CN102757936A - Proliferation accelerator for human adipose-derived stem cells and application method thereof - Google Patents
Proliferation accelerator for human adipose-derived stem cells and application method thereof Download PDFInfo
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- CN102757936A CN102757936A CN2012101974172A CN201210197417A CN102757936A CN 102757936 A CN102757936 A CN 102757936A CN 2012101974172 A CN2012101974172 A CN 2012101974172A CN 201210197417 A CN201210197417 A CN 201210197417A CN 102757936 A CN102757936 A CN 102757936A
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Abstract
The invention discloses a proliferation accelerator for human adipose-derived stem cells and an application method thereof. The proliferation accelerator for the human adipose-derived stem cells comprises insulin, hydrocortisone, parathyroid hormone, estradiol, testosterone, a platelet-derived growth factor-AB, an epidermal growth factor, a basic fibroblast growth factor, L-glutamine, 2-mercaptoethanol, a leukemia inhibitory factor, L-ascorbic acid, biotin, dexamethasone, an IGF-I (Insulin-like Growth Factor-I) and a SCF (Stem Cell Factor). The proliferation accelerator for the human adipose-derived stem cells, disclosed by the invention, is applied to proliferation and subculture of the adipose-derived stem cells, can be used to not only specifically promote the growth sped and the proliferation quantity of the adipose-derived stem cells to increase in the form of geometric progression, but also suppress early differentiation and maturity of the adipose-derived stem cells so as to prevent apoptosis or premature senility of the stem cells during culturing and keep the vigorous and active differentiation potentials of the adipose-derived stem cells. The proliferation accelerator for the human adipose-derived stem cells, disclosed by the invention, has good application prospect in the field of culture of the adipose-derived stem cells.
Description
Technical field
The present invention relates to biotechnology and cell engineering field, particularly a kind of human adipose-derived stem cell enhancer of proliferation.
Background technology
Human adipose-derived stem cell is body fat mescenchymal stem cell (adipose-derived stem cells; ADSCs) be a kind of adult stem cell that is widely used in organizational project and regenerative medicine field at present, the same with mesenchymal stem cells MSCs have a multidirectional differentiation potential.ADSCs is the growth of inoblast appearance, and endochylema and kernel are abundant, is parallel or the arrangement of whirlpool appearance.Cell cycle analysis shows that the cell of G0/G1 phase accounts for 69%, and the S phase accounts for 24%, and the G2/M phase accounts for 8%.Under the existence condition of foetal calf serum, go down to posterity and cultivate 1 times of cell proliferation in 2-3 days.Repeatedly go down to posterity after (10-20 generation), cell proliferation rate does not have obviously and slows down, and senile cell occurs in the cell colony after 6 times going down to posterity, and senile cell accounts for 15% [3] in the 15th generation cell colony.ADSCs has and the essentially identical cellular immunization phenotype of mesenchymal stem cells MSCs, through flow cytometry analysis CD9, CD10, CD13, CD29; CD44, CD49d, CD49e, CD54, CD55; CD59, CD90, CD105, CD117, CD146; CD166, STRO-1 are all positive, and CD31, CD34, CD45 are all negative.Maximum difference is that ADSCs expresses CD49d, does not express CD106; And just in time opposite, BMSCs expresses CD106, does not express CD49d.
ADSCs isolation cultivation method at present commonly used is to separate from fatty tissue to obtain cell, remove mature cell such as red corpuscle through machinery and method of enzymatically treating after, the substratum that utilizes feeder layer cells and employing to contain 10% foetal calf serum is cultivated.The shortcoming of this culturing stem cells method is that method is complicated, and the stem cell population of extraction is few, and purity is not high, and shoot proliferation is slow.
Summary of the invention
The purpose of this invention is to provide a kind of human adipose-derived stem cell enhancer of proliferation, be used to improve shoot proliferation efficient.
The shortcoming of culturing stem cells method commonly used is that method is complicated, and the stem cell population of extraction is few, and purity is not high, and shoot proliferation is slow.
