CN104004713A - Method for inducing and culturing umbilical cord mesenchymal stem cells into nerve cells - Google Patents
Method for inducing and culturing umbilical cord mesenchymal stem cells into nerve cells Download PDFInfo
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Abstract
The invention provides a method for inducing and culturing umbilical cord mesenchymal stem cells into nerve cells. According to the invention, a combined method of two steps of pre-induction and induction is adopted, and an optimized pre-induction culture solution and an induction culture solution are prepared. The induction mode is short in induction period and high in cell differentiation homogeneity. Electrophysiological detection finds that the obtained nerve cells have the function of induced electric discharge, which indicates that the induced nerve cells are obtained successfully.
Description
Technical field
The present invention relates to neurobiology field, particularly a kind of method that is neurocyte by umbilical cord mesenchymal stem cells inducing culture, the invention still further relates to and cultivate the neurocyte obtaining.
Background technology
Umbilical cord mesenchymal stem cells (MSC) is mesoderm origin, can carry out self, and possesses to skeletonization, cartilage and three kinds of a kind of adult stem cells that mesoderm is cytodifferentiation potential of fat.Recent study finds that MSC, outside mesoderm, can also carry out transdifferentiation across germinal layer, as is divided into the neurocyte of ectoderm system and liver cell and the pancreatic cell of epithelial cell and entoderm system.Because MSC wide material sources, are easy to separation and Culture, immunogenicity is lower, and the ethnics Problem that does not exist embryonic stem cell to face, and this makes its application aspect regenerative medicine possess the unexistent advantage of other stem cells.Therefore the long-term puzzlement such as the nerve injury that the nerve degenerative diseases of central nervous system causes as Parkinson and the exterior trauma mankind's problem also will have more real feasible solution.
In the past few years, domestic and international many research groups have reported that external is the method for neurone, spongiocyte by MSC Induction of committed differentiation.
MSC Induction of committed differentiation is that the experimental study of neurocyte has minority foreign study mechanism to carry out, and domestic research seldom, and the method for inducing differentiation induction duration of existing report is long, and the neurocyte differentiation efficiency that obtains is low, cytodifferentiation is all once low.
Summary of the invention
The object of the invention is to provide a kind of novel method that in vitro human umbilical cord mesenchymal stem cells inducing culture is divided into neurocyte, prepares the neurocyte of differentiation simultaneously.
For achieving the above object, the present invention is by the following technical solutions: a kind of method that umbilical cord mesenchymal stem cells inducing culture is neurocyte, is characterized in that comprising the following steps:
(1), cultivation and the amplification of umbilical cord mesenchymal stem cells: obtain umbilical cord mesenchymal stem cells monomer, umbilical cord mesenchymal stem cells monomer is pressed to 1-5 * 10
5/ ml is inoculated in umbilical cord mesenchymal stem cells nutrient solution, and every 3-8 days changes liquid and goes down to posterity once, obtains cultured cells;
Described umbilical cord mesenchymal stem cells nutrient solution is containing DMEM/F12,2%B27,2-4mM L-glutaminate, 1mM thioglycerin (MTG), 1% non-essential amino acid, EGF20-40ng/ml, bFGF20-30ng/ml, 80-90U/ml penicillin, 60-70 μ g/ml Streptomycin sulphate;
(2), pre-induction differentiation: the cell that step (1) is obtained is by 1～5 * 10
4adherent inducing culture is carried out in the cell density inoculation of/ml, and adherent culture is changed pre-inducing culture liquid and cultivated after 3 days, and within 3 days, later half amount is changed liquid, stops induction in the time of 3-4 days;
Described pre-inducing culture liquid is that DMEM is as basic medium, also contain on this basis the vitamins C of 20nmol～50nmol, 10-20U/ml human leukocyte supressor, 1-8mML-glutamine, 2-5mM thioglycerin (MTG), 80-100U/ml penicillin, 80-100 μ g/ml Streptomycin sulphate;
Step (3) induction differentiation: the cell that step (2) is obtained is by 1～5 * 10
4adherent inducing culture is carried out in the cell density inoculation of/ml, and adherent culture is changed inducing culture liquid and carried out inducing culture after 2 days, and within 3 days, later half amount is changed liquid, stops induction in the time of 3-6 days, obtains neurocyte;
Described inducing culture liquid is that DMEM is as basic medium, also contain on this basis the vitamins C of 20nmol～50nmol, the VITMAIN B1 of 30nmol～40nmol, the DMSO of 0.01-0.02%, 0.1mM2-beta-mercaptoethanol, 10-20U/ml human leukocyte supressor, 10-30U/ml Prostatropin, 1-8mML-glutamine, 2-5mM thioglycerin (MTG), 80-100U/ml penicillin, 80-100 μ g/ml Streptomycin sulphate.
