CN106434539A - Osteogenic induction medium and osteogenic differentiation method - Google Patents
Osteogenic induction medium and osteogenic differentiation method Download PDFInfo
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Abstract
The invention relates to the field of the biotechnology, in particular to an osteogenic induction medium and an osteogenic differentiation method. The osteogenic induction medium is prepared from vitamin C, dexamethasone, beta-sodium glycerol-phosphate, bone morphogenetic protein-2, a vascular endothelial growth factor and a basic medium. Multiple inducing factors in the osteogenic induction medium are combined to act on GMSCs (gingival mesenchymal cells) and have a synergistic effect, the osteogenic differentiation of MSCs can be effectively induced, and the osteogenic differentiation effect is remarkably superior to that of a conventional induction method.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of Osteogenic Induction Medium and bone are to differentiation method.
Background technology
Periodontal is not only the main cause for causing adult to lose tooth, or harm Human Oral Cavity or even whole body health
Main oral disease.Periodontal disease is the infectious disease under the chronic inflammatory disease of periodontal tissue stimulates, and is the microorganism in dental plaque
The inflammation for causing under corresponding microenvironment, it damages the destruction of periodontal tissue, especially alveolar bone and the forfeiture of periodontal tissue,
Ultimately result in odontoseisiies come off and alveolar bone absorption.And the target of periodontal treatment seeks to treat periodontal disease, improve
Microenvironment regenerates new connective tissue attachment and supporting tissue again, completes alveolar bone, cementum and pericemental structure.Often
Periodontal regenerative method, such as guided tissue regeneration, root planing etc., such method is simple, but can not be complete
Feature and structural paradenlal tissue regeneration is realized, with certain limitation.The appearance of periodontal tissue engineering technology and development
Brand-new thinking and method are provided for periodontal regenerative.
Gingiva belongs to periodontal tissue, is attached to the periodontal mucosa screen for separating lower section periodontal tissue and space outerpace
Barrier.Gingiva includes epithelium and lamina propria two parts, from development source, epithelium from primitive oral cavity epithelium, on oral mucosa
Skin-deep continuity；Lamina propria is then developed and migrates the mesenchyme to be formed from early stage neural crest cell, epithelium and the continuous phase interaction of mesenchyme
With ultimately forming gingiva tissue.One typical feature of gingiva tissue is that have stronger wound healing ability, shows as cutting
Remove, damage after can no scar healing, recover gingival contour.
Gingiva mescenchymal stem cell (gingival mesenchymal stem cells, GMSCs) is composition gingiva connective
The main mesenchymal cell of tissue, from mesoblastic fibroblast, not only has the ability of active self renewal, and
And the function also with the extracellular matrix such as synthesis and degraded collagen：I type and III Collagen Type VI, fibronectin splicing variants etc..Between gingiva
Mesenchymal stem cells draw materials conveniently, cell content is big in gingiva tissue, value-added speed is fast, used as in adult mesenchymal stem cells
Member, is equally had using suitable derivant in vitro and is divided into cemental ability.This function of gingiva mescenchymal stem cell
Characteristic has greatly attracted from preclinical medicine and clinical medical researcher, provides one kind to the researchers of basic science
The biological pattern of research and development, reparation, replacement and the regeneration capacity that gingiva mescenchymal stem cell has is also clinical medicine
Chance there is provided a development new therapy.Although have study using becoming including the Various Tissues source including fat, bone marrow
Somatic stem cell carries out periodontal regenerative, but draw materials invasive and moral check the problems such as still constrain its development.Therefore, GMSCs
It is expected to become the seed cell of paradenlal tissue regeneration.
Research currently for the induction of GMSCs Osteoblast Differentiation focuses primarily upon different cytokines to its biological behaviour
The Different Effects of generation, but often it is confined to the research of its effect of single cell factor pair.As organism is a complexity
The many inducements of presence organism, therefore in the microenvironment of periodontal tissue's complexity, multiple inducible factors are in periodontal regenerative
During may play a part of mutual collaboration or antagonism.Therefore it provides a kind of have synergistic inducible factor group
Compound has important practical significance.