According to an aspect of the present invention; A kind of human adipose-derived stem cell enhancer of proliferation is provided, it is characterized in that: form by Regular Insulin, HYDROCORTISONE INJECTIONS, Rat parathyroid hormone 1-34, Theelin,dihydro-, testosterone, Thr6 PDGF BB-AB, Urogastron, Prostatropin, L-glutaminate, 2 mercapto ethanol, LIF, L-xitix, vitamin H, DEXAMETHASONE BP98, insulin-like growth factor I GF-I and human stem cell factor SCF.After adding the human adipose-derived stem cell substratum in the human adipose-derived stem cell enhancer of proliferation each composition activity to be followed successively by insulin concentration be that 0.1 ~ 10 μ g/mL, HYDROCORTISONE INJECTIONS concentration are 10
-10~ 5 * 10
-9Mol/L, Rat parathyroid hormone 1-34 concentration are 2 * 10
-10~ 7 * 10
-10Mol/L; Estradiol concentration 0.2 ~ 3.5ng/mL; Testosterone concentration is 0.8 ~ 15ng/mL; Thr6 PDGF BB-AB concentration is 1 ~ 8ng/mL; Urogastron concentration is 2 ~ 16ng/mL; Prostatropin concentration is 2 ~ 20ng/mL; The L-glutaminate preferred concentration is 0.4 ~ 4mmol/L; 2 mercapto ethanol concentration is 0.07 ~ 0.15mmol/L; LIF concentration is 1 ~ 7ng/mL; The L-ascorbic acid concentrations is 10 ~ 80 μ g/mL; Biotin concentration is 10 ~ 45 μ g/mL; Dexamethasone concentration is 10
-8~ 5 * 10
-8Mol/L, IGF-I concentration are that 1 ~ 10ng/mL and SCF concentration are 0.8 ~ 15ng/mL.Wherein the Regular Insulin most preferable concentrations is 6.25 μ g/mL, and the HYDROCORTISONE INJECTIONS most preferable concentrations is 1 * 10
-9Mol/L, the Rat parathyroid hormone 1-34 most preferable concentrations is 5 * 10
-10Mol/L, the Theelin,dihydro-most preferable concentrations is 1ng/mL, the testosterone most preferable concentrations is 4ng/mL; Thr6 PDGF BB-AB most preferable concentrations is 4ng/mL, and the Urogastron most preferable concentrations is 10ng/mL, and the Prostatropin most preferable concentrations is 10ng/mL; The L-glutaminate most preferable concentrations is 2mmol/L; The 2 mercapto ethanol most preferable concentrations is 0.1mmol/L, and the LIF most preferable concentrations is 5ng/mL, and L-xitix most preferable concentrations is 50 μ g/mL; The vitamin H most preferable concentrations is 33 μ g/mL, and the DEXAMETHASONE BP98 most preferable concentrations is 10
-8Mol/L, the IGF-I most preferable concentrations is 5ng/mL, it is 5ng/mL that SCF forms most preferable concentrations.
It is following to hormone and the various growth factor effect of keeping the cell index growth that the present invention provides:
Insulin Regular Insulin: can promote cell to utilize glucose and amino acid
Hydrocortisone HYDROCORTISONE INJECTIONS: stimulate the growth of stem cell, guarantee cell function
Parathyroid hormone Rat parathyroid hormone 1-34: the growth that stimulates stem cell
Estradiol Theelin,dihydro-: stimulate the growth of stem cell, guarantee cell function
Testosterone testosterone: stimulate the growth of stem cell, guarantee cell function
PDGF-AB Thr6 PDGF BB-AB: mitogen can stimulate inoblast and other various kinds of cell division growths.
EGF Urogastron: have intensive to urge splitting action to multiple histocyte
The bFGF Prostatropin: can stimulate cellular proliferation, move, induce plasminogen activator and collagenase activities, be the cell mitogen that high-affinity is arranged with heparin.
L-Glutamine L-glutaminate: increase the volume of cell, promote cell enlargement
2-Mercaptoethanol 2 mercapto ethanol: can protect the sulfydryl in desmo enzyme and the protein not oxidized, promote the adherent of cell and propagation.
The LIF LIF: have functions such as suppressing differentiation of stem cells, the stem cell that is used for keeping cultivation is in undifferentiated state.
L-ascorbic acid L-xitix: inhibitor all can promote the growth of stem cell, increases surviving rate.
The biotin vitamin H: vitamin H is known as vitamin H again, belongs to water-soluble vitamin B group family, participates in helping the synthetic normally and metabolism of material in the adipocyte.Promote stem cell growth.