The present invention's nutrient solution used can supplement one or more compositions and obtain on the basis of conventional cell based basal culture medium.Can be used for cell based basal culture medium of the present invention can include but are not limited to: DMEM, DMEM/F12,2%B27.The formula of these substratum is known in the field, not only in common textbook and laboratory manual, has a detailed description, but also can directly with the form of finished product, from company, buy and obtain.
The composition of thing can be any maintaining or the composition of Promote cell's growth as a supplement.They can include but not limited to: amino acid, VITAMIN, albumen, hormone, metal ion, trace element, lipid acid, sugar etc.
The present invention's all reagent used all can be bought and be obtained by commercial sources.
The neurocyte that the method for the invention obtains at least has one of following feature:
(a) there is the cellularstructures such as typical cell paste, aixs cylinder and dendron;
(b) expressing protein at least: β-tublin III;
(c) neurocyte accounts for the 86-91% of inducing cell colony left and right;
(d) stimulate and there is discharging function to external world.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the method for an one-step inducing neurocyte compared to existing technology, the present invention adopts pre-induction and induction two step combined techniqueses, and prepared pre-inducing culture liquid and the inducing culture liquid of optimizing, adopt this induction mode, induction duration is short 6～10 days, cytodifferentiation all once high (ratio is up to more than 90%), apparently higher than the induction efficiency of neurocyte in currently available technology, and electric physiological detection finds that this neurocyte has induced discharge function.
Unexposed the delivering of combination of the optimization nutrient solution of two one-step inducing methods of the present invention and use, those skilled in the art can not infer the method for the present invention that obtains according to existing technology if do not spent creative work at all.
Accompanying drawing explanation
Fig. 1 is the immunocytochemistry result of neurocyte of the present invention, and A is neurocyte β-tublin III immunocytochemistry result of the present invention, and B is for differing figure;
Fig. 2 is the flow cytometry of the neurocyte of embodiment 2 inductions, and B is neurocyte of the present invention, and A is homotype contrast;
Fig. 3 is the flow cytometry of the neurocyte of embodiment 3 inductions, and B is neurocyte of the present invention, and A is homotype contrast;
Fig. 4 is the electric physiological detection of the neurocyte of embodiment 2 inductions;
Fig. 5 is the electric physiological detection of the neurocyte of embodiment 3 inductions.
Embodiment
Embodiment 1, umbilical cord mesenchymal stem cells obtain and cultivate
Use pluripara to agree to that the neonatal umbilical cord of mandate is as the source of mescenchymal stem cell.
Neonatal umbilical cord is washed three times in containing 1% dual anti-physiological saline, remove surperficial blood stains, be then cut into the segment of approximately 1 cm long.With eye scissors, umbilical cord is longitudinally cut open along blood vessel parallel direction, 2 Umbilical artery and 1 umbilical vein blood vessel are peeled off totally from umbilical cord.Peel surperficial amnion off, the logical glue of China, partly with fully washing 3 times containing 1% dual anti-physiological saline, shreds to about 1mm
3size.The tissue block shredding is laid in to 75cm uniformly
2in culturing bottle, room temperature is placed 5-10min, and tissue block is adjacent to.Add 5ml substratum, substratum is containing DMEM/F12,2%B27,4mM L-glutaminate, 1mM thioglycerin (MTG), 1% non-essential amino acid, EGF20ng/ml, bFGF20ng/ml, 90U/ml penicillin, 70 μ g/ml Streptomycin sulphates, 37 ℃ of 5%CO
2incubator is cultivated.Every three days, change a subculture, Growth of Cells is to the cultivation of going down to posterity after confluent culture ware.
