CN107904202B - A kind of method preparing multipotential stem cell like cell, composition and application - Google Patents
A kind of method preparing multipotential stem cell like cell, composition and application Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
- A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
Abstract
The present invention relates to a kind of method for preparing multipotential stem cell like cell, composition and application, the method for the invention to include the following steps：1) monocyte is cultivated in the culture medium containing Porcine HGF M-CSF, the working concentration of the M-CSF in the medium is 10~50ng/ml, and the time of the culture is 3~5 days；2) after step 1), the monocyte is cultivated in the culture medium containing M-CSF and Radix Ophiopogonis homoisoflavone, wherein, the working concentration of the M-CSF and Radix Ophiopogonis homoisoflavone in the medium is respectively 10~30ng/ml and 30~100 μ g/ml, the incubation time is 4~8 days to get multipotential stem cell like cell.Compared with traditional stem cell induction mode, the method for the invention has the advantages that easy to operate, induced efficiency is high and cost performance is high.
Description
Technical field
The present invention relates to stem cell repair field, in particular to a kind of method for preparing multipotential stem cell like cell,
Composition and application.
Background technique
Stem cell is the neoblast before differential period, according to the difference of differentiation capability, can be substantially divided into complete
Energy stem cell (Totipotent stem cells), multipotential stem cell (Pluripotent stem cells) and single ability are thin
Born of the same parents (Monopotent Sterm Cells).Wherein, myeloid-lymphoid stem cell refers to all thin before fertilized eggs to 32 cell of cleavage stage
Born of the same parents have and form complete individuals differentiation potential；And multipotential stem cell has the ability for being divided into various kinds of cell, can be formed each
Kind cell and organ, but complete individuals can not be formed；And unipotent stem cell, also known as specially energy stem cell or partially energy stem cell, it can only
To a seed type or closely related two kinds of cell differentiation.
Myeloid-lymphoid stem cell is generated by that can develop adult embryo, is easy to cause ethics problem.Point of unipotent stem cell
Change ability is limited.And multipotential stem cell shorter mention ethics problem and have a variety of differentiation potentials, therefore become current stem cell
The hot spot and focus of research.Multipotential stem cell research not only have important theory significance, but also disease treatment, tissue and
Organ reparation and regeneration aspect have great application value.
Currently, country Liu Guang is intelligent equal using gene editing technological transformation anti contravariance related gene, it is high to obtain degeneration-resistant specificity
Genes amplification type mescenchymal stem cell, for resisting cell ageing and tissue repair.Columbia Univ USA and Iowa
The scientist of university realizes eye illness color by way of CRISPR/Cas9 gene editing technological guide sufferer pluripotent stem cell differentiation
The reparation of disposition retinitis related defects gene.Recently, studies in China personnel induce the Skin Cell of patient at iPS cell
Afterwards, by CRISPR/Cas9 technology mediate homologous recombination in the way of repair the hemoglobin gene of mutation, then by the iPS of reparation
Cell directional is induced to differentiate into candidate stem cell, is transplanted in patient body, treats the sickle-shaped anemia of the mankind.
Although obtaining encouraging progress in all respects, existing multipotential stem cell technology still have all multi-risk Systems and
Deficiency interferes the application of multipotential stem cell technology, including：
(1) so far, it is dry thin to be still difficult to the multipotencys for largely meeting clinical treatment requirement with reasonable expense acquisition for this field
Born of the same parents：
1), source of human stem cell is restricted：Currently, multipotency is dry thin mainly from organizers such as marrow, peripheral blood, liver and placentas
It is separated in official, and quantity of the stem cell in marrow is only 1/10000, the accounting in peripheral blood is only 1/250000, in liver
Dirty middle accounting is 1/100000, and the stem cell amount that obtain clinical treatment needs has pressure to donor body；It does in placenta source
Although cell quantity is better than marrow, peripheral blood and liver, limited by ethics and oncogenicity；
2), induction type multipotential stem cell (iPS) needs are transferred to the genes such as Oct4, sox2, c-Myc, KLF4 in body cell,
Mode of operation is complicated, at high cost and have the defects that cell carcinogenesis risk is high；
3), using the combination of M-CSF, IL-3, IL-6, IL-2 and anti-CD-13 antibody etc., induced synthesis multipotential stem cell,
The combination using cytokine profiles or antibody is needed in preparation process, it is at high cost, and adult cell dedifferentes that form multipotency dry
The low efficiency of cell.
(2) for therapeutic effect, reparation of the multipotential stem cell to damage or necrotic tissue, the especially damage of inflammation part
The repair ability of wound or necrotic tissue is poor, and therapeutic effect is undesirable.
Therefore, this field is it is necessary to improve existing multipotential stem cell preparation method and therapeutic modality, to simplification
The preparation efficiency and therapeutic efficiency operate, reduce cost, improving multipotential stem cell.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of method of induced synthesis multipotential stem cell like cell, and the method is logical
The combination of M-CSF and M-CSF and Radix Ophiopogonis homoisoflavone are crossed, effectively can form multipotential stem cell sample by induced monocyte
Cell has the advantages that easy to operate, induced efficiency is high and cost performance is high compared with traditional stem cell induction mode.
The second object of the present invention is to provide a kind of while induced synthesis multipotential stem cell like cell and M2 type macrophage is thin
The method of born of the same parents, the method pass through the combination of M-CSF and M-CSF, Radix Ophiopogonis homoisoflavone and ganodenic acid, can obtain simultaneously
Much can stem cell-like cell and M2 type macrophage, without inducing respectively, operating method is simple, quick.
The third object of the present invention is to provide a kind of composition, and the composition includes more according to made of preceding method
Energy stem cell-like cell or multipotential stem cell like cell and M2 type macrophage, can be effectively to damage or non-viable non-apoptotic cell, group
It knits or organ is repaired.
The fourth object of the present invention is to provide aforementioned composition in the drug of preparation treatment rheumatoid arthritis
Using.
The fifth object of the present invention is to provide application of the aforementioned composition in the drug of preparation treatment ischemic lesions.
The sixth object of the present invention is to provide aforementioned composition in the drug of preparation treatment neurodegenerative diseases
In application.
The seventh object of the present invention is to provide aforementioned composition in the drug of preparation treatment autoimmune disease
Using.
The eighth object of the present invention is to provide composition above-mentioned in preparation treatment cirrhosis or the drug of chronic hepatitis
In application.
The ninth object of the present invention is to provide composition above-mentioned in preparation treatment chronic renal insufficiency or glomerulus
Application in the drug of ephritis.
The tenth object of the present invention be to provide composition above-mentioned preparation treatment diabetes, diabetic complication and
Application after Severe metabolic syndrome in the drug of group.
Of the invention the 11st is designed to provide composition above-mentioned in the drug of preparation treatment disease consumption syndrome
In application.