Dexamethasone DEXAMETHASONE BP98: belong to adrenal cortex hormones drug, in cell cultivation process, play the effect of growth factor, promote the proliferation and differentiation of cell.
IGF-I rhIGF-1: be one type of multi-functional cell proliferation regulatory factor.In the differentiation of cell, propagation, individual growing, has important promoter action.
SCF human stem cell factor: be a kind of I type transmembrane glycoprotein of wide expression, can promote stem cell to become to live, quicken propagation.
According to an aspect of the present invention, a kind of human adipose-derived stem cell enhancer of proliferation that provides comprises keeping the hormone and the various growth factor of cell index growth; The propagation that the is used for fat stem cell cultivation of going down to posterity; Not only can specificity promote the fat stem cell speed of growth and amplification quantity to increase by geometric progression, suppress its too early differentiation and maturation again, avoid stem cell apoptosis or early ageing in cultivation; Keep the potential of the vigorous active differentiation of fat stem cell, promote the stem cell that extracts to become to live.
Description of drawings
Fig. 1 is the basic medium and the base culture base human adipose-derived stem cell growth curve chart that does not add the human adipose-derived stem cell enhancer of proliferation that adds the human adipose-derived stem cell enhancer of proliferation among the present invention;
Fig. 2 is the form of cell when using common basic medium group culturing human fat stem cell to cultivate 6 days among the present invention;
Fig. 3 is the form of cell when end user's fat stem cell enhancer of proliferation culturing human fat stem cell is cultivated 6 days among the present invention;
Fig. 4 is end user's fat stem cell enhancer of proliferation culturing human fat stem cell human adipose-derived stem cell telomerase activation detected result among the present invention.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.Testing used utensil instrument reagent all can obtain through commercial sources.
Embodiment 1: the preparation of the general population's fat stem cell basic medium:
Get DMEM/F12 cell culture fluid (buying) added with antibiotic of 900mL: penicillium mould 90000U, Streptomycin sulphate 90000U from TAKARA BIO INC..
Adjust pH: use massfraction 5%NaHCO
3Regulate pH to 7.2.
Filtration sterilization: adopt each one of aperture 0.45 μ m and 0.22 μ m filter membrane, the upper strata is 0.45 μ m, and lower floor is 0.22um, to guarantee filter effect.
Add calf serum: face with before will add calf serum and be settled to 1000mL (buying) from TAKARA BIO INC..
Embodiment 2: prepare a kind of proliferated culture medium A that contains the human adipose-derived stem cell enhancer of proliferation
Predetermined amount is the DMEM/F12 cell culture fluid (TAKARA BIO INC.) that 1000mL then gets 900mL, added with antibiotic: penicillium mould 90000U, Streptomycin sulphate 90000U.
Adjust pH: use massfraction 5%NaHCO
3Regulate pH to 7.2.
Add enhancer of proliferation: composition shown in the according to the form below 1 also joins in the above-mentioned DMEM/F12 cell culture fluid for preparing with the amount weighing successively, and vibration or ultrasonic hydrotropy do not heat hydrotropy.
Table 1
Then go up each composition concentration in proliferated culture medium A shown in the table and be followed successively by Regular Insulin 0.1 μ g/mL, HYDROCORTISONE INJECTIONS 10
-10Mol/L, Rat parathyroid hormone 1-34 2 * 10
-10Mol/L, Theelin,dihydro-0.2ng/mL, testosterone 0.8ng/mL, Thr6 PDGF BB-AB 1ng/mL, Urogastron 2ng/mL, Prostatropin 2ng/mL, L-glutaminate 0.4mmol/L, 2 mercapto ethanol 0.07mmol/L, LIF 1ng/mL, L-xitix 10 μ g/mL, vitamin H 10 μ g/mL, DEXAMETHASONE BP98 10
-8Mol/L, IGF-I 1ng/mL and SCF 0.8ng/mL
Filtration sterilization: adopt each one of aperture 0.45um and 0.22um filter membrane, the upper strata is 0.45um, and lower floor is 0.22um, to guarantee filter effect.
Add calf serum: face with before will add calf serum and be settled to 1000mL (buying) from TAKARA BIO INC..