Content of the invention
In view of this, the invention provides a kind of Osteogenic Induction Medium and bone are to differentiation method.The osteogenic induction culture
Base includes vitamin C, dexamethasone, sodium β-glycerophosphate, BMP-2 (BMP-2), VEGF
(VEGF) inducible factor such as, multiple inducible factor use in conjunction act on the mescenchymal stem cells such as GMSCs, between effectively inducing
Mesenchymal stem cells bone is to differentiation, and its Osteoblast Differentiation effect is significant is better than existing conventional abductive approach.
In order to realize foregoing invention purpose, the present invention provides technical scheme below：
The invention provides a kind of Osteogenic Induction Medium, including vitamin C, dexamethasone, sodium β-glycerophosphate, bone shape
Become albumen -2, VEGF and basal medium.
At present, existing conventional Osteogenic Induction Medium includes vitamin C, dexamethasone and sodium β-glycerophosphate, this
The Osteogenic Induction Medium of bright offer adds BMP-2 and blood vessel endothelium life on the basis of conventional inducible factor is combined
The long factor, multiple inducible factor use in conjunction act on the mescenchymal stem cells such as GMSCs, more efficiently between induction GMSCs etc.
The bone of mesenchymal stem cells is to differentiation.
Preferably, the consumption of each component is in Osteogenic Induction Medium：
Vitamin C：10～100 μ g/L；
Dexamethasone：(1～10) × 10-8mol/L；
Sodium β-glycerophosphate：1～20mol/L；
BMP-2：50～150 μ g/L；
VEGF：10～30 μ g/L；
Basal medium：Supply.
Preferably, in Osteogenic Induction Medium, the consumption of each component is：
Vitamin C：50μg/L；
Dexamethasone：1×10-8mol/L；
Sodium β-glycerophosphate：10mol/L；
BMP-2：50～150 μ g/L；
VEGF：10～30 μ g/L；
Basal medium：Supply.
It is highly preferred that the consumption of each component is in Osteogenic Induction Medium：
Vitamin C：50μg/L；
Dexamethasone：1×10-8mol/L；
Sodium β-glycerophosphate：10mol/L；
BMP-2：100μg/L；
VEGF：20μg/L；
Basal medium：Supply.
In the embodiment that the present invention is provided, basal medium is the culture of low sugar DMEM containing 10% volume fraction FBS
Base.
Present invention also offers a kind of method of inducing mesenchymal stem cell bone to differentiation, is trained using osteogenic induction of the present invention
Foster base culture mescenchymal stem cell.
In the embodiment that the present invention is provided, mescenchymal stem cell is gingiva mescenchymal stem cell.
Preferably, mescenchymal stem cell is the 2nd～5 generation mescenchymal stem cell.
Preferably, mescenchymal stem cell is the 3rd generation mescenchymal stem cell.
Preferably, the inoculum density of mescenchymal stem cell is (1～10) × 104cell/mL.
Preferably, the inoculum density of mescenchymal stem cell is 1 × 104cell/mL.
Preferably, the cultivation temperature of mescenchymal stem cell is 37 DEG C.
Preferably, the incubation time of mescenchymal stem cell is not less than 14 days.
Preferably, the incubation time of mescenchymal stem cell is not less than 21 days.
It is highly preferred that the incubation time of mescenchymal stem cell is not less than 28 days.
The invention provides a kind of Osteogenic Induction Medium and bone are to differentiation method.The Osteogenic Induction Medium includes dimension life
Plain C, dexamethasone, sodium β-glycerophosphate, BMP-2, VEGF and basal medium.The present invention is extremely
There is one of following advantage less：
1) present invention provides one kind and acts on the mescenchymal stem cells such as GMSCs by multiple inducible factor use in conjunction, has
Synergism, effectively inducing mesenchymal stem cell bone is to differentiation for energy, and its Osteoblast Differentiation effect is significant is lured better than existing conventional
Guiding method.
2) GMSCs autologous draw materials conveniently, be easy to amplification in vitro, can be used for autotransplantation, so as to avoid immunologic rejection
Reaction.