Also can use the umbilical cord mesenchymal stem cells in commercially available or the legal source that business sells directly to cultivate by the method for embodiment 1.
Embodiment 2, umbilical cord mesenchymal stem cells directional induction in vitro differentiation 1
The fresh P3-6 going down to posterity for umbilical cord mesenchymal stem cells with 5 * 10
4/ ml is inoculated in 24 orifice plates that are placed with coated poly-lysine cover glass, 1ml/ hole, and adherent culture is changed pre-inducing culture liquid and is cultivated after 3 days, and within 3 days, later half amount is changed liquid, stops induction in the time of the 3rd day.
Pre-inducing culture liquid be DMEM as basic medium, on this basis, also contain the vitamins C of 50nmol, 20U/ml human leukocyte supressor, 8mM L-glutaminate, 5mM thioglycerin (MTG), 80U/ml penicillin, 80 μ g/ml Streptomycin sulphates.
The cell that pre-inducing culture is obtained is by 5 * 10
4monolayer adherence inducing culture is carried out in the cell density inoculation of/ml, and adherent culture is changed inducing culture liquid and carried out inducing culture after 2 days, and within 3 days, later half amount is changed liquid, stops induction in the time of the 3rd day, obtains neurocyte;
Inducing culture liquid is that DMEM is as basic medium, also contain on this basis the vitamins C of 50nmol, the VITMAIN B1 of 30nmol, 0.01% DMSO, 0.1mM2-beta-mercaptoethanol, 10U/ml human leukocyte supressor, 30U/ml Prostatropin, 8mM L-glutaminate, 5mM thioglycerin (MTG), 100U/ml penicillin, 80 μ g/ml Streptomycin sulphates.
Immunocytochemistry result, flow cytometry result and the electric physiological detection result of the umbilical cord mesenchymal stem cells that induction in embodiment 2 is obtained, result shows that inducing culture induces and has produced neurocyte, and cytodifferentiation ratio reaches 91.43%, and the neurocyte obtaining stimulates to external world and has discharging function.
Embodiment 3, umbilical cord mesenchymal stem cells directional induction in vitro differentiation 2
The fresh P3-6 going down to posterity for umbilical cord mesenchymal stem cells with 5 * 10
4/ ml is inoculated in 24 orifice plates that are placed with coated poly-lysine cover glass, lml/ hole, and adherent culture is changed pre-inducing culture liquid and is cultivated after 3 days, and within 3 days, later half amount is changed liquid, stops induction in the time of the 4th day.
Pre-inducing culture liquid be DMEM as basic medium, on this basis, also contain the vitamins C of 20nmol, 10U/ml human leukocyte supressor, 1mM L-glutaminate, 2mM thioglycerin (MTG), 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates.
The cell that pre-inducing culture is obtained is by 5 * 10
4monolayer adherence inducing culture is carried out in the cell density inoculation of/ml, and adherent culture is changed inducing culture liquid and carried out inducing culture after 2 days, and within 3 days, later half amount is changed liquid, stops induction in the time of the 6th day, obtains neurocyte;
Inducing culture liquid is that DMEM is as basic medium, on this basis, also contain the vitamins C of 20nmol, the VITMAIN B1 of 40nmol, 0.02% DMSO, 0.1mM2-beta-mercaptoethanol, 20U/ml human leukocyte supressor, 10U/ml Prostatropin, 1mM L-glutaminate, 2mM thioglycerin (MTG), 80U/ml penicillin, 80 μ g/ml Streptomycin sulphates.
The neurocyte that induction in embodiment 3 is obtained carries out immunocytochemistry, flow cytometry and electric physiological detection, result shows that induction has produced neurocyte, and cytodifferentiation ratio reaches 86.64%, and the neurocyte obtaining stimulates to external world and has discharging function.