In order to realize above-mentioned purpose of the invention, spy uses following technical scheme：
A kind of method of induced synthesis multipotential stem cell like cell, which is characterized in that the described method comprises the following steps：
1) monocyte is cultivated in the culture medium containing Porcine HGF M-CSF, the M-CSF is in the medium
Working concentration is 10~50ng/ml, and the time of the culture is 3~5 days；
2) after step 1), the monocyte is cultivated in the culture medium containing M-CSF and Radix Ophiopogonis homoisoflavone,
In, the working concentration of the M-CSF and Radix Ophiopogonis homoisoflavone in the medium is respectively 10~30ng/ml and 30~100 μ
G/ml, the incubation time are 4~8 days to get multipotential stem cell like cell.
Flavones (flavone) refers to two phenyl ring with phenolic hydroxyl group, made of being connected with each other by central thricarbon atom
A series of compounds, basic parent nucleus are 2- phenyl chromone.Existing some results of study show flavone compound to dry
Regulating and controlling effect is played in proliferation, apoptosis and the differentiation of cell, but has not yet to see and go point about flavone compound and monocyte
Change the relevant report for forming stem cell-like cell.
For probe into flavones and monocyte dedifferente between relationship, and obtaining for screening being capable of induced monocyte shape
At the drug of stem cell-like cell, the present invention stimulates monocyte using a variety of different types of flavones.As a result make us
It surprisinglys found that, Radix Ophiopogonis general flavone (that is, Radix Ophiopogonis homoisoflavone) and M-CSF cooperate, and can effectively inhibit monocyte mark
The expression of object CD14 promotes the expression of stem cell markers CD90；Meanwhile the form of cell also accordingly changes, cell body
Product becomes larger, and pseudopodium occur in cell edges, show the morphological feature of stem cell.Subsequent induction differentiation test is also further demonstrate,proved
Real, the stem cell of the method preparation being capable of induced synthesis quasi-liver cell, class nerve cell at different conditions according to the present invention
With class cardiac muscle cell, " multipotential stem cell " characteristic for being divided into a variety of specialized cells is shown.And other kinds of flavones, such as
Soybean general flavone, barren wort total chromocor etc. can not inhibit the expression of CD14, promote the expression of CD90, also monokaryon can not be stimulated thin
Born of the same parents show stem cell-like cell form.
Compared with the method for conventional transgenic induced synthesis induction type multipotential stem cell (iPS), induction side of the present invention
Method does not need to import the genes such as Oct4, sox2, c-Myc, KLF4 in monocyte, can be obtained multipotency by simply stimulating
Stem cell has the advantages that easy to operate, canceration risk is low etc..In addition, the method for the invention exists compared with combination of cytokines
It only needs to reduce the cost induced using combination of cytokines using individual cells factor M-CSF in Induction Process.
The invention further relates to：A kind of method of while induced synthesis multipotential stem cell like cell and M2 type macrophage, institute
The method of stating includes the following steps：
1) monocyte is cultivated in the culture medium containing Porcine HGF M-CSF, the M-CSF is in the medium
Working concentration is 10~50ng/ml, and the time of the culture is 3~5 days；
2) after step 1), the list is cultivated in the culture medium containing M-CSF, Radix Ophiopogonis homoisoflavone and ganodenic acid
Nucleus, wherein the M-CSF, the working concentration of Radix Ophiopogonis homoisoflavone and ganodenic acid in the medium are respectively 10
~30ng/ml, 30~100 μ g/ml and 100~1000ng/ml, the incubation time are 4~8 days to get multipotential stem cell sample
Cell and M2 type macrophage.
The method of the invention stimulates monocyte by the combination of M-CSF, Radix Ophiopogonis homoisoflavone and ganodenic acid,
Multipotential stem cell like cell and M2 type macrophage can be obtained simultaneously.And other drug candidates can not be in M-CSF and Radix Ophiopogonis
Induced synthesis M2 type macrophage while homoisoflavone induced synthesis multipotential stem cell like cell, some of them drug candidate,
Such as lentinan or polysaccharides, it is not only unable to induced synthesis M2 type macrophage, instead to the shape of multipotential stem cell like cell
At inhibited.
In some specific embodiments, the method also includes：3) harvest step 2) the multipotential stem cell sample is thin
The thin of like cell containing multipotential stem cell and M2 type macrophage is made in born of the same parents or multipotential stem cell like cell and M2 type macrophage
Born of the same parents' suspension.
In some specific embodiments, step 3) the multipotential stem cell like cell or multipotential stem cell like cell
With M2 type macrophage, mechanically or mode of action is removed from the solid phase carrier that it adheres to.
In some specific embodiments, the method further includes the step 3) cell suspension is frozen or matched
Pharmaceutical composition is made.
In some specific embodiments, the multipotential stem cell expresses pluripotency marker CD90.
In some specific embodiments, in described M2 type Expression of Macrophages CD68, CD163, CD14 and CD16
It is one or more, it is preferable that M2 type the Expression of Macrophages CD68 and CD163.
In some specific embodiments, the Radix Ophiopogonis homoisoflavone is selected from 6-aldehydoisoophiopogonanone A, methyl wheat
One of the different ophiopogonone A of winter flavanones B, methyl ophiopogon flavonoid A, 6- aldehyde radical and the different ophiopogonone B of 6- aldehyde radical or a variety of.
In some specific embodiments, the ganodenic acid is selected from ganoderic acid, red sesame acid methyl esters, ganoderic acid, ganoderma lucidum
Alcohol, ganoderma lucidum oxalic acid, ganoderma lucidum olefin(e) acid, red sesame alcohol, ganoderma lucidum alcohol, ganoderal, epoxy ganoderma lucidum alcohol, ganoderma lucidum sterone, ganoderma lucidum terpinum, ganoderma lucidum
Terpene triol, ganoderma lucidum terpene ketone glycol, ganoderma lucidum terpene ketone triol, Reishi sporule acid, red sesame acid, red sesame terpene ketone, red sesame alcohol, red sesame terpene alcohol,
One of ganosporelactjone A, Ganolactone are a variety of.
In some specific embodiments, the working concentration of M-CSF described in step 1) is 10,15,20,25,30,
35,40,45 or 50ng/ml；Preferably, the working concentration of the M-CSF is 30ng/ml.
In some specific embodiments, the working concentration of M-CSF described in step 2) is 10,11,12,13,14,
15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30ng/ml；Preferably, the work of the M-CSF
Concentration is 20ng/ml.
In some specific embodiments, in step 2), the working concentration of the Radix Ophiopogonis homoisoflavone is 30,35,40,
45,50,55,60,65,70,75,80,85,90,95 or 100 μ g/ml；Preferably, the concentration of the Radix Ophiopogonis homoisoflavone is 60 μ
g/ml。
In some specific embodiments, in step 2), the working concentration of the ganodenic acid in the medium is
100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850 or 900ng/ml；It is excellent
Selection of land, the working concentration of the ganodenic acid in the medium are 800ng/ml.