Embodiment 3: prepare a kind of proliferated culture medium B that contains the human adipose-derived stem cell enhancer of proliferation
Preparing method and predetermined amount such as embodiment 2, but during weighing with each composition in the table 1 by making its concentration in proliferated culture medium B be followed successively by Regular Insulin 10 μ g/mL, HYDROCORTISONE INJECTIONS 5 * 10
-9Mol/L, Rat parathyroid hormone 1-34 7 * 10
-10Mol/L, Theelin,dihydro-3.5ng/mL, testosterone 15ng/mL, Thr6 PDGF BB-AB8ng/mL, Urogastron 16ng/mL, Prostatropin 20ng/mL, L-glutaminate 4mmol/L, 2 mercapto ethanol 0.15mmol/L, LIF 7ng/mL, L-xitix 80 μ g/mL, vitamin H 45 μ g/mL, DEXAMETHASONE BP98 5 * 10
-8The amount weighing of mol/L, IGF-I10ng/mL and SCF 15ng/mL.
Embodiment 4: the proliferated culture medium C of preparation human adipose-derived stem cell enhancer of proliferation
Preparing method and predetermined amount such as embodiment 2, but during weighing with each composition in the table 1 by making its concentration in proliferated culture medium C be followed successively by Regular Insulin 6.25 μ g/mL, HYDROCORTISONE INJECTIONS 1 * 10
-9Mol/L, Rat parathyroid hormone 1-34 5 * 10
-10Mol/L, Theelin,dihydro-1ng/mL, testosterone 4ng/mL; Thr6 PDGF BB-AB 4ng/mL, Urogastron 10ng/mL, Prostatropin 10ng/mL; L-glutaminate 2mmol/L, 2 mercapto ethanol 0.1mmol/L, LIF 5ng/mL; L-xitix 50 μ g/mL, vitamin H 33 μ g/mL, DEXAMETHASONE BP98 10
-8Mol/L, IGF-I5ng/mL, the amount weighing of SCF 5ng/mL.
Embodiment 5: utilize multiplication agent culturing cell of the present invention and test
Use above-mentioned 4 kinds of culture medium culturing human adipose-derived stem cells
1. with 1 * 10
6Individual fat stem cell is inoculated in four groups of diameter 100mm petridish that fill different culture medium respectively and mixing, and every group of 10 petridish place observation inverted microscope under;
2. adding substratum:
10 petridish of control group add ordinary culture medium 5mL among the embodiment 1;
A organizes 10 petridish, adds proliferated culture medium A5mL;
B organizes 10 petridish, adds proliferated culture medium B5mL;
C organizes 10 petridish, adds proliferated culture medium C5mL.
3. cell is placed at 37 ℃ 5%CO
2Incubator in cultivate; Inverted microscope is observed, and whenever removes the upper strata substratum at a distance from 3 days with the transfer pipet suction, adds new substratum, continues to cultivate.
4. cell counting: get a petridish and put into clean bench every day in each group, inhales with transfer pipet then and abandon substratum.PBS damping fluid washing 2 times, adding 1mL trypsinase complex body Trypsin-EDTA after inverted microscope finds that down attached cell separates, adds the basic medium termination pancreatin effect that 4mL contains 10% Ox blood serum FBS.
Use trypan blue (Typan Blue) dyeing blood cell counting count board living cell counting number.
5. experimental result is as shown in Figure 1, uses proliferated culture medium C can very quicken the breeding of fat stem cell effectively.Use the approximate serpentine of cell growth curve of common basic medium and proliferated culture medium A, be in residence time, then get into logarithmic phase, reached the climax about the 6th day at the preceding 3d cell of inoculating.Cell enlargement deceleration afterwards gets into platform area, increases to the 9th day apoptotic cells, and cell dishful rate reaches more than 90%, but uses proliferated culture medium A still to be superior to common basic medium.Use the human adipose-derived stem cell of high dosage (B group) and most preferred dose (C group) cell proliferating agent to breed from becoming to live and quickening very soon; Show that residence time is very short; Only just begin rapid division growth less than time of two days; Get into increased logarithmic phase in advance than ordinary culture medium group cell, the growth number is 5-6 times of ordinary culture medium group approximately.The cell enlargement rate of high dose group and most preferred dose group that rose by the 5th day is distinguished, 7 days values of peaking of the cell sustainable growth to the of most preferred dose group, and cell quantity reaches 7x10
7, after this getting into platform area, 9 days beginnings were seen the minority apoptotic cell.And the cell of high dose group is at most only long to 5x10
7, after this apoptotic cell constantly increases, cell quantity decrescence, later this group cell quantity and low dose group and common basic medium did not have significant difference by the 8th day.So the high dosage multiplication agent can not obtain the effect of best human adipose-derived stem cell propagation, C group dosage promotes the stem cell breeding to increase and did not occur a large amount of apoptosis in 10 days in vitro culture.