Embodiment 4 comparative examples
The fresh P3-5 going down to posterity for umbilical cord mesenchymal stem cells with 5 * 10
4/ ml is inoculated in 24 orifice plates that are placed with coated poly-lysine cover glass, 1ml/ hole, and adherent culture is changed nutrient solution after 3 days be DMEM/F12,2%B27 also contains 8mM L-glutaminate on this basis, 80U/ml penicillin, 80 μ g/ml Streptomycin sulphates.Within 3 days, later half amount is changed liquid, stops induction in the time of the 6th day.
The detection of embodiment 5 neurocyte
(1) immunohistochemical methods is identified
Step: the neurocyte that (1) obtains embodiment 2 methods adds 4ml to be preheated to 30 ℃, with Du Shi phosphoric acid buffer (DPBS) rinsing cell twice, each 4min; (2) add 2mi3% paraformaldehyde stationary liquid, room temperature is 20min fixedly; (3) DPBS rinsing is 4 times, each 2min.With containing the DPBS of 1%Triton X-100 35 ℃ of incubations 20 minutes.(4) add 2ml to contain the DPBS of 6% lowlenthal serum, under room temperature, act on 20min.(5) with rifle head, inhale and abandon after confining liquid, add mouse-anti people β-tublin III monoclonal antibody of dilution in 1: 500,4 ℃ are spent the night.(6) with rifle head, inhale and abandon primary antibodie reaction solution, with DPBS rinsing 4 times, each 3min.(7) add the sheep anti mouse fluorescence two of dilution in 1: 200 anti-, 37 ℃ of lucifuge incubation 1h.(8) with rifle head, inhale and abandon after second antibody reaction solution, with DPBS rinsing twice, each 4min.(9) distilled water rinsing is 3 times, and fluorescent microscope (Nikon) is lower to be observed and Taking Pictures recording, as Fig. 1 shows, and neurocyte β-tublin III immunocytochemistry result of A embodiment 2 inductions, B is for differing figure.Result shows successfully to induce and has produced neurocyte.
The neurocyte that embodiment 3 methods are obtained repeats the method, and result shows equally successfully to induce and produced neurocyte.
(2) flow cytometry
Step: (1) by the neurocyte of embodiment 2-4 method induction, respectively with after the centrifugal 4min of 300 * g with twice of 4 ℃ of DPBS rinsing.(2) be resuspended in 4% paraformaldehyde of 4 ℃ of 0.5ml 18 ℃ of incubations 10 minutes.Vortex pipe is to maintain single cell suspension off and on.(3) DPBS rinsing is 2 times, each 2min.The centrifugal 4min of 300 * g.(4) with containing the DPBS of 1%Triton X-100 37 ℃ of incubations 20 minutes.The centrifugal 4min of (5) 300 * g, adds 2ml to contain the DPBS (confining liquid) of 5% lowlenthal serum after abandoning supernatant, under room temperature, acts on 20min.The centrifugal 5min of (6) 300 * g, abandons the β-tublin III monoclonal antibody that adds after supernatant the FITC mark of dilution in 1: 50 to cross; Adding antibody diluent contrasts as homotype.Incubated at room 2 hours.The centrifugal 5min of (7) 300 * g, abandons supernatant, with DPBS rinsing 3 times, and each 3min.The centrifugal 5min of (8) 300 * g, abandons the DPBS re-suspended cell of every effective 200ul after supernatant, and upflowing cell instrument detects analysis.In Fig. 2, B shows in embodiment 2 inducing cells that it is that embodiment 4 contrasts that neurocyte ratio accounts for 91.43%, A figure.In Fig. 3, B shows in embodiment 3 inducing cells that it is that embodiment 4 contrasts that neurocyte ratio accounts for 86.64%, A figure.
(3) electric physiological detection
In cultivation testing chamber in culture dish, add the 10 μ g/ml poly-lysine surface treatment liquids of 150 μ l, in 37 ℃ of incubators, place 20 hours, then use sterilizing distilled water by culture dish rinsing 4 times, spread the neurocyte obtaining into embodiment 2,37 ℃, 5% ℃ O
2cultivate 2 days, then neurocyte is carried out processing 4h without 37 ℃ of magnesium extracellular fluids.Adopt LAPS chip system to detect the neurocyte current potential inducing, result is as shown in Fig. 4-5.In Fig. 4, show neurocyte that embodiment 2 obtains through without magnesium induction electric potential signal (on) and spontaneous potential signal (under), in Fig. 5, show neurocyte that embodiment 3 obtains through without magnesium induction electric potential signal (on) and spontaneous potential signal (under), result shows that the present invention cultivates the neurocyte obtaining and stimulates to external world and have discharging function.