In some specific embodiments, in step 1), the incubation time is 3,4 or 5 days, it is preferable that the training
Supporting the time is 3 days.
In some specific embodiments, incubation time described in step 2) is 4,5,6,7 or 8 days；Preferably, described
Incubation time is 4 days.
Preceding method of the present invention is also to parameters such as the dosages of the specific type of Radix Ophiopogonis homoisoflavone, dosage and ganodenic acid
It is optimized, the method after optimization is either in induced synthesis multipotential stem cell like cell, still induced synthesis at the same time
Multipotential stem cell like cell and the invention of M2 type macrophage, induced efficiency improve.
In some specific embodiments, step 1) and the step 2) culture medium are selected from RPMI culture medium, DMEM
One of culture medium, IMDM culture medium, MEM culture medium, F12 culture medium, MCDB131 culture medium and PCM culture medium are a variety of.
In some specific embodiments, the monocyte is isolated from peripheral blood, Cord blood, lymph and marrow
It is one or more, it is preferable that the monocyte is isolated from peripheral blood.
The invention further relates to a kind of composition, the composition includes that the multipotential stem cell sample according to made of preceding method is thin
Born of the same parents and M2 type macrophage.
Composition of the present invention includes stem cell-like cell and M2 type macrophage.Wherein, the stem cell-like cell
Can be divided into specific cell type under specific ecological microenvironment, and M2 type macrophage have to inflammatory reaction it is potent
Inhibiting effect removes necrotic tissue and plays antioxidation, guide the starting and extension of Regeneration and Repair process, is stem-like cell
The differentiation of cell provides good physiological environment, and the stem cell-like cell is promoted to start the reparation of cell and tissue.To sum up institute
It states, the stem cell-like cell and M2 type macrophage in composition of the present invention cooperate, and have the function of synergy.
Further include drug in the composition in some specific embodiments, the drug include selected from anti-inflammatory agent,
Antioxidant and treatment cell, tissue or organ dysfunction, damage or the drug of necrosis.
In some specific embodiments, the anti-inflammatory agent is selected from steroidal anti-inflammatory drugs or non-steroid anti-inflammatory drug, excellent
Selection of land, the steroidal anti-inflammatory drugs are selected from one of hydrocortisone, dexamethasone or prednisone or a variety of, the non-steroidal
Anti-inflammatory drugs is selected from one of aspirin, Diclofenac or brufen or a variety of.
In some specific embodiments, the antioxidant be selected from selenium, ascorbic acid, vitamin E, catechuic acid, kind
One of Lycopene, beta carotene, coenzyme q-10, EPA and DHA or a variety of.
In some specific embodiments, treatment cell, tissue or the organ dysfunction, damage or the medicine of necrosis
Object is selected from insulin, Insulin-like growth factor II, secretin, melbine, hyaluronic acid, Simvastatin, epidermal growth factor, white
One of interleukin -11, salinomycin and adriamycin are a variety of.
It further include pharmaceutically acceptable carrier in the composition in some specific embodiments, the carrier
Selected from one of blood plasma, serum, albumin, gel and physiological saline or a variety of.
The invention further relates to application of the composition above-mentioned in the drug of preparation treatment rheumatoid arthritis.
The invention further relates to application of the composition above-mentioned in the drug of preparation treatment ischemic lesions, it is preferable that institute
It states ischemic lesions and is selected from the athero- ischemic lesions of extremity arterial, ischemic cardiovascular disease, ischemic cerebrovascular disease or outer
All artery sclerosis ischemic lesions；It is highly preferred that the ischemic cardiovascular disease is heart infarction, the ischemic cerebrovascular disease
For cerebral infarction.
It is excellent the invention further relates to application of the composition above-mentioned in the drug of preparation treatment neurodegenerative diseases
Selection of land, the neurodegenerative diseases are selected from ALS, MS, MSA, Parkinson's disease, cerebellar atrophy, vascular senile dementia
Or one of partial motor meta function obstacle or a variety of.
The invention further relates to composition above-mentioned preparation treatment autoimmune disease drug in application, it is described from
Body immunity disease is selected from one of rheumatoid, Crohn enteritis, lupus erythematosus or transplant organ repulsion or a variety of.
The invention further relates to application of the composition above-mentioned in preparation treatment cirrhosis or the drug of chronic hepatitis.
The invention further relates to compositions above-mentioned in the drug of preparation treatment chronic renal insufficiency or glomerulonephritis
Application.
It is comprehensive in preparation treatment diabetes, diabetic complication and Severe metabolism that the invention further relates to compositions above-mentioned
Application after simulator sickness in the drug of group
The invention further relates to application of the composition above-mentioned in the drug of preparation treatment disease consumption syndrome, preferably
Ground, the disease consumption syndrome is cancer of late stage dyscrasia.
In order to which the present invention is more easily to understand, following term is defined first：
Term " stem cell-like cell " refers to self-renewal capacity and forms at least one or more of specialized cell class
The cell of the ability of type；
Term " versatility " refers to that stem cell has the ability for being capable of forming a variety of specialized cells；
Term " M2 type macrophage " refers to macrophage (the alternatively activated of alternative activation
macrophage)；
Term " M-CSF " refers to Macrophage-Colony stimulating factor (macrophage colony-stimulating
factor)；
Term " CD90 " refers to " Cluster of Differentiation 90 ", also known as Thy-1, and it is viscous to belong to cell
Attached molecule immunoglobulin superfamily member is stem cell surface marker；
Term " CD14 " refers to " Cluster of Differention 14 ", is LPS receptor, belongs to one kind and be present in
The leukocyte differentiation antigen of the cell surfaces such as monocyte, macrophage；
Term " CD16 " refers to " Cluster of Differentiation 16 ", is antibody dependent cellular mediation
Fc γ RIII Fc needed for cytotoxicity (Antibody-Dependent Cell-mediated Cytotoxicity, ADCC)
Receptor；
Term " CD68 " refers to " Cluster of Differentiation 68 ", is special in M2 type macrophage
Property expression cell surface antigen；
Term " CD163 " refers to " Cluster of Differentiation 163 ", is special in M2 type macrophage
The cell surface antigen of opposite sex expression；
Term " ALS " refers to " amyotrophic lateral sclerosis ", that is, amyotrophic lateral sclerosis；
Term " MS " refers to " multiple sclerosis, MS ", that is, multiple sclerosis；
Term " MSA " refers to " multiple system atrophy, MSA ", that is, multi-system atrophy；
Term " EPA " refers to " Eicosapentaenoic Acid ", that is, it is more not to belong to Ω -3 series for eicosapentaenoic acid
Saturated fatty acid；
Term " DHA " refers to " Docose Hexaenoie Acid ", that is, it is more to belong to Ω -3 series for docosahexaenoic acid
Unsaturated fatty acid.