The security test of cell proliferating agent culturing human fat stem cell
1 cellular form:
Beginning is adherent behind the 4h of isolated cells inoculation just, and the cellular form of which group cell in lag phase all is small circular, and nucleus is placed in the middle, double-core or multinuclear can occur.Cell occurs stretching growth after getting into the rise period, and cell enlargement is obviously accelerated, and is changes such as fusiform, polygon, and fusiformis and fibrocyte shape form appear in visible attached cell when then getting into the peak period.Four groups of cells show cell quantity notable difference (Fig. 2, Fig. 3) identical only on sometime on the form, and it is intensive and active still to highlight the fat stem cell cell growth of using C dosage of cells multiplication agent.
2 surface antigen analyses:
The lipfanogen of the getting cultivation foster stem cell of being commissioned to train is removed nutrient solution, and 2.5% trypsin solution and the mixture slaking of 0.02%EDTA solution with volume ratio 1: 1 contain 1 * 10 with processing every 100ul after the PBS washing that contains 1% bovine serum albumin (BSA)
6The single cell suspension of individual cell; Be divided into 7 parts; Add respectively in 7 Eppendorf pipes; Pipe add that FITC Mouse IgG1, APC-CY7Mouse IgG2b and the dyeing damping fluid of totally 20 μ L are used to detect since the background that the antibody non-specific binding produces as contrast, other test tubes add CD29, CD34, CD44, CD45, CD105, each 20 μ L of HLA.DR monoclonal antibody respectively, every pipe adds cell suspension 100 μ L respectively and (contains 1 * 10
6Individual cell), incubated at room 25min, after the PBS washing that contains 1%BSA, flow cytometer detects.
Flow cytometry analysis human adipose-derived stem cell phenotype of former generation: CD29+ (77.5%), CD34-(3.3%), CD44+ (26.8%), CD45-(3.8%), CD105+ (30.1%), HLA.DR-(2.0%) shows that this cell has the stem cell characteristic.HLA.DR expresses not obvious, and getting rid of such cell is inoblast.
The mensuration of 3 propagation Telomerase Activity:
Detect the human adipose-derived stem cell of vitro culture more than 6 days with TeloTAGGG Telomerase PCR ELISA test kit (Chemocon Inc.) by specification (TRAP method); Simultaneously with the positive contrast of HEKC's 293 cell strain cells (293T), the negative contrast of Interferon, rabbit (providing in the test kit).Detection method is following, gets 2 * 10
6Individual cell; Lysing cell is got the PCR that centrifugal supernatant 2 μ L carry out the reaction of telomerase catalytic specific substrate, and the specific probe with the DIG mark after the sex change of PCR product is hybridized; Then, measure ELISA result at ELIASA with the capable ELISA reaction of anti-DIG antibody of coupling px.Measuring wavelength is 450nm and 690nm absorbancy (A) value, absorbance difference △ A=A450-A690, and △ A represents telomerase activation.Judgement criteria as a result: positive control △ A>1.5, negative control △ A < 0.25, testing sample △ A value deducts negative control △ A value, △ A>0.2 is positive.Adopt the SAS9.0 software package to carry out interpretation of result, measured value is represented with means standard deviation.
The human adipose-derived stem cell sample measured value of using optimum dosage of cells multiplication agent to cultivate is that the 6th day human adipose-derived stem cell is (0.050 ± 0.002); Human adipose-derived stem cell was (0.055 ± 0.012) in the 10th day; Positive control 293 cells are 2.1, and the negative control Interferon, rabbit is 0.048 (Fig. 4).
Telomere is the special construction that end of chromosome is made up of TTAGGG Tumor-necrosis factor glycoproteins and relevant conjugated protein, and is closely related with the multiple fission ability of cell.Proved that about 80% malignant tumour and immortalized cell line cell (like positive control 293 cells of this experiment) express high-caliber telomerase activation, and normal somatic cell Telomerase Expression level is very low.Therefore, no matter what proliferated culture medium was cultivated is the most vigorous the 6th day and the 10th day of continuing to breed in growth, and the cell telomerase activation remains normal level, explains that cultured cells is normal, does not have canceration.
