CN110042079A - It is a kind of for cultivating the culture medium of mescenchymal stem cell - Google Patents
It is a kind of for cultivating the culture medium of mescenchymal stem cell Download PDFInfo
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- CN110042079A CN110042079A CN201910305686.8A CN201910305686A CN110042079A CN 110042079 A CN110042079 A CN 110042079A CN 201910305686 A CN201910305686 A CN 201910305686A CN 110042079 A CN110042079 A CN 110042079A
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Abstract
The present invention discloses a kind of for cultivating the culture medium of mescenchymal stem cell, and the culture medium includes DL- alpha-tocopherol, DL- alpha-tocopherol acetate, catalase, superoxide dismutase, reduced glutathione, vitamin C, transferrins, insulin, epidermal growth factor, fibroblast growth factor 2, biotin, D- galactolipin, cortex rouge ketone, L-carnitine, linoleic acid, linolenic acid, sodium selenite, progesterone, catalase, putrescine and trilute.Culture medium provided by the invention is full artificial synthetic medium, it is free of any animal sources or source of people substance, immunological rejection caused by animal sources and/or source of people substance can be effectively avoided, while the full artificial synthetic medium is more conducive to quality control and large scale preparation；Culture medium of the invention can be used for cultivating mescenchymal stem cell, use for the research of basic regenerativ biology and clinical regenerative medicine research.
Description
Technical field
The present invention relates to technical field of stem cell culture more particularly to a kind of for cultivating the culture of mescenchymal stem cell
Base.
Background technique
Stem-cell research and regenerativ biology research have great theoretical value and application prospect, and adult stem cell is
One kind is present in organism, is a kind of cell of specific cells and tissue with self-renewing and directed differentiation.Stem cell can
To supplement the cell and tissue of damage, body repair function is played, regenerativ biology and regenerative medicine are therefore widely used in
Field.Have that the adult stem cell of value is widely applied includes candidate stem cell and mescenchymal stem cell (MSC) in human body, wherein
Mescenchymal stem cell is mainly derived from viviparous tissue (such as umbilical cord and placenta) and fat.
At present in basic research and clinical application research field, the culture medium for cultivating mescenchymal stem cell is source of people blood platelet
Lysate or human serum.It is limited however, these source of people substances usually have source and easily causes the immunological rejection of host anti-
The problems such as answering, therefore, research and development be free of any animal and people source full-synthetic culture medium, for mescenchymal stem cell basis and
Application study is crucial.
Therefore, the existing technology needs to be improved and developed.
Summary of the invention
In view of above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a kind of for cultivating mescenchymal stem cell
Culture medium, it is intended to solve the problems, such as that existing stem cell media easily causes immunological rejection.
Technical scheme is as follows:
It is a kind of for cultivating the culture medium of mescenchymal stem cell, wherein the culture medium includes DL- α-life of 10-100ug/ml
Educate phenol, the DL- alpha-tocopherol acetate of 10-100ug/ml, the catalase of 50-200ug/ml, the super oxygen of 100-900U/ml
Compound mutase, the reduced glutathione of 10-200ug/ml, the vitamin C of 1-10mg/ml, 2-20mg/ml's turns iron egg
It is white, the insulin of 200-800ug/ml, the epidermal growth factor of 1-10mg/ml, the fibroblast growth factor of 1-20mg/ml
2,50-200ug/ml biotin, the D- galactolipin of 200-1500ug/ml, the cortex rouge ketone of 0.5-5ug/ml, 50-500ug/
The L-carnitine of ml, the linoleic acid of 20-500ug/ml, the linolenic acid of 20-500ug/ml, the sodium selenite of 300-3000ng/ml,
The progesterone of 200-2000ng/ml, the catalase of 50-200ug/ml, the putrescine of 0.5-5mg/ml, 20-500ng/ml's
Trilute.
The culture medium for being used to cultivate mescenchymal stem cell, wherein the culture medium includes the DL- α-of 30-80ug/ml
Tocopherol, the DL- alpha-tocopherol acetate of 30-80ug/ml, the catalase of 100-150ug/ml, 300-700U/ml's is super
Superoxide dismutase, the reduced glutathione of 50-150ug/ml, the vitamin C of 4-8mg/ml, 6-15mg/ml's turns iron egg
It is white, the insulin of 400-600ug/ml, the epidermal growth factor of 4-8mg/ml, the fibroblast growth factor of 6-15mg/ml
2,100-150ug/ml biotin, the D- galactolipin of 600-1000ug/ml, the cortex rouge ketone of 2-4ug/ml, 200-400ug/
The L-carnitine of ml, the linoleic acid of 150-350ug/ml, the linolenic acid of 200-400ug/ml, the selenous acid of 1000-2000ng/ml
Sodium, the progesterone of 600-1500ng/ml, the catalase of 80-120ug/ml, the putrescine of 2-4mg/ml, 100-400ng/ml
Trilute.