Compared with prior art, beneficial effects of the present invention are：
(1) it has been found, unexpectedly, the combination of M-CSF and Radix Ophiopogonis homoisoflavone being capable of effective stimulus monocyte
It dedifferentes to form stem cell-like cell, the stem cell-like cell has the potential for being divided into a variety of specialized cells；
(2) different from the method for conventional induced synthesis multipotential stem cell, the method for the invention is without passing through genetic engineering
Method import the genes such as Oct4, sox2, c-Myc, KLF4, it is easy to operate, canceration risk is low；Meanwhile the method for the invention
Only needed in Induction Process using individual cells factor M-CSF, and usage amount is low, induction time is short, significantly reduce induction at
This；
(3) ganodenic acid is added on the basis of M-CSF and Radix Ophiopogonis homoisoflavone by largely screening discovery in the present invention,
M2 type macrophage can be formed, the method for the invention is by once inducing while induced synthesis stem cell-like cell
It can be obtained stem cells and M2 type macrophage, it is easy to operate without separately induction；
(4) composition of the present invention includes stem cell-like cell and M2 type macrophage, wherein M2 type macrophage pair
Inflammatory reaction has potent inhibiting effect, removes necrotic tissue and plays antioxidation, guides the starting of Regeneration and Repair process
And extension, provide good physiological environment for the differentiation of stem cell-like cell, promote the stem cell-like cell start cell and
The reparation of tissue, the stem cell-like cell and M2 type macrophage play synergistic effect in repair process.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the cellular morphology figure (40 ×) under the stimulation of different type general flavone, wherein：
Figure 1A is negative control；
Figure 1B is Radix Ophiopogonis general flavone；
Fig. 1 C is soybean general flavone；
Fig. 1 D is barren wort total chromocor；
Fig. 1 E is wild pepper seed total flavonoid；
Fig. 1 F is tartary buckwheat general flavone；
Fig. 1 G is Pueraria Flavonid；
Fig. 1 H is hawthorn fruit total flavone；
Fig. 1 I is honeysuckle general flavone；
Fig. 1 J is ginkgo leaf total flavonoid；
Fig. 1 K is eucommiae total flavones；
Fig. 1 L is total flavonoids in leonurus；
Fig. 2 is the cellular morphology figure (40 ×) under 60 μ g/ml methyl ophiopogon flavonoid A stimulation, wherein：
Fig. 2A is negative control；Fig. 2 B is the different ophiopogonone A of 6- aldehyde radical；
Fig. 3 is under 60 μ g/ml methyl ophiopogon flavonoid A stimulation, and the flow cytometer detection of CD14 and CD90 are as a result, wherein：
Fig. 3 A is negative control；Fig. 3 B is 60 μ g/ml methyl ophiopogon flavonoid A；
Fig. 4 is the flow cytometer detection knot of CD 14, CD90, CD16, CD68 and CD163 under the stimulation of 800ng/ml ganodenic acid
Fruit, wherein：
Fig. 4 A is control；Fig. 4 B is 800ng/ml ganodenic acid.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products obtained can be bought by city.
Embodiment 1
The monocyte of derived from peripheral blood is prepared in the following manner：
1) peripheral blood blood preparation is acquired.4 sterile 50ml centrifuge tubes are taken, sample are carefully poured into centrifuge tube, every pipe
Put 50ml blood preparation.900g is centrifuged 10min, slow to rise slow drop.
2) take upper layer yellow blood plasma into new 50ml centrifuge tube (at most take to the 3/4 of serum layer, so as not to loss cell
Yield), 56 DEG C of inactivation 15min.3500rpm is centrifuged 10min.
3) it is stand-by to be placed in 4 DEG C of refrigerators to new 50ml centrifuge tube for transfer upper plasma.
4) remaining blood sample is resuspended with PBS to original volume, prepares several sterile 50ml centrifuge tubes, people's monokaryon is added in every pipe
Cell separating liquid 20ml is sure not to splash lymphocyte separation medium centrifugation tube wall (by monocyte separation medium：Blood=1:1, example
Such as 80ml whole blood, point 4 pipes carry out separation of lymphocytes).
5) 900g is centrifuged 20min, slow to rise slow drop.
6) prepare several new 50ml centrifuge tubes.After density gradient centrifugation, with 10ml pipette by upper layer supernatant
Gentle aspiration is to from tunica albuginea layer 0.5cm and discarding.
7) careful again to draw intermediate tunica albuginea confluent monolayer cells into new 50ml centrifuge tube.
8) cell suspension of tunica albuginea layer is blown even, and the cell suspension of every 10ml is dispensed into 50ml centrifuge tube.
9) plus PBS is mixed into cell suspension, until at 50ml scale.
10) 1600rpm is centrifuged 10min, abandons supernatant.PBS to 45ml is added to be resuspended, 1200rpm is centrifuged 10min, abandons supernatant.
11) plus PBS to 45ml is resuspended, and takes 100ul sample count, takes appropriate cell suspension to carry out streaming according to count results
Detection supplies PBS to 45ml, 1200rpm, is centrifuged 10min, abandons supernatant.
Embodiment 2
Screening induced monocyte dedifferentes the drug to form multipotential stem cell like cell in the following way：
1)Day 0：Monocyte prepared by embodiment 1 is resuspended, and is 1 × 10 by the density of monocyte6/ ml, inoculation
Into 24 orifice plates, in 37 DEG C, 5%CO2Under conditions of adhere-wall culture 3h；Later, it inhales and abandons suspension cell, added in every hole
0.5ml RPMI1640 culture medium, and by 10% supplement autoserum, in 37 DEG C, 5%CO2Under conditions of continue to cultivate, it is described
M-CSF and autoserum, the final concentration of 30ng/ml of the M-CSF in the medium are added in culture medium；
2) 1 Day, Day 2 and Day3：Observe cellular morphology under the microscope daily.
Day3：The culture medium for abandoning 1/2 volume is inhaled in every hole, supplements the fresh culture of 1/2 volume, in the fresh culture
Containing M-CSF (drug to be screened not being added, as control) or M-CSF and drug to be screened, and by the 10% self blood of supplement
Clearly, wherein the working concentration of the M-CSF is 20ng/ml, and the concentration of the drug to be screened is 50 μ g/ml.
3) 4 Day, Day5 and Day6：Cellular morphology is observed under the microscope.
4)Day 7：The form of cell is observed under the microscope, and specific cellular morphology result is referring to Fig. 1.Cell is harvested, is mentioned
MRNA, reverse transcription cDNA are taken, monocyte marker CD14 and stem cell labeling in cell are detected by way of quantitative PCR
The expression of object CD90, the expression quantity of described CD14, CD90 pass through house-keeping gene GAPDH homogenization, specific testing result
As shown in table 1.
