CN109022357A - A kind of dedicated estradiol culture medium of culture mescenchymal stem cell and its application - Google Patents
A kind of dedicated estradiol culture medium of culture mescenchymal stem cell and its application Download PDFInfo
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Abstract
The invention discloses a kind of dedicated estradiol culture medium of culture mescenchymal stem cell and its applications.The culture medium further includes estradiol.Based on the mescenchymal stem cell that this culture medium culture obtains, the significant raising of enzyme HADCY2 expression of cAMP is synthesized.Experiment in vitro confirms that its phenotype of the MSC of Estrogenization is constant, but the speed of growth obviously increases, and immune suppression function and adjusting endothelial cell are significantly enhanced at vessel patency.Therefore, estradiol can be used for placenta source MSC culture and for placenta source MSC function enhance the pre-stimulation factor, make its clinical treatment and in terms of play more effectively effect.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of dedicated estradiol culture medium of culture mescenchymal stem cell and
It is applied.
Background technique
Mescenchymal stem cell (MSC) is widely used in clinical tissue reparation, clinic organ transplantation, autoimmune disease treatment and beauty at present
Etc. industries.And placenta is main source for mesenchymal stem cells organ, including amnion MSC, decidua MSC and villus center
(Villous core) MSC etc..Although the mescenchymal stem cell of separate sources has similitude in terms of molecular marker expression,
It is that there are apparent organ specificities in terms of many functions.This shows that the microenvironment of mescenchymal stem cell existence is dry to mesenchyma
The function of cell has vital influence.
Placenta derives from gravid woman.And during pregnancy, the lasting raising of the estrogen level of women.Some pregnant
It is pregnent in related disease (such as preeclampsia), the estrogen level of women is significantly lower than the estrogen level of normal pregnancies.With this
The consistent variation of phenomenon is, in placenta in preeclampsia, the mescenchymal stem cell in placenta source growth, immune suppression function and
It is significantly reduced in terms of adjusting vascularization ability.This shows that estrogen may be grown between the mescenchymal stem cell for maintaining placenta
With Function important function.
Application number 201310180222.1, a kind of entitled human mesenchymal stem cell culture medium.Culture medium of the invention,
Following components is added in basal medium, each component is final concentration of: 1~2g/L of human serum albumins, transferrins 5~
10mg/L, 2~8mg/L of fibronectin, laminin 1~4mg/L, Fe (NO3) 39H2O50g/L, FeSO4
7H2O417g/L, 1~3 μ g/L of estradiol, 2~5 μ g/L of testosterone, 1~3 μ g/L of progesterone, 39.25~117.74 μ g/ of dexamethasone
L, 5~10mg/L of insulin, riboflavin 376.36mg/L, 80.96~242.87mg/L of coacetylase, 4.41~6.17mg/ of butanediamine
L, 1~2mg/L of taurine, 0.61~1.85mg/L of ethylaminoethanol, 8.81~26.42mg/L of pyruvic acid, sodium selenate 3.78~
7.56 μ g/L, 292.3~584.6mg/L of L-Glutamine, 2~8 μ g/L of vascular endothelial growth factor, epidermal growth factor 4~
10 μ g/L, 4~10 μ g/L of basic fibroblast growth factor, 1~5 μ g/L of LIF ELISA, insulin-like growth factor
μ g/L of son-I1~5,2~8 μ g/L of stem cell factor composition.Culture medium of the invention is free of animal blood serum, eliminates animal blood
Potential animal derived endotoxin or virus in clear, it is convenient to be applied to clinic.The purpose of this patent is to provide one for mescenchymal stem cell
The culture medium of kind serum-free.There are many ektogenic of addition, but do not have theoretical foundation, and it is dry may to be not suitable for all mesenchymas
Cell type.Such as.Although also there is estradiol addition in the inside, there are also progestin components.It is well known that estradiol and progesterone are
A pair has the hormone of antagonism, and the two is made an addition to simultaneously in culture medium may be improper.In addition, the hormone of above-mentioned patent addition
And there are many cell factor type, considerably increase cell culture cost, limit its extensive use.
But we but often ignore the addition of estrogen when being separately cultured placenta derived mesenchymal stem cell in vitro,
Caused result may be to slow down the growth of placenta derived mesenchymal stem cell, cause cell senescence, or even change mesenchyma
The biological function of stem cell.We have found that the mescenchymal stem cell of Estrogenization synthesizes cAMP by gene expression profile
Enzyme HADCY2 expression significant increase.We experiment in vitro confirm that its phenotype of the mescenchymal stem cell of estradiol processing is constant, but
It is that the speed of growth obviously increases, immune suppression function and adjusting endothelial cell are significantly enhanced at vessel patency.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of dedicated estradiol of culture mescenchymal stem cell
Culture medium and its application, the growth of mesenchymal stem cells speed that the culture medium culture containing estradiol obtains is fast, and comprehensive performance is strong,
Gained mescenchymal stem cell is suitable for preparing clinical treatment or beauty class product.