The culture medium for being used to cultivate mescenchymal stem cell, wherein the culture medium further includes 0.01-0.1ug/ml second
Hydramine, the putrescine of 0.5-5mg/ml.
The culture medium for being used to cultivate mescenchymal stem cell, wherein the culture medium includes DL- α-life of 50ug/ml
Educate phenol, the DL- alpha-tocopherol acetate of 50ug/ml, the catalase of 100ug/ml, the superoxide dismutase of 500U/ml,
The reduced glutathione of 100ug/ml, the vitamin C of 5mg/ml, the transferrins of 10mg/ml, the insulin of 500ug/ml,
The epidermal growth factor of 5mg/ml, the fibroblast growth factor 2 of 10mg/ml, the biotin of 100ug/ml, 800ug/ml's
D- galactolipin, the cortex rouge ketone of 3ug/ml, the L-carnitine of 300ug/ml, the linoleic acid of 300ug/ml, flax of 300ug/ml
Acid, the sodium selenite of 1500ng/ml, the progesterone of 1000ng/ml, the catalase of 100ug/ml, the putrescine of 3mg/ml,
The trilute of 300ng/ml, 0.05ug/ml ethanol amine, the putrescine of 3mg/ml.
The utility model has the advantages that the culture medium that the present invention can provide includes DL- alpha-tocopherol, DL- alpha-tocopherol acetate, peroxide
Change hydrogen enzyme, superoxide dismutase, reduced glutathione, vitamin C, transferrins, insulin, epidermal growth factor, at
Fibroblast growth factor 2, biotin, D- galactolipin, cortex rouge ketone, L-carnitine, linoleic acid, linolenic acid, sodium selenite,
Progesterone, catalase, putrescine and trilute.Culture medium provided by the invention is complete artificial synthesized training
Base is supported, any animal sources or source of people substance is free of, can effectively avoid immunological rejection caused by animal sources and/or source of people substance anti-
It answers, while the full artificial synthetic medium is more conducive to quality control and large scale preparation；Culture medium of the invention can be used for training
Mescenchymal stem cell is supported, is used for the research of basic regenerativ biology and clinical regenerative medicine research.
Detailed description of the invention
Fig. 1 is that third generation mankind MSC cell is incubated at FBS culture medium respectively, in 5% PL culture medium and NBVbe culture medium
Doubling time comparison diagram when five generations is persistently cultivated in passage.
Fig. 2 is that MSC cell cultivates the cellular morphology after 5 generations in NBVbe culture medium.
Fig. 3 is that MSC cell cultivates the cellular morphology after 5 generations in 5%PL culture medium.
Fig. 4 is after MSC cell cultivated for 5 generations in NBVbe culture medium, after CD73-FITC ice bath label 30 minutes, with stream
Formula cell instrument detects the result figure of cell expression MSC molecular labeling.
Fig. 5 is after MSC cell cultivated for 5 generations in NBVbe culture medium, after CD90-FITC ice bath label 30 minutes, with stream
Formula cell instrument detects the result figure of cell expression MSC molecular labeling.
Fig. 6 is after CD105-FITC ice bath label 30 minutes, to use after MSC cell cultivated for 5 generations in NBVbe culture medium
The result figure of flow cytomery cell expression MSC molecular labeling.
Fig. 7 is after HLADR-FITC ice bath label 30 minutes, to use after MSC cell cultivated for 5 generations in NBVbe culture medium
The result figure of flow cytomery cell expression MSC molecular labeling.
Fig. 8 is after MSC cell cultivated for 5 generations in NBVbe culture medium, after CD45-FITC ice bath label 30 minutes, with stream
Formula cell instrument detects the result figure of cell expression MSC molecular labeling.
Fig. 9 is after MSC cell cultivated for 5 generations in NBVbe culture medium, after CD34-FITC ice bath label 30 minutes, with stream
Formula cell instrument detects the result figure of cell expression MSC molecular labeling.
Figure 10 is after CD19-FITC ice bath label 30 minutes, to use after MSC cell cultivated for 5 generations in NBVbe culture medium
The result figure of flow cytomery cell expression MSC molecular labeling.