The chromosome karyotype analysis of 4 human adipose-derived stem cells:
Respectively get the 6th day, the 10th day the fat stem cell that multiplication agent is cultivated, add the NST-757 (whole mass concentration is 0.04mg/L) of 5g/L, place 37 ℃, 5% (volume(tric)fraction) CO then
2Cultivate 5h in the incubator, digestion, centrifugal adds 0.075%kcl, leaves standstill 20min, and adding a little stationary liquid (10% Superlysoform), to pre-fix the back centrifugal, and fixing 30min is centrifugal then, drips sheet, the Giemsa dyeing of dry back, oven dry, microscopically observation.More than each method with different sample cell repeated experiments 3 times.The result shows: use this patent cell proliferating agent under the condition of cultured and amplified in vitro, ANOMALOUS VARIATIONS such as the karyotype of human adipose-derived stem cell transposition, inversion does not take place, lack or duplicates, fusion.The abnormal change of karyotype and cancer substantial connection arranged.Present research has confirmed that the generation of tumour and cell chromosome reciprocity, disappearance, insertion and inversion cause oncogene activation expression of chromosome specific breaking point zone or cancer suppressor gene disappearance, cause existing necessary relation between the change of correlative coding abnormal protein and cytobiology proterties.Therefore, after separation and Culture, amplification number generation, whether its karyotype abnormal change takes place human adipose-derived stem cell under vitro conditions, this and its as the security of tissue engineering seed cell important relation is arranged.This patent experimental result shows: cultivated in many days through cell proliferating agent, ANOMALOUS VARIATIONS such as the human adipose-derived stem cell caryogram transposition, inversion does not all take place, lack or duplicates, fusion are normal cell caryogram.
Therefore; Use the cell proliferating agent of this patent invention stimulate human adipose-derived stem cell external breed with fission process in its Telomerase be to be negative or to be lower than the low expression level of TRAP method detection level, and unlike the such high expression level telomerase activation that continues of tumour cell.In addition, also no abnormality seen change of its karyotype.The human adipose-derived stem cell of this research indication vitro culture possibly be safe, for it further is applied to studies safety indexes and theoretical basis is provided.
Above-described only is embodiments more of the present invention.For the person of ordinary skill of the art, under the prerequisite that does not break away from the invention design, can also make some changes and improvement, these all belong to protection scope of the present invention.
Claims (8)
1. a human adipose-derived stem cell enhancer of proliferation is characterized in that: be made up of Regular Insulin, HYDROCORTISONE INJECTIONS, Rat parathyroid hormone 1-34, Theelin,dihydro-, testosterone, Thr6 PDGF BB-AB, Urogastron, Prostatropin, L-glutaminate, 2 mercapto ethanol, LIF, L-xitix, vitamin H, DEXAMETHASONE BP98, insulin-like growth factor I GF-I and human stem cell factor SCF.
2. human adipose-derived stem cell enhancer of proliferation as claimed in claim 1 is characterized in that: each components in proportions is that 0.1 ~ 10 μ g/mL, HYDROCORTISONE INJECTIONS concentration are 10 for making after adding the human adipose-derived stem cell substratum in the human adipose-derived stem cell enhancer of proliferation each composition activity be followed successively by insulin concentration
-10~ 5 * 10
-9Mol/L, Rat parathyroid hormone 1-34 concentration are 2 * 10
-10~ 7 * 10
-10Mol/L; Estradiol concentration 0.2 ~ 3.5ng/mL; Testosterone concentration is 0.8 ~ 15ng/mL; Thr6 PDGF BB-AB concentration is 1 ~ 8ng/mL; Urogastron concentration is 2 ~ 16ng/mL; Prostatropin concentration is 2 ~ 20ng/mL; The L-glutaminate preferred concentration is 0.4 ~ 4mmol/L; 2 mercapto ethanol concentration is 0.07 ~ 0.15mmol/L; LIF concentration is 1 ~ 7ng/mL; The L-ascorbic acid concentrations is 10 ~ 80 μ g/mL; Biotin concentration is 10 ~ 45 μ g/mL; Dexamethasone concentration is 10
-8~ 5 * 10
-8Mol/L, IGF-I concentration are that 1 ~ 10ng/mL and SCF concentration are 0.8 ~ 15ng/mL.