A kind of dedicated estradiol culture medium of culture mescenchymal stem cell, including basal medium, further include estradiol, institute
The concentration for stating estradiol is 2.7-27.3 μ g/L.
It is that it further includes nutridoma 0.2- that the basal medium, which is DMEM-F12 basal medium, as improved
1mg/ml, dual anti-100 units of mycillin/ml, pyruvic acid 5-20mg/L, L-Glutamine 3-6mg/ml.
It is as improved, the source for mesenchymal stem cells is in placenta.
Application of the above-mentioned mescenchymal stem cell in preparation treatment eclampsia drug or beauty product.
A kind of drug for treating eclampsia, active constituent are the obtained mescenchymal stem cell of above-mentioned culture, concentration is 1 ×
105-1×107A/ml.
A kind of anti-aging beauty-care liquid, active constituent are the mescenchymal stem cell that above-mentioned culture obtains, and concentration is 1 × 105-
1×107A/ml.
The utility model has the advantages that
Compared with prior art, estradiol is added after the MSC culture medium of placenta, the speed of growth of MSC is accelerated, and table
Reveal and stronger immunosuppression capability and be adjusted to vessel patency, in addition, the clinical treatment drug made of MSC of the invention or
Effect on beauty product is obvious.Function of the estrogen for placenta source MSC enhances the pre-stimulation factor, promotes MSC cell
Immunosuppression capability and rush vascularization ability.
Detailed description of the invention
Fig. 1 is that gene microarray analysis E2 handles changes in gene expression situation after MSC, wherein A is differential gene
Heatmap figure, B are that the cyberrelationship of differential gene is analyzed；
Fig. 2 is the expression of MSC surface marker after E2 processing, wherein A CD11b, B CD14, C CD19, D CD106, E
For CD29, F CD44, G CD7, H CD90；
Fig. 3 is the performance change that E2 handles MSC, wherein A is MSC proliferative capacity, and B is MSC cell cycle analysis, and C is on MSC
Clearly to HUVEC at the influence of blood vessel, D is mixing lymph experiment, inhibiting effect of the analysis MSC to T cell.
Specific embodiment
1 gene microarray analysis E2 of embodiment handles changes in gene expression situation after MSC.
1, material and method
1.1 main agents
Hank ' s balanced salt solution, estradiol, pancreatin, clostridiopetidase A, hyaluronidase, DNase, cell separating liquid, Percoll,
Trizol, library kit is built；Reagent used is conventional reagent.
1.2 key instrument
Agilent 2100 bioanalyzer、 RNA LabChip® kits、 Agilent microarray scanner
1.3 main method
1.3.1 placenta source MSC is separately cultured
Placenta tissue derives from Nanjing drum tower hospital gynemetrics Normal Parturients.Experimental program is in 2010 through Nanjing drum tower hospital human relations
The reason committee ratifies and signs informed consent form.The aseptically placenta between clip placenta tissue decidua layer and rete layer
Tissue, lightly to scrape chorionic villi after sterile PBS repeated flushing placenta tissue.With sterile PBS repeated flushing chorionic villi
To pink colour, it is eliminated as much as macroscopic thin vessels and fiber coordinator.
Treated chorionic villi with 800rpm is centrifuged 2 min, discards supernatant, then with sterile PBS repeated flushing,
It is centrifuged again, abandons supernatant.Clostridiopetidase A is added according to the amount that chorionic villi and the mass volume ratio (g/ml) of collagenase type I are 1:6, is set
25 min are digested under 37 °C of water-baths, the revolving speed of 175rpm.It inhales and abandons supernatant, according to chorionic villi and 0.05g/100ml
The amount that the mass volume ratio (g/mL) of Trypsin is 1:6 adds 0.05% Trypsin, is placed in 37 °C of water-baths, 175 rpm/
Min is incubated for until there is interstitial cell, and then Aspirate supernatant, 0.05% Trypsin is added again and is digested, until occurring
A large amount of interstitial cell, after transfer supernatant, centrifugation, resuspension, 100 mesh stainless steel mesh sievings processing is spread after Percoll sorting
Plate culture.
1.3.2 gene microarray analysis after estradiol processing MSC
After equal mescenchymal stem cells reach the third generation, streaming confirms that its purity is 99%.With 10-8M estradiol (E2, Sigma,
USA) after processing for 24 hours, total serum IgE is collected.Unused E2 processing as a control group, each 4 repetitions of E2 processing group.RNA Quality Identification
Afterwards, subsequent gene microarray analysis is carried out.After obtaining data, bioinformatic analysis is carried out.