Figure 11 is after CD11b-FITC ice bath label 30 minutes, to use after MSC cell cultivated for 5 generations in NBVbe culture medium
The result figure of flow cytomery cell expression MSC molecular labeling.
Figure 12 is that the violet staining after MSC cell is cultivated 3 days in 5%PL culture medium is taken pictures figure.
Figure 13 is that violet staining is taken pictures figure after MSC cell is cultivated 3 days in NBVbe culture medium.
Figure 14 is after MSC cell cultivated for 5 generations in NBVbe culture medium, to be divided into the development figure of fat cell.
Figure 15 is after MSC cell cultivated for 5 generations in NBVbe culture medium, to be divided into the development figure of osteoblast.
Figure 16 is after MSC cell cultivated for 5 generations in NBVbe culture medium, to be divided into the development figure of cartilage cell.
Figure 17 is to extract genomic DNA after MSC cell cultivated for 5 generations respectively in NBVbe and 5%PL culture medium and be sequenced
When exon introne shearing site error event comparison diagram.
Figure 18 is to extract genomic DNA after MSC cell cultivated for 5 generations respectively in NBVbe and 5%PL culture medium and be sequenced
When site mutation event comparison diagram.
Specific embodiment
The present invention provide it is a kind of for cultivating the culture medium of mescenchymal stem cell, to make the purpose of the present invention, technical solution
And effect is clearer, clear, the present invention is described in more detail below.It should be appreciated that specific implementation described herein
Example is only used to explain the present invention, is not intended to limit the present invention.
Embodiment of the present invention provides a kind of for cultivating the culture medium of mescenchymal stem cell, and the culture medium includes 10-
The DL- alpha-tocopherol of 100ug/ml, the DL- alpha-tocopherol acetate of 10-100ug/ml, the catalase of 50-200ug/ml,
The superoxide dismutase of 100-900U/ml, the reduced glutathione of 10-200ug/ml, the vitamin C of 1-10mg/ml, 2-
The transferrins of 20mg/ml, the insulin of 200-800ug/ml, the epidermal growth factor of 1-10mg/ml, 1-20mg/ml at
Fibroblast growth factor 2, the biotin of 50-200ug/ml, the D- galactolipin of 200-1500ug/ml, the skin of 0.5-5ug/ml
Matter rouge ketone, the L-carnitine of 50-500ug/ml, the linoleic acid of 20-500ug/ml, the linolenic acid of 20-500ug/ml, 300-
The sodium selenite of 3000ng/ml, the progesterone of 200-2000ng/ml, the catalase of 50-200ug/ml, 0.5-5mg/ml
Putrescine, the trilute of 20-500ng/ml.
In the present embodiment, the ng/ml refers to nanograms/milliliter, and ug/ml refers to mcg/ml, and mg/ml refers to milli
Grams per milliliter, the U/ml are enzyme activity units；The DL- alpha-tocopherol acetate, DL- alpha-tocopherol, catalase surpass
Superoxide dismutase, reduced glutathione, vitamin C and transferrins can be used for activating mescenchymal stem cell incubation
In DNA superoxides injury repair approach；The insulin, epidermal growth factor, fibroblast growth factor 2 facilitate
Activate the insulin signaling pathway in mescenchymal stem cell incubation；The biotin, D- galactolipin, cortex rouge ketone are left-handed
Carnitine, linoleic acid, linolenic acid and sodium selenite then facilitate activate mescenchymal stem cell incubation in glycolipid metabolism it is normal
It carries out.
In the present embodiment, since 5% platelet cracking content can promote the fast breeder of MSC, present embodiment
MSC is cultivated respectively and containing five days in 0%, 1%, 2% and 5% platelet cracking content, is then extracting RNA progress high-flux sequence, is led to
Be overexpressed characteristic spectrum, time series, signal path enrichment and protein-protein interaction network prediction, and combine literature search and
Extensive potential nutriment test, finally show that the in-vitro multiplication that MSC can be maintained in the present embodiment expands, and have MSC
Cells and characteristic of stem and differentiation potential culture medium.Compared with existing stem cell media, the culture medium of present embodiment offer
Without any animal sources or source of people substance, it is conducive to quality control and large scale preparation, also avoids animal sources or source of people substance can
Immunological rejection caused by energy.