Wherein specific drug to be screened is as follows：
Soybean general flavone, barren wort total chromocor, wild pepper seed total flavonoid, tartary buckwheat general flavone, Pueraria Flavonid, Radix Ophiopogonis are always yellow
Ketone, hawthorn fruit total flavone, honeysuckle general flavone, ginkgo leaf total flavonoid, eucommiae total flavones, total flavonoids in leonurus.
According to data shown in table 1 it is found that soybean general flavone, barren wort total chromocor, wild pepper seed total flavonoid, tartary buckwheat are always yellow
Ketone, Pueraria Flavonid, hawthorn fruit total flavone, honeysuckle general flavone, ginkgo leaf total flavonoid, eucommiae total flavones, total flavonoids in leonurus are simultaneously
The expression of stem cell labeling object cannot be stimulated, and Radix Ophiopogonis general flavone can stimulate the expression of stem cell labeling object CD90, press down simultaneously
The expression of monocyte marker CD14 processed shows that Radix Ophiopogonis general flavone may be that monocyte can be stimulated to form luring for stem cell
Lead object.
Meanwhile cellular morphology is it is found that compared with negative control according to Fig. 1, and under the stimulation of Radix Ophiopogonis general flavone, cell
It is rendered obvious by out volume increase, there is the morphological feature of pseudopodium in cell surface, show class stem cell morphology feature, and other objects
Matter cannot stimulate cell similar class stem cell morphology feature occur.
1 different pharmaceutical of table stimulates the expression of lower CD14 and CD90
| CD14 | CD90 | |
| Control | 1.0±0.05 | 1.0±0.07 |
| Soybean general flavone | 1.1±0.06 | 1.2±0.09 |
| Barren wort total chromocor | 0.9±0.07 | 1.1±0.05 |
| Wild pepper seed total flavonoid | 0.9±0.05 | 1.5±0.12 |
| Tartary buckwheat general flavone | 1.4±0.11 | 0.9±0.04 |
| Pueraria Flavonid | 1.5±0.10 | 0.7±0.05 |
| Radix Ophiopogonis general flavone | 0.3±0.04 | 5.4±0.27 |
| Hawthorn fruit total flavone | 0.7±0.07 | 1.8±0.11 |
| Honeysuckle general flavone | 2.1±0.15 | 0.9±0.06 |
| Ginkgo leaf total flavonoid | 2.2±0.12 | 1.0±0.04 |
| Eucommiae total flavones | 1.2±0.11 | 1.4±0.09 |
| Total flavonoids in leonurus | 0.9±0.09 | 1.1±0.06 |
Embodiment 3
Detect the ability that different types of ophiopogonone induced monocyte forms stem cell-like cell：
Specific method is referring to embodiment 2, and difference is only that, drug to be measured is respectively 6-aldehydoisoophiopogonanone A, methyl
The different ophiopogonone A of ophiopogon flavonoid B, methyl ophiopogon flavonoid A, the 6- aldehyde radical and different ophiopogonone B of 6- aldehyde radical, specific testing result
Referring to table 2.According to data shown in table 2 it is found that above-mentioned five kinds of ophiopogonones are able to suppress the expression and up-regulation of CD14 gene
The expression of CD90 gene forms the ability of stem cell-like cell with induced monocyte, wherein the different ophiopogonone A of 6- aldehyde radical
Inducibility it is most strong.
The different ophiopogonone induced monocytes of table 2 form the ability of stem cell-like cell
| CD14 | CD90 | |
| Control | 1.0±0.05 | 1.0±0.07 |
| 6-aldehydoisoophiopogonanone A | 0.35±0.06 | 4.31±0.21 |
| Methyl ophiopogon flavonoid B | 0.41±0.04 | 4.93±0.25 |
| Methyl ophiopogon flavonoid A | 0.25±0.02 | 5.84±0.44 |
| The different ophiopogonone A of 6- aldehyde radical | 0.50±0.07 | 5.19±0.37 |
| The different ophiopogonone B of 6- aldehyde radical | 0.33±0.04 | 4.75±0.35 |
Embodiment 4
It detects under various concentration, the different ophiopogonone A induced monocyte of 6- aldehyde radical forms the ability of stem cell-like cell：
Referring to embodiment 2, difference is only that specific detection method, and drug to be measured is the different ophiopogonone A of 6- aldehyde radical, the 6-
The concentration of the different ophiopogonone A of aldehyde radical is respectively 0,10,20,30,40,50,60,70,80,90,100 μ g/ml, specific testing result
Referring to table 3.According to the testing result of table 3 it is found that the inducing effect of the different ophiopogonone A of 6- aldehyde radical is most when concentration is 60 μ g/ml
By force.
Under the different inducer concentration of table 3, the expression of CD14 and CD90
| The different ophiopogonone A of 6- aldehyde radical | CD14 | CD90 |
| 0μg/ml | 1.0±0.07 | 1.0±0.09 |
| 10μg/ml | 0.50±0.09 | 4.31±0.15 |
| 20μg/ml | 0.43±0.07 | 4.79±0.21 |
| 30μg/ml | 0.37±0.05 | 4.97±0.25 |
| 40μg/ml | 0.33±0.06 | 5.11±0.29 |
| 50μg/ml | 0.25±0.03 | 5.67±0.37 |
| 60μg/ml | 0.22±0.02 | 6.13±0.42 |
| 70μg/ml | 0.31±0.05 | 5.36±0.34 |
| 80μg/ml | 0.36±0.06 | 5.11±0.27 |
| 90μg/ml | 0.41±0.09 | 5.40±0.18 |
| 100μg/ml | 0.43±0.10 | 5.67±0.29 |
Embodiment 5
The methyl ophiopogon flavonoid A for the use of concentration being 60 μ g/ml, referring to 2 the method induced monocyte shape of embodiment
At stem cell-like cell, at the 6th day, cellular morphology is observed, and harvest cell, pass through Flow cytometry monocyte mark
Object CD14, stem cell labeling object CD90 expression.Wherein, the specific testing result of cellular morphology A- Fig. 2 B referring to fig. 2, flow cytometry
Testing result is referring to Fig. 3 A- Fig. 3 B.
Cellular morphology shown in A- Fig. 2 B is yellow by the methyl Radix Ophiopogonis of 60 μ g/ml it is found that compared with negative control according to fig. 2
After alkanone A stimulation, obvious transformation occurs for cellular morphology, and volume significantly increases, and pseudopodium occurs, shows the cell of class stem cell
Form.Meanwhile streaming result shown in Fig. 3 A-3B also indicates that, the expression quantity decline of onthe surface of monocytes mark CD14, and stem cell
The expression quantity of surface marker CD90 rises.Comprehensive aforementioned testing result, the present invention are expressed from the mRNA of cellular morphology, marker
The different aspects such as horizontal and protein expression level, it was demonstrated that different ophiopogonone A of 6- aldehyde radical etc. can induced monocyte to form class dry
Cell-like cell.