2, result
Above-mentioned processing is analyzed, as a result as shown in 1A, after E2 handles mescenchymal stem cell, there is series of genes expression hair
It is raw to change；Show that HADCY2 mediates effect of the E2 to MSC from the bioinformatic analysis of Figure 1B again.
2 E2 of embodiment does not influence the expression of mescenchymal stem cell surface marker.
1, material, reagent, equipment
1.1 main agents
Pancreatin, formula antibody (anti-CD11b, anti-CD14, anti-CD19, anti-CD106, anti-CD29, anti-
CD44, anti-CD73, anti-CD90)
The culture medium of E2 processing group: DMEM-F12 basal medium basal medium (GIBCO, Cat#11330057) further includes
Nutridoma (Roche) 0.2-1mg/ml, dual anti-100 units of mycillin/ml, pyruvic acid 5-20mg/L, L-Glutamine
3-6mg/ml, estradiol 2.7-27.3 μ g/L.
The culture medium of contrast groups: except without in addition to estradiol, remaining culture medium with E2 processing group.
1.2 key instrument
Flow cytometer
1.3 main method
After E2 handles mescenchymal stem cell, pancreatin digestion, is centrifuged and mescenchymal stem cell is resuspended in PBS.Between unused E2 processing
Mesenchymal stem cells are as a control group and E2 processing group mescenchymal stem cell is respectively classified into 9 pipes, every pipe of control group and E2 processing group
Anti-CD11b, anti-CD14, anti-CD19, anti-CD106, anti-CD29, anti-CD44, anti-is added in cell
CD73, anti-CD90 antibody and isotype control Ab are protected from light incubation at 4 DEG C overnight, then carry out each mark point of flow cytometer detection
The expression of son.
2, result
Flow cytometer showed MSC surface marker CD11b(A), CD14(B), CD19(C), CD106(D), CD29(E), CD44(F),
CD73(G), CD90(H) expression.The results show that E2 processing will not change the expression (Fig. 2) of MSC surface marker.
3 E2 of embodiment promotes MSC proliferative capacity, vascularization regulating power and immune suppression function
1, material, reagent, equipment
1.1 main agents
Pancreatin (GIBCO, Cat# 25200056), DMEM-F12 basal medium (GIBCO, Cat#11330057), CCK8 examination
Agent box (Dojindo, Cat# CK04), PI reagent (Sigma, Cat#P4170), matrigel(BD, Cat#354230),
3H- thymidine (Cat#157049-39-3).
1.2 key instrument
Microscope, microplate reader, flow cytometer and liquid scintillation instrument
1.3 main method
1.3.1 cell-proliferation activity
MSC is laid on 96 orifice plates (10000, every hole cell), and after E2 handles MSC 48h, CCK-8 reagent is added.At 450nm wavelength
Readings is detected, and is statisticallyd analyze.
1.3.2 cell cycle analysis
Collected by trypsinisation logarithmic growth phase cell is drawn in old culture medium to a new centrifuge tube；A small amount of room temperature PBS(washes 2
Secondary, the PBS of cleaning will be also collected into above-mentioned centrifuge tube；Then cell precipitation is collected in pancreatin digestion centrifugation (300g, 5min).With
The PBS cleaning cell of pre-cooling twice, is resuspended cell precipitation with the PBS of 0.5ml pre-cooling, cell is made sufficiently to suspend at unicellular, then
99.7% dehydrated alcohol (ethyl alcohol final concentration 70%) that cell will be resuspended 1.2ml pre-cooling is added, piping and druming mix in order to avoid aggregation, and 4
DEG C fixed 2h.It is centrifuged (1000r/300g 5min) and collects fixed cell, cell is washed with the PBS of 1.8mL primary, centrifugation, again
100 μ lRNaseA are added in 37 DEG C of water-bath 30min after obtaining cell precipitation, is eventually adding 400 μ l PI and is protected from light dyeing 30min.With
Standardization program flow cytomery, it is general to count ten thousand cells of 2-3, as a result software ModFit points are fitted with the cell cycle
Analysis.It when analysis, is shown using FL2-w and FL2-A, removes conjuncted cell.
1.3.3 vascular endothelial cell cyclization is tested
Vascular endothelial cell (HUVEC) is laid in 12 well culture plates for being coated with matrigel.It collects from embodiment 1
The culture supernatant of MSC.Supernatant is added in the culture solution of HUVEC.After for 24 hours, microscopically observation HUVEC cyclization situation.