In some embodiments, the culture medium includes the DL- alpha-tocopherol of 30-80ug/ml, 30-80ug/ml's
DL- alpha-tocopherol acetate, the catalase of 100-150ug/ml, the superoxide dismutase of 300-700U/ml, 50-
The reduced glutathione of 150ug/ml, the vitamin C of 4-8mg/ml, the transferrins of 6-15mg/ml, 400-600ug/ml's
Insulin, the epidermal growth factor of 4-8mg/ml, the fibroblast growth factor 2 of 6-15mg/ml, the life of 100-150ug/ml
Object element, the D- galactolipin of 600-1000ug/ml, the cortex rouge ketone of 2-4ug/ml, the L-carnitine of 200-400ug/ml, 150-
The linoleic acid of 350ug/ml, the linolenic acid of 200-400ug/ml, the sodium selenite of 1000-2000ng/ml, 600-1500ng/ml
Progesterone, the catalase of 80-120ug/ml, the putrescine of 2-4mg/ml, the triiodo thyroid gland original ammonia of 100-400ng/ml
Acid.
In some embodiments, the culture medium further includes 0.01-0.1ug/ml ethanol amine, the corruption of 0.5-5mg/ml
Amine.The putrescine can promote the synthesis of RNA in cell, rotten when nuclei dyeing chromaticness or nucleohistone are by as primer
Amine can promote the synthesis of DNA.The ethanol amine is a kind of compound of important stimulating cellular growth, is the conjunction of brain phosphoric acid
At premise.
It is imitated below by the specific embodiment culture medium and its culture for cultivating mescenchymal stem cell a kind of to the present invention
Fruit is further explained explanation:
Embodiment 1
It is a kind of for cultivating the culture medium of mescenchymal stem cell, wherein the culture medium includes the DL- alpha-tocopherol of 50ug/ml,
The DL- alpha-tocopherol acetate of 50ug/ml, the catalase of 100ug/ml, the superoxide dismutase of 500U/ml,
The reduced glutathione of 100ug/ml, the vitamin C of 5mg/ml, the transferrins of 10mg/ml, the insulin of 500ug/ml,
The epidermal growth factor of 5mg/ml, the fibroblast growth factor 2 of 10mg/ml, the biotin of 100ug/ml, 800ug/ml's
D- galactolipin, the cortex rouge ketone of 3ug/ml, the L-carnitine of 300ug/ml, the linoleic acid of 300ug/ml, flax of 300ug/ml
Acid, the sodium selenite of 1500ng/ml, the progesterone of 1000ng/ml, the catalase of 100ug/ml, the putrescine of 3mg/ml,
The trilute of 300ng/ml, 0.05ug/ml ethanol amine, the putrescine of 3mg/ml.
Embodiment 2
It is a kind of for cultivating the culture medium of mescenchymal stem cell, wherein the culture medium includes the DL- alpha-tocopherol of 20ug/ml,
The DL- alpha-tocopherol acetate of 20ug/ml, the catalase of 50ug/ml, the superoxide dismutase of 100U/ml, 20ug/
The reduced glutathione of ml, the vitamin C of 2mg/ml, the transferrins of 4mg/ml, the insulin of 200ug/ml, 1mg/ml's
Epidermal growth factor, the fibroblast growth factor 2 of 2mg/ml, the biotin of 50ug/ml, the D- galactolipin of 200ug/ml,
The cortex rouge ketone of 0.5ug/ml, the L-carnitine of 100ug/ml, the linoleic acid of 20ug/ml, the linolenic acid of 20ug/ml, 300ng/
The sodium selenite of ml, the progesterone of 200ng/ml, the catalase of 50ug/ml, the putrescine of 0.5mg/ml, the three of 20ng/ml
Iodine thyronine, 0.01ug/ml ethanol amine, the putrescine of 0.5mg/ml.
Embodiment 3
It is a kind of for cultivating the culture medium of mescenchymal stem cell, wherein the culture medium includes DL- α-fertility of 100ug/ml
Phenol, the DL- alpha-tocopherol acetate of 100ug/ml, the catalase of 200ug/ml, the superoxide dismutase of 800U/ml,
The reduced glutathione of 200ug/ml, the vitamin C of 10mg/ml, the transferrins of 20mg/ml, the pancreas islet of 700ug/ml
Element, the epidermal growth factor of 10mg/ml, the fibroblast growth factor 2 of 20mg/ml, the biotin of 150ug/ml,
The D- galactolipin of 1000ug/ml, the cortex rouge ketone of 4ug/ml, the L-carnitine of 400ug/ml, the linoleic acid of 400ug/ml,
The linolenic acid of 400ug/ml, the sodium selenite of 2000ng/ml, the progesterone of 2000ng/ml, the catalase of 200ug/ml,
The putrescine of 5mg/ml, the trilute of 400ng/ml, 0.1ug/ml ethanol amine, the putrescine of 5mg/ml.