6 multipotential stem cell like cell of embodiment induction differentiation test (induced synthesis quasi-liver cell)
1, multipotential stem cell like cell is formed using the methyl ophiopogon flavonoid A induced monocyte of 60 μ g/ml (specifically to lure
Guiding method is referring to embodiment 5).
2, under given conditions, inducing aforementioned pluripotent stem cell differentiation is quasi-liver cell, wherein induced synthesis class liver is thin
The method of born of the same parents is as follows：
The multipotential stem cell like cell is inoculated into and is covered with fibronectin (50 μ g/cm in advance2), contain Fiber differentiation
In the 50ml plastic culture bottle of base, it is placed in 37 DEG C, 5%CO2, saturated humidity incubator in carry out Fiber differentiation, change liquid within every 3 days
1 time, coinduction 28 days.Wherein, the culture medium is that the DMEM high glucose medium for having FGF4 and HGF is added, and wherein FGF4 is being trained
Supporting the working concentration in base is 40ng/ml, and the working concentration of HGF in the medium is 70ng/ml.
3, induced synthesis quasi-liver cell is detected whether successfully, the specific detection method is as follows：
(1) cellular morphology before observation induction, after induction the 14th day and induction the 28th day；
(2) before induction, induction the 14th day and induction the 28th day, cell total rna is extracted, institute is detected by way of PCR
MRNA expression (be result through house-keeping gene GAPDH homogenization after) of the cell about AFP, FGFR2 and albumin is stated,
Specific testing result is referring to table 4.
According to experimental result described in table 4 it is found that multipotential stem cell like cell of the present invention is gradually in Induction Process
Reveal epithelial cell sample feature, meanwhile, molecular labeling AFP, FGFR2 of liver cell specificity, albumin, carbamyl phosphate synthesis
Expression quantity of the enzyme I in mRNA level in-site significantly rises, and shows that there is multipotent stem cells like cell of the present invention differentiation to become
The ability of liver cell.
4 multipotential stem cell like cell of table induction front and back cellular morphology and gene expression dose variation
7 multipotential stem cell like cell of embodiment induction differentiation test (induced synthesis class nerve cell)
1, multipotential stem cell like cell is formed using the methyl ophiopogon flavonoid A induced monocyte of 60 μ g/ml (specifically to lure
Guiding method is referring to embodiment 5).
2, under given conditions, inducing aforementioned pluripotent stem cell differentiation is class nerve cell, wherein induced synthesis class mind
Method through cell is as follows：
Aforementioned multipotential stem cell like cell is inoculated in 6 orifice plates and (is pre-placed sterile cover slips in hole), it is raw to cell
When length reaches 80% Fusion Strain, culture solution is discarded, the pre-induced culture solution (DMEM/ containing 20%FBS, 1mmol/L β-ME is added
F12 complete culture solution) culture for 24 hours after, remove pre-induced culture solution, PBS wash 3 times, add induction liquid (1mmol/L β-
The serum-free DMEM/F12 of ME, 2%DMSO, 200 μm of ol/L BHA) induction is for 24 hours.
3, induced synthesis class nerve cell is detected whether successfully, the specific detection method is as follows, and specific testing result is referring to table
2：
(1) under the microscope before observation induction, after induction for 24 hours, the growthform of cell；
(2) it takes growth to have the coverslip of cell, immunocytochemical stain, detection cell expression is carried out using SABC method
The case where NSE, wherein positive cell percentage is calculated according to the following formula in piece 5 times again, every meter positive cell number and total cell number
Rate：Positive percentage=positive cell number/total number of cells × 100%.
According to experimental result described in table 5 it is found that stem cell-like cell of the present invention is showing class mind after induction
Through cell sample form, show that stem cell-like cell of the present invention has the ability for inducing differentiation into nerve cell.
5 multipotential stem cell like cell of table induction front and back cellular morphology and the variation of NSE expression
8 multipotential stem cell like cell of embodiment induction differentiation test (induced synthesis class cardiac muscle cell)
1, multipotential stem cell like cell is formed using the methyl ophiopogon flavonoid A induced monocyte of 60 μ g/ml (specifically to lure
Guiding method is referring to embodiment 5).
2, under given conditions, inducing aforementioned pluripotent stem cell differentiation is class nerve cell, wherein induced synthesis class mind
Method through cell is as follows：
Aforementioned multipotential stem cell like cell is inoculated in 6 orifice plates, when cell growth reaches 60~70% Fusion Strain,
Culture solution is discarded, inducer induction is added, the inducer is DMSO, and specific abductive approach is：Liquid is induced with 8mL/L DMSO
Induction for 24 hours, sucks DMSO induction liquid, and the DMEM culture medium for being changed to the FBS containing 100mL/L continues to cultivate 21d.
4, induced synthesis class cardiac muscle cell is detected whether successfully, the specific detection method is as follows, and specific testing result is referring to table
6：
(1) under the microscope observation induction before, after 21 days of induction, the growthform of cell；
(2) extract total serum IgE, reverse transcription cDNA, by PCR detect cardiomyocyte marker object Desmin, Nkx2.5 and
The expression of Tropnin I.
According to experimental result described in table 6 it is found that stem cell-like cell of the present invention is showing the class heart after induction
Myocyte's sample form, and the expression quantity of marker Desmin, Nkx2.5 and Tropnin I of cardiac muscle cell significantly rises, and shows
Stem cell-like cell of the present invention has the ability for inducing differentiation into cardiac muscle cell.
6 multipotential stem cell like cell of table induction front and back cellular morphology and gene expression dose
The drug of the screening of embodiment 9 while induced synthesis multipotential stem cell like cell and M2 type macrophage
Screening induction in the following way can induce the drug for generating stem cell-like cell and M2 type macrophage simultaneously：
1)Day 0：Monocyte prepared by embodiment 1 is resuspended, by monocyte with 1 × 106The density of/ml, inoculation
Into 24 orifice plates, in 37 DEG C, 5%CO2Under conditions of adhere-wall culture 3h；Later, it inhales and abandons suspension cell, added in every hole
0.5ml RPMI1640 culture medium, and by 10% supplement autoserum, in 37 DEG C, 5%CO2Under conditions of continue to cultivate, it is described
M-CSF and autoserum, the final concentration of 30ng/ml of the M-CSF in the medium are added in culture medium；
2) 1 Day, Day 2 and Day3：Observe cellular morphology under the microscope daily.
