO again
2Cultivate the mesenchymal stem cells MSCs that obtains purifying under the condition.
10. method as claimed in claim 9 is characterized in that described 15% stem cell nutrient solution is low sugar DMEM liquid and 250: 44.117 mixed solution of foetal calf serum liquid volume ratio.
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