1.3.4 MSC inhibits T cell proliferation experiment
The pretreated MSC of MSC and E2 and T cell will be compareed to co-culture in the ratio of 1:5,1:10 and 1:20.3H- is added in every hole
20 μ L of TdR makes its final concentration of (3.7-18.5) × 104Bq/mL.Cell is taken with bull cell harvestor and combines in glass fibers
It ties up on filter paper.In the sufficiently dry postposition measurement bottle of filter paper, 7mL scintillation solution is added, measures umber of pulse per minute with liquid scintillation instrument
(cpm).
2, result
It can be seen that E2 is obviously promoted the proliferation activity of MSC from Fig. 3 A；It can be seen that E2 promotes MSC cell week from Fig. 3 B
Phase shows as the reduction of cell cycle G0/G1 cell proportion, the increase of its cell proportion of S phase and M；As shown in Figure 3 C, MSC is trained
After supporting supernatant processing HUVEC, compared compared with control group, the pretreated MSC culture supernatant of E2 significantly increases the cyclic ability of HUVEC；
Mixing lymph experimental result such as Fig. 3 D shows that in 1:5 and 1:10, T cell proliferation activity is obviously pressed down for MSC and T cell ratio
System.The pretreated MSC of E2 and T cell train ratio in 1:5 and 1:10 altogether, also obviously inhibit T cell proliferative capacity, and than control
MSC shows more significant inhibiting effect.In 1:20, control MSC is proliferated without obvious inhibiting effect T cell, but E2 is pre-
The MSC of processing still keeps efficient rejection ability.
These results indicate that estradiol can be used as the nutrient media components of culture placenta source MSC.And through containing estradiol
Culture medium culture after, the growth of MSC is accelerated, and function enhancing can be used in beauty or drug.
The MSC of 4 E2 of embodiment processing is used to prepare treatment eclampsia drug
Choose 60 suffer from preeclampsia patients, be divided into placebo, the MSC group that the culture medium of no added E2 is cultivated and
The MSC group that the culture medium of addition E2 is cultivated, every group of 20 people, each taking cell quantity 106It is a, it takes within every 5 days once, simultaneously
Carry out the detection of blood pressure and albuminuria.The result shows that being that the mesenchyma that culture medium culture of the present invention obtains is dry taking active constituent
After cell, patients' blood is obviously improved, and albumen urine concentration is substantially reduced, hence it is evident that is better than the culture medium of placebo and no added E2
The MSC group cultivated.
The MSC of 5 E2 of embodiment processing is used to prepare beauty product
60 healthy elder people are chosen, the age is 50 years old ± 2 years old, is divided into placebo, the culture medium of no added E2 is cultivated
MSC group and add the MSC group cultivated of culture medium of E2, every group of 20 people.In culture 106 After MSC 48 hours, dried noodle
Film is infiltrated into cells and supernatant 2 hours, then spreads on face.Once a day, 1 hour every time after continuing one month, is seen
Examining each group skin of face improves situation.The result shows that the mescenchymal stem cell group that culture medium culture of the present invention obtains is to skin
Improvement is significantly better than the MSC group that the culture medium of placebo and no added E2 is cultivated.
In conclusion the proliferation of placenta derived mesenchymal stem cell, regulation of blood vessels can be remarkably reinforced in culture medium of the present invention
And immunosuppression capability.And the mescenchymal stem cell cultivated through culture medium of the present invention have in clinical treatment and beauty industry it is latent
In application value.
Claims (6)
1. a kind of dedicated estradiol culture medium of culture mescenchymal stem cell, including basal medium, which is characterized in that further include female
Glycol, the concentration of the estradiol are 2.7-27.3 μ g/L.
2. a kind of dedicated estradiol culture medium of culture mescenchymal stem cell according to claim 1, which is characterized in that described
Basal medium is DMEM-F12 basal medium, further includes nutridoma 0.2-1mg/ml, and mycillin dual anti-100 is single
Position/ml, pyruvic acid 5-20mg/L, L-Glutamine 3-6mg/ml.
3. a kind of dedicated estradiol culture medium of culture mescenchymal stem cell according to claim 1, which is characterized in that described
Source for mesenchymal stem cells is in placenta.
4. mescenchymal stem cell the answering in preparation treatment eclampsia drug or beauty product obtained based on claim 1 culture
With.
5. a kind of treat eclampsia drug, active constituent is the mescenchymal stem cell that claim 1 is cultivated, and concentration is 1 × 105-1
×107A/ml.
6. a kind of anti-aging beauty-care liquid, active constituent is the mescenchymal stem cell that claim 1 is cultivated, and concentration is 1 × 105-1
×107A/ml.
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