3)Day3：The culture medium for abandoning 1/2 volume is inhaled in every hole, supplements the fresh culture of 1/2 volume, the fresh culture
In containing M-CSF (do not add drug and 6-aldehydoisoophiopogonanone A to be screened, 1) or M-CSF and 6- aldehyde radical as control
Different ophiopogon flavonoid A (does not add drug to be screened, 2) or M-CSF, 6-aldehydoisoophiopogonanone A and to be screened as control
Drug, and by 10% supplement autoserum, wherein the working concentration of the M-CSF is 20ng/ml, the 6- aldehyde radical different Radix Ophiopogonis
The concentration of flavanones A is 60 μ g/ml, and the concentration of the drug to be screened is 0.5 μ g/ml, and the drug to be screened is specific as follows
It is shown：
Lentinan, ganoderma lucidum polysaccharide, astragalus polyose, polysaccharides, general ginsenoside, arasaponin, total saponins of tribulus,
Sea cucumber total saposins, glycyrrhizin, oleanane-type triterpene saponin and ganodenic acid.
4) 4 Day, Day5 and Day6：Cellular morphology is observed under the microscope.
5)Day 7：Cell is harvested, mRNA, reverse transcription cDNA is extracted, passes through monocyte in quantitative PCR detection cell
The expression of marker CD14, stem cell labeling object CD90, M1 type macrophage marker CD16 and M2 type macrophage mark
The expression of will object CD68, the expression quantity of said gene are the result after house-keeping gene GAPDH homogenization.
Specific testing result is as shown in table 7.According to result shown in table 7 it is found that ganodenic acid and the different wheat of M-CSF, 6- aldehyde radical
Winter, flavanones A was applied in combination, and can make monocyte while forming stem cell-like cell, induced synthesis macrophage, and
The macrophage is M2 type macrophage, rather than M1 type macrophage, and polysaccharide or other terpenoids can not be same
When induction M2 type macrophage formation in addition certain polysaccharide can also hinder the formation of stem cell-like cell to a certain extent.
The inducing effect of 7 polysaccharide of table and terpene to monocyte
The energy of ganodenic acid induced synthesis multipotential stem cell like cell and M2 type macrophage under 10 various concentration of embodiment
Power
1, it detects under various concentration, ganodenic acid induced monocyte forms stem cell-like cell and M2 type macrophage
Ability, referring to embodiment 7, difference is only that specific detection method, and drug to be measured is ganodenic acid, the concentration of the ganodenic acid
Respectively 100,200,300,400,500,600,700,800,900,1000ng/ml, specific testing result is referring to table 5.According to
For the testing result of table 5 it is found that when concentration is 800ng/ml, the inducing effect of ganodenic acid is most strong.
Under 8 various concentration of table, inducibility of the ganodenic acid to monocyte
2, the ganodenic acid for the use of concentration being 800ng/ml, forms dry referring to 7 the method induced monocyte of embodiment
Cell-like cell and M2 type macrophage pass through Flow cytometry monocyte marker in the 6th day harvest cell
CD14, stem cell labeling object CD90, M1 type macrophage marker CD16, M2 type macrophage marker CD68 and CD163
Expression, specific testing result is referring to fig. 4.The testing result of Fig. 4 proves that after ganodenic acid is added, monokaryon is thin from protein level
Born of the same parents can be induced to form the composition of stem cell-like cell and M2 type macrophage simultaneously.
The effect of 11 cell composition of embodiment treatment rheumatoid arthritis
1, the composition containing stem cell-like cell is prepared referring to 5 the method for embodiment；Reference 7 the method for embodiment,
The group containing stem cell-like cell and M2 type macrophage is formed using the ganodenic acid induced monocyte that concentration is 800ng/ml
Close object；48h is stimulated using 100 μ g/L IL-4, induction human peripheral blood mononuclear cell forms M2 type macrophage.
2,12 patients are convened, wherein male patient 6, female patient 6, average age is 59 years old, and the patient presses
According to rheumatism association of the U.S. (ACR) in 2009 and European rheumatism alliance (EULAR) diagnostic criteria, score value at 6 points or more, is made a definite diagnosis
For rheumatoid disease patient.
3, patient is randomly divided into 3 groups, wherein the 1st group of patient treats only with the composition containing stem cell-like cell, the 2nd
Group patient treats only with the composition of the macrophage of type containing M2, and the 3rd group of patient, which uses, contains stem cell-like cell and M2 type macrophage
The composition of cell is treated.In therapeutic process, in the 1st group, the dosage of the stem cell-like cell is 1 × 106A cell/
Kg；In 2nd group, the dosage of the M2 type macrophage is 1 × 106A cell/Kg；In 3rd group, the stem cell-like cell and
The dosage of M2 type macrophage is 1 × 106A cell/Kg.Treatment time is 5 months, at the 1st, 2,3,4 month point 4 of the course for the treatment of
Secondary input stem cell-like cell, M2 type macrophage or stem cell-like cell and M2 type macrophage.
4, before the course for the treatment of starts, the course for the treatment of after, the disease activity for carrying out 28 joints to patient scores (DAS28),
Specific testing result is referring to table 9.According to result shown in table 9 it is found that with stem cell-like cell or M2 type macrophage is used alone
It compares, the combination of stem cell-like cell and M2 type macrophage can significantly increase stem cell-like cell to rheumatoid arthritis
Repair.
The variation of the pretherapy and post-treatment DAS value of table 9
| Group | n | x±s | P |
| Before 1st group for the treatment of | 4 | 4.36±0.56 | |
| After 1st group for the treatment of | 4 | 3.95±0.49 | P < 0.05 |
| Before 2nd group for the treatment of | 4 | 4.47±0.67 | |
| After 2nd group for the treatment of | 4 | 4.21±0.34 | P < 0.05 |
| Before 3rd group for the treatment of | 4 | 4.60±0.37 | |
| After 3rd group for the treatment of | 4 | 3.17±0.23 | P < 0.05 |
Note, compared with pre-treatment, P < 0.05, difference is statistically significant.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that：Its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
Claims (32)
1. a kind of method of induced synthesis multipotential stem cell like cell, which is characterized in that the described method comprises the following steps：
1）Monocyte, the work of the M-CSF in the medium are cultivated in the culture medium containing Porcine HGF M-CSF
Concentration is 10 ~ 50ng/ml, and the time of the culture is 3 ~ 5 days；
2）In step 1）Later, the monocyte is cultivated in the culture medium containing M-CSF and Radix Ophiopogonis homoisoflavone, wherein
The working concentration of the M-CSF and Radix Ophiopogonis homoisoflavone in the medium is respectively 10 ~ 30ng/ml and 30 ~ 100 μ g/ml,
The incubation time is 4 ~ 8 days to get multipotential stem cell like cell.