The measure of merit experiment of culture medium is as follows:
1, third generation mankind MSC cell is plated on 6 orifice plates culture plate by 40,000 cells/wells, is then incubated at FBS culture respectively
Base (DMEM+10%FBS), 5% PL culture medium (DMEM+10%FBS) and NBVbe culture medium (culture medium group in DMEM+ embodiment 1
Point) culture to cell density reaches 80-90%, cell digests with TrypLE (Gibco company), cell count, then thin by 40,000
Born of the same parents/hole is plated on 6 orifice plates culture plate, and passage persistently cultivated for five generations.Cell times is calculated according to cell generation time and cell number
Increase the time, it is as shown in Figure 1 to obtain result.It can be seen from figure 1 that being cultivated using the NBVbe comprising nutrient media components in embodiment 1
Base culture mescenchymal stem cell, growth rate and widely used human blood platelets lysate (PL) are close, but are better than tire ox blood
(FBS) clearly.Fig. 2 is that MSC cell cultivates the cellular morphology after 5 generations in NBVbe culture medium；Fig. 3 is that MSC cell is cultivated in 5%PL
Cellular morphology after cultivating for 5 generations in base；Fig. 4-Figure 11 is respectively to use after MSC cell cultivated for 5 generations in NBVbe culture medium
TrypLE (Gibco company) digestion be it is unicellular, with CD73-FITC, CD90-FITC, CD105-FITC, HLADR-
FITC, CD45-FITC, CD34-FITC, CD19-FITC and CD11b-FITC ice bath label used fluidic cell after 30 minutes
Instrument detects the case where cell expression MSC molecular labeling.
After MSC cell cultivated for 5 generations in NBVbe culture medium, it is unicellular for being digested with TrypLE (Gibco company), then
It is plated on non-attaching tissue culture plate, after being cultivated 3 days in NBVbe and 5%PL culture medium, attaching tissue culture plate is transferred to and holds
Continuous culture 2 days.After cell is determined with 4% paraformaldehyde state, taken pictures with violet staining, the MSC cell is trained in 5%PL culture medium
Support 3 days after figure of taking pictures it is as shown in figure 12, the MSC cell cultivated 3 days in NBVbe culture medium after figure such as Figure 13 institute of taking pictures
Show.It can be seen from the figure that the culture medium (NBVbe) using in the present embodiment 1 cultivates mescenchymal stem cell, can be formed
Stem cell spheres.
After MSC cell cultivated for 5 generations in NBVbe culture medium, it to be used for fat cell, the orientation of osteoblast and cartilage cell
Differentiation.Differentiation step and authentication step are carried out according to universal kit and standardization program；Used kit is StemPro
Adipogenesis Differentiation Kit (Gibco), StemPro® Osteogenesis
Differentiation Kit (Gibco), StemPro® Chondrogenesis Differentiation Kit
(Gibco), and Human Mesenchymal Stem Cell Functional Identifcation Kit (R&D
Systems).The noble cells development figure as shown in Figure 14-Figure 16 can be respectively obtained by Analytical Chemical Experiment.As shown, described
MSC cell can be divided into fat cell, osteoblast and cartilage cell after cultivating for 5 generations in NBVbe culture medium.
After MSC cell cultivated for 5 generations respectively in NBVbe and 5%PL culture medium, extracts genomic DNA and shown outside high-throughput
Son sequencing, sequencing result contrast table is as shown in Figure 17-Figure 18, wherein the end A5SS:5' shearing site error event； A3SS:
The end 3' shearing site error event；MXE: mutual exclusion exon error event；RI: introne residual errors event；SE: outer
Aobvious son jump error event；SNV: single base difference mutation；InDel: insertion and deletion mutation；Total: event summation.
Alternative splicing events: exon introne shearing site error event；Counts: site mutation thing
Part.It can be seen that by Figure 17 and Figure 18, compared with source of people platelet cracking content (PL), contain 1 nutrient media components of the present embodiment
The cell that NBVbe culture medium is turned out has less genome mutation, possesses higher Genome stability.