3. human adipose-derived stem cell enhancer of proliferation as claimed in claim 1; It is characterized in that: each components in proportions is 6.25 μ g/mL for making after adding the human adipose-derived stem cell substratum in the human adipose-derived stem cell enhancer of proliferation each composition activity be followed successively by insulin concentration, and HYDROCORTISONE INJECTIONS concentration is 1 * 10
-9Mol/L, Rat parathyroid hormone 1-34 concentration is 5 * 10
-10Mol/L, estradiol concentration is 1ng/mL, testosterone concentration is 4ng/mL; Thr6 PDGF BB-AB concentration is 4ng/mL, and Urogastron concentration is 10ng/mL, and Prostatropin concentration is 10ng/mL; L-glutaminate concentration is 2mmol/L, and 2 mercapto ethanol concentration is 0.1mmol/L, and LIF concentration is 5ng/mL; The L-ascorbic acid concentrations is 50 μ g/mL, and biotin concentration is 33 μ g/mL, and dexamethasone concentration is 10
-8Mol/L, IGF-I concentration is 5ng/mL, SCF concentration is 5ng/mL.
4. the application method of each described human adipose-derived stem cell enhancer of proliferation among the claim 1-3 is characterized in that: each composition of human adipose-derived stem cell enhancer of proliferation is added respectively in the substratum of human adipose-derived stem cell.
5. the application method of a kind of human adipose-derived stem cell enhancer of proliferation according to claim 4 is characterized in that adding in the substratum of human adipose-derived stem cell the human adipose-derived stem cell of its cultivation is bred rapidly.
6. the application method of a kind of human adipose-derived stem cell enhancer of proliferation according to claim 4 is characterized in that: to be followed successively by insulin concentration be that 0.1 ~ 10 μ g/mL, HYDROCORTISONE INJECTIONS concentration are 10 to each composition activity in the human adipose-derived stem cell enhancer of proliferation
-10~ 5 * 10
-9Mol/L, Rat parathyroid hormone 1-34 concentration are 2 * 10
-10~ 7 * 10
-10Mol/L; Estradiol concentration 0.2 ~ 3.5ng/mL; Testosterone concentration is 0.8 ~ 15ng/mL; Thr6 PDGF BB-AB concentration is 1 ~ 8ng/mL; Urogastron concentration is 2 ~ 16ng/mL; Prostatropin concentration is 2 ~ 20ng/mL; The L-glutaminate preferred concentration is 0.4 ~ 4mmol/L; 2 mercapto ethanol concentration is 0.07 ~ 0.15mmol/L; LIF concentration is 1 ~ 7ng/mL; The L-ascorbic acid concentrations is 10 ~ 80 μ g/mL; Biotin concentration is 10 ~ 45 μ g/mL; Dexamethasone concentration is 10
-8~ 5x10
-8Mol/L, IGF-I concentration are that 1 ~ 10ng/mL and SCF concentration are 0.8 ~ 15ng/mL.
7. the application method of a kind of human adipose-derived stem cell enhancer of proliferation according to claim 4 is characterized in that: the Regular Insulin optimal concentration is 6.25 μ g/mL, and the HYDROCORTISONE INJECTIONS optimal concentration is 1 * 10
-9Mol/L, the Rat parathyroid hormone 1-34 optimal concentration is 5 * 10
-10Mol/L, the Theelin,dihydro-optimal concentration is 1ng/mL, the testosterone optimal concentration is 4ng/mL; Thr6 PDGF BB-AB optimal concentration is 4ng/mL, and the Urogastron optimal concentration is 10ng/mL, and the Prostatropin optimal concentration is 10ng/mL; The L-glutaminate optimal concentration is 2mmol/L; The 2 mercapto ethanol optimal concentration is 0.1mmol/L, and the LIF optimal concentration is 5ng/mL, and L-xitix optimal concentration is 50 μ g/mL; The vitamin H optimal concentration is 33 μ g/mL, and the DEXAMETHASONE BP98 optimal concentration is 10
-8Mol/L, the IGF-I optimal concentration is 5ng/mL, it is 5ng/mL that SCF forms optimal concentration.
8. the application method of a kind of human adipose-derived stem cell enhancer of proliferation according to claim 4 is characterized in that: the substratum of described human adipose-derived stem cell is the DMEM/F12 cell culture medium.
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