Finally, it is also to be noted that, what more than enumerate is only specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, can also have many distortion.
Claims (4)
1. the method that umbilical cord mesenchymal stem cells inducing culture is neurocyte, is characterized in that comprising the following steps:
(1), cultivation and the amplification of umbilical cord mesenchymal stem cells: obtain umbilical cord mesenchymal stem cells monomer, umbilical cord mesenchymal stem cells monomer is pressed to 1-5 * 10
5/ ml is inoculated in umbilical cord mesenchymal stem cells nutrient solution, and every 3-8 days changes liquid and goes down to posterity once, obtains cultured cells;
Described umbilical cord mesenchymal stem cells nutrient solution is containing DMEM/F12,2%B27,2-4mM L-glutaminate, 1mM thioglycerin, 1% non-essential amino acid, EGF20-40ng/ml, bFGF20-30ng/ml, 80-90U/ml penicillin, 60-70 μ g/ml Streptomycin sulphate;
(2), pre-induction differentiation: the culturing cell that step (1) is obtained is by 1～5 * 10
4adherent inducing culture is carried out in the cell density inoculation of/ml, and adherent culture is changed pre-inducing culture liquid and cultivated after 3 days, and within 3 days, later half amount is changed liquid, stops induction in the time of 3-4 days;
Described pre-inducing culture liquid be DMEM as basic medium, also contain on this basis the vitamins C of 20nmol～50nmol, 10-20U/ml human leukocyte supressor, 1-8mM L-glutaminate, 2-5mM thioglycerin, 80-100U/ml penicillin, 80-100 μ g/ml Streptomycin sulphate.
2. the method that umbilical cord mesenchymal stem cells inducing culture according to claim 1 is neurocyte, is characterized in that: also comprise step (3) induction differentiation: the cell that step (2) is obtained is by 1～5 * 10
4adherent inducing culture is carried out in the cell density inoculation of/ml, and adherent culture is changed inducing culture liquid and carried out inducing culture after 2 days, and within 3 days, later half amount is changed liquid, stops induction in the time of 3-6 days, obtains neurocyte; Described inducing culture liquid is that DMEM is as basic medium, also contain on this basis the vitamins C of 20nmol～50nmol, the VITMAIN B1 of 30nmol～40nmol, the dimethyl sulfoxide (DMSO) of 0.01-0.02%, 0.1mM2-beta-mercaptoethanol, 10-20U/ml human leukocyte supressor, 10-30U/ml Prostatropin, 1-8mM L-glutaminate, 2-5mM thioglycerin, 80-100U/ml penicillin, 80-100 μ g/ml Streptomycin sulphate.
3. the method that umbilical cord mesenchymal stem cells inducing culture according to claim 2 is neurocyte, it is characterized in that: described umbilical cord mesenchymal stem cells nutrient solution is containing DMEM/F12,2%B27,4mM L-glutaminate, 1mM thioglycerin, 1% non-essential amino acid, EGF20ng/ml, bFGF20ng/ml90U/ml penicillin, 70 μ g/ml Streptomycin sulphates; Described pre-inducing culture liquid be DMEM as basic medium, also contain on this basis the vitamins C of 50nmol, 20U/ml human leukocyte supressor, 8mM L-glutaminate, 5mM thioglycerin, 80U/ml penicillin, 80 μ g/ml Streptomycin sulphates; Described inducing culture liquid is that DMEM is as basic medium, also contain on this basis the vitamins C of 50nmol, the VITMAIN B1 of 30nmol, 0.01% dimethyl sulfoxide (DMSO), 0.1mM2-beta-mercaptoethanol, 10U/ml human leukocyte supressor, 30U/ml Prostatropin, 8mM L-glutaminate, 5mM thioglycerin, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates.
4. according to the method for one of claim 1-3, cultivate the neurocyte obtaining.
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