In conclusion the culture medium that the present invention can provide includes DL- alpha-tocopherol, DL- alpha-tocopherol acetate, peroxide
Change hydrogen enzyme, superoxide dismutase, reduced glutathione, vitamin C, transferrins, insulin, epidermal growth factor, at
Fibroblast growth factor 2, biotin, D- galactolipin, cortex rouge ketone, L-carnitine, linoleic acid, linolenic acid, sodium selenite,
Progesterone, catalase, putrescine and trilute.Culture medium provided by the invention is complete artificial synthesized training
Base is supported, any animal sources or source of people substance is free of, can effectively avoid immunological rejection caused by animal sources and/or source of people substance anti-
It answers, while the full artificial synthetic medium is more conducive to quality control and large scale preparation；Culture medium of the invention can be used for training
Mescenchymal stem cell is supported, is used for the research of basic regenerativ biology and clinical regenerative medicine research.
It should be understood that the application of the present invention is not limited to the above for those of ordinary skills can
With improvement or transformation based on the above description, all these modifications and variations all should belong to the guarantor of appended claims of the present invention
Protect range.
Claims (4)
1. a kind of for cultivating the culture medium of mescenchymal stem cell, which is characterized in that the culture medium includes 10-100ug/ml's
DL- alpha-tocopherol, the DL- alpha-tocopherol acetate of 10-100ug/ml, the catalase of 50-200ug/ml, 100-900U/
The superoxide dismutase of ml, the reduced glutathione of 10-200ug/ml, the vitamin C of 1-10mg/ml, 2-20mg/ml's
The fibroblast of transferrins, the insulin of 200-800ug/ml, the epidermal growth factor of 1-10mg/ml, 1-20mg/ml is raw
The long factor 2, the biotin of 50-200ug/ml, the D- galactolipin of 200-1500ug/ml, the cortex rouge ketone of 0.5-5ug/ml, 50-
The L-carnitine of 500ug/ml, the linoleic acid of 20-500ug/ml, the linolenic acid of 20-500ug/ml, the Asia of 300-3000ng/ml
Sodium selenate, the progesterone of 200-2000ng/ml, the catalase of 50-200ug/ml, the putrescine of 0.5-5mg/ml, 20-
The trilute of 500ng/ml.
2. according to claim 1 for cultivating the culture medium of mescenchymal stem cell, which is characterized in that the culture medium includes
The DL- alpha-tocopherol of 30-80ug/ml, the DL- alpha-tocopherol acetate of 30-80ug/ml, the hydrogen peroxide of 100-150ug/ml
Enzyme, the superoxide dismutase of 300-700U/ml, the reduced glutathione of 50-150ug/ml, the vitamin C of 4-8mg/ml,
The transferrins of 6-15mg/ml, the insulin of 400-600ug/ml, the epidermal growth factor of 4-8mg/ml, 6-15mg/ml at
Fibroblast growth factor 2, the biotin of 100-150ug/ml, the D- galactolipin of 600-1000ug/ml, the cortex of 2-4ug/ml
Rouge ketone, the L-carnitine of 200-400ug/ml, the linoleic acid of 150-350ug/ml, the linolenic acid of 200-400ug/ml, 1000-
The sodium selenite of 2000ng/ml, the progesterone of 600-1500ng/ml, the catalase of 80-120ug/ml, 2-4mg/ml's
Putrescine, the trilute of 100-400ng/ml.
3. -2 is any described for cultivating the culture medium of mescenchymal stem cell according to claim 1, which is characterized in that the culture
Base further includes 0.01-0.1ug/ml ethanol amine, the putrescine of 0.5-5mg/ml.
4. according to claim 3 for cultivating the culture medium of mescenchymal stem cell, which is characterized in that the culture medium includes
The DL- alpha-tocopherol of 50ug/ml, the DL- alpha-tocopherol acetate of 50ug/ml, the catalase of 100ug/ml, 500U/ml
Superoxide dismutase, the reduced glutathione of 100ug/ml, the vitamin C of 5mg/ml, the transferrins of 10mg/ml,
The insulin of 500ug/ml, the epidermal growth factor of 5mg/ml, the fibroblast growth factor 2 of 10mg/ml, 100ug/ml's
Biotin, the D- galactolipin of 800ug/ml, the cortex rouge ketone of 3ug/ml, the L-carnitine of 300ug/ml, the Asia oil of 300ug/ml
Acid, the linolenic acid of 300ug/ml, the sodium selenite of 1500ng/ml, the progesterone of 1000ng/ml, the hydrogen peroxide of 100ug/ml
Enzyme, the putrescine of 3mg/ml, the trilute of 300ng/ml, 0.05ug/ml ethanol amine, the putrescine of 3mg/ml.
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