2. a kind of method of induced synthesis multipotential stem cell like cell and M2 type macrophage simultaneously, which is characterized in that the side
Method includes the following steps：
1）Monocyte, the work of the M-CSF in the medium are cultivated in the culture medium containing Porcine HGF M-CSF
Concentration is 10 ~ 50ng/ml, and the time of the culture is 3 ~ 5 days；
2）In step 1）Later, it is thin that the monokaryon is cultivated in the culture medium containing M-CSF, Radix Ophiopogonis homoisoflavone and ganodenic acid
Born of the same parents, wherein the M-CSF, the working concentration of Radix Ophiopogonis homoisoflavone and ganodenic acid in the medium be respectively 10 ~
30ng/ml, 30 ~ 100 μ g/ml and 100 ~ 1000ng/ml, the incubation time be 4 ~ 8 days to get multipotential stem cell like cell and
M2 type macrophage.
3. method according to claim 1 or 2, which is characterized in that the method also includes：3）Harvest step 2）It is described more
Energy stem cell-like cell or multipotential stem cell like cell and M2 type macrophage, are made cell suspension.
4. according to the method described in claim 3, it is characterized in that, step 3）The multipotential stem cell like cell or described
Multipotential stem cell and M2 type macrophage, mechanically or mode of action is removed from the solid phase carrier that it adheres to.
5. according to the method described in claim 3, it is characterized in that, the method further includes by step 3）The cell is outstanding
Liquid freezes or is configured to pharmaceutical composition.
6. according to claim 1 ~ 2,4 ~ 5 described in any item methods, which is characterized in that the multipotential stem cell expresses versatility
Mark CD90.
7. according to claim 2,4 ~ 5 described in any item methods, which is characterized in that the M2 type Expression of Macrophages CD68,
One of CD163, CD14 and CD16 or a variety of.
8. the method according to the description of claim 7 is characterized in that M2 type the Expression of Macrophages CD68 and CD163.
9. according to claim 1 ~ 2,4 ~ 5 described in any item methods, which is characterized in that the Radix Ophiopogonis homoisoflavone is selected from 6- aldehyde
The different ophiopogon flavonoid A of base, methyl ophiopogon flavonoid B, the different ophiopogonone A of methyl ophiopogon flavonoid A, 6- aldehyde radical and the different wheat of 6- aldehyde radical
One of winter flavones B or a variety of.
10. according to the method described in claim 2, it is characterized in that, the ganodenic acid be selected from ganoderic acid, red sesame acid methyl esters,
Ganoderma lucidum alcohol, ganoderma lucidum oxalic acid, ganoderma lucidum olefin(e) acid, red sesame alcohol, ganoderma lucidum alcohol, ganoderal, epoxy ganoderma lucidum alcohol, ganoderma lucidum sterone, ganoderma lucidum terpinum,
Ganoderma lucidum terpene triol, ganoderma lucidum terpene ketone glycol, ganoderma lucidum terpene ketone triol, Reishi sporule acid, red sesame acid, red sesame terpene ketone, red sesame alcohol, red sesame terpene
One of alcohol, ganosporelactjone A, Ganolactone are a variety of.
11. according to claim 1 ~ 2,4 ~ 5 described in any item methods, which is characterized in that step 1）Described in M-CSF work
Concentration is 10,15,20,25,30,35,40,45 or 50ng/ml.
12. according to the method for claim 11, which is characterized in that the working concentration of the M-CSF is 30ng/ml.
13. according to claim 1 ~ 2,4 ~ 5 described in any item methods, which is characterized in that step 2）Described in M-CSF work
Concentration is 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30ng/ml.
14. according to the method for claim 13, which is characterized in that the working concentration of the M-CSF is 20ng/ml.
15. according to claim 1 ~ 2,4 ~ 5 described in any item methods, which is characterized in that step 2）In, the Radix Ophiopogonis Gao Yihuang
The working concentration of ketone is 30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 μ g/ml.
16. according to the method for claim 15, which is characterized in that the concentration of the Radix Ophiopogonis homoisoflavone is 60 μ g/ml.
17. according to the method described in claim 2, it is characterized in that, step 2）In, the work of the ganodenic acid in the medium
Making concentration is 100,150,200,250,300,350,400,450,500,600,650,700,750,800,850 or 900 ng/
ml。
18. according to the method for claim 17, which is characterized in that the working concentration of the ganodenic acid in the medium is
800ng/ml。
19. according to claim 1 ~ 2,4 ~ 5 described in any item methods, which is characterized in that step 1）In, the incubation time is
3,4 or 5 days.
20. according to the method for claim 19, which is characterized in that step 1）Described in incubation time be 3 days.
21. according to claim 1 ~ 2,4 ~ 5 described in any item methods, which is characterized in that step 2）Described in incubation time be
4,5,6,7 or 8 days.
22. according to the method for claim 21, which is characterized in that step 2）Described in incubation time be 4 days.
23. according to claim 1 ~ 2, any one of 4 ~ 5 the methods, which is characterized in that the monocyte be isolated from peripheral blood,
One of Cord blood, lymph and marrow are a variety of.
24. according to the method for claim 23, which is characterized in that the monocyte is isolated from peripheral blood.
25. a kind of composition, which is characterized in that the composition includes being made according to any one of claim 2 ~ 24 the method
Multipotential stem cell like cell and M2 type macrophage.
26. composition according to claim 25, which is characterized in that it further include drug in the composition, the drug
Selected from anti-inflammatory agent, antioxidant and one for the treatment of cell, tissue or organ dysfunction, damage or the drug of necrosis or
It is a variety of.
27. composition according to claim 26, which is characterized in that the anti-inflammatory agent is selected from steroidal anti-inflammatory drugs or non-steroid
Body anti-inflammatory drugs.
28. composition according to claim 27, which is characterized in that the steroidal anti-inflammatory drugs be selected from hydrocortisone,
One of dexamethasone or prednisone are a variety of, and the non-steroid anti-inflammatory drug is selected from aspirin, Diclofenac or cloth Lip river
One of sweet smell is a variety of.
29. composition according to claim 26, which is characterized in that the antioxidant is selected from selenium, ascorbic acid, dimension life
One of plain E, catechuic acid, lycopene, beta carotene, coenzyme q-10, EPA and DHA or a variety of.
30. composition according to claim 26, which is characterized in that the treatment cell, tissue or organ dysfunction,
Damage or necrosis drug be selected from insulin, Insulin-like growth factor II, secretin, melbine, hyaluronic acid, Simvastatin,
One of epidermal growth factor, interleukin-11, salinomycin and adriamycin are a variety of.
31. composition according to claim 25, which is characterized in that further include pharmaceutically acceptable in the composition
Carrier, the carrier are selected from one of blood plasma, serum, albumin, gel and physiological saline or a variety of.
32. application of the composition described in claim 25 in the drug of preparation treatment rheumatoid arthritis.
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