Description of the drawings
Fig. 1 shows GMSCs aspect graph；Wherein, 1-1 shows that GMSCs amplifies 40 times of pictures, and 1-2 shows that GMSCs amplifies 100 times of pictures；
Fig. 2 shows GMSCs cell surface marker flow cytometer detection result figure；Wherein, 2-1～2-6 shows CD146, HLA- respectively
The positive expression rate of DR, CD105, CD34, CD90, CD45；
Fig. 3 shows matched group and experimental group cell Alizarin red staining design sketch；Wherein, 3-1～3-2 shows in matched group 1 respectively
Cell induction amplifies 40 times, 100 times of picture after 28 days；
3-3～3-4 shows that in matched group 2, cell induction amplifies 40 times, 100 times of picture after 28 days respectively；
3-5～3-6 shows that in matched group 3, cell induction amplifies 40 times, 100 times of picture after 28 days respectively；
3-7～3-8 shows that in matched group 4, cell induction amplifies 40 times, 100 times of picture after 28 days respectively；
3-9～3-10 shows that in experimental group 1, cell induction amplifies 40 times, 100 times of picture in 28 days respectively；
3-11～3-12 shows that in experimental group 2, cell induction amplifies 40 times, 100 times of picture in 28 days respectively；
3-13～3-14 shows that in experimental group 3, cell induction amplifies 40 times, 100 times of picture in 28 days respectively；
Fig. 4 shows matched group and experimental group cell mineralising Area comparison (p<0.01)；
Fig. 5 shows the Cellular alkaline phosphatase expression activitiy of different time points matched group and experimental group；
Fig. 6 shows that matched group and OCN, Col-I, Runx2 expression of experimental group compare.
Specific embodiment
The invention discloses a kind of Osteogenic Induction Medium and bone are to differentiation method, those skilled in the art can use for reference this
Literary content, is suitably modified technological parameter realization.Specifically, all similar replacements and change are to art technology
For personnel it is it will be apparent that they are considered as including in the present invention.The method of the present invention and application have passed through preferably
Embodiment is described, and related personnel substantially can be to side as herein described in without departing from present invention, spirit and scope
Method and application are modified or suitably change and combine, and realize and apply the technology of the present invention.
Osteogenic Induction Medium and bone biomaterial used, reagent and instrument in differentiation method that the present invention is provided
Buied by market.
With reference to embodiment, the present invention is expanded on further：
The primary separation and Culture of 1 GMSCs of embodiment, pass on and identify
1st, the primary separation and Culture of GMSCs
1) raw material sources：Taking gingiva that clinically orthodontic uprighting art cuts or impaction exodontia needs and cuts off and surround perseverance
Periodontal gingiva tissue.It is required that patient does not have gingival hyperplasia, inflammation and using the medicine for causing excessively gingival hyperplasia.The tooth for cutting
In PBS of the quick immersion of gum tissue containing 3 times of dual anti-4 DEG C pre-coolings.
Wherein, 3 times of dual anti-concentration are 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL.
2) rinse：Rinsed 3 times with the PBS dual anti-containing 3 times, then gingiva is placed in mesenchymal stem cell serum-free culture medium
Soak 5min；
Wherein, 3 times of dual anti-concentration are 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL streptomycin 300 μ/mL penicillin, 300 μ/mL.
Wherein, 100 μ/mL penicillin, 100 μ/mL streptomycin are contained in mesenchymal stem cell serum-free culture medium.
3) digest for the first time：Gingiva tissue is cut into 1cm3Size, adds appropriate NTx enzyme-Dispase enzyme mixing to disappear
Change liquid, 37 DEG C are put, in 200R constant-temperature table, digest 45min.After digestion terminates, 1000r/min is centrifuged 5min, abandons supernatant.Add
Appropriate PBS is resuspended, and 100 μm of cell strainer filter suspension, the cell suspension 1000r/min centrifugation 5min after filtration, abandons supernatant.
Wherein, the ratio of NTx enzyme-Dispase enzyme is 1:1, collagenase concentration is collagenase 3g/L, Dispase enzyme
Concentration is Dispase enzyme 4g/L；
Digestive environments are 37 DEG C, and in 200R constant-temperature table, digestion time is 45min；
Cell strainer aperture used is 100 μm；
Centrifugal speed is 1000r/min, and centrifugation time is 5min.
4) digest for second：Step 3) in filter under get off indigested tissue add isopyknic 0.125% pancreas
Protease, puts 37 DEG C, continues digestion 15min in 200R constant-temperature table.After digestion terminates, 1000r/min is centrifuged 5min, abandons
Clearly.Add appropriate PBS resuspended, 70 μm of cell strainer filter suspension, the cell suspension 1000r/min centrifugation 5min after filtration, abandons
Supernatant.
Wherein, the ratio of trypsin and gingiva tissue is volume ratio 1:1, tryptic concentration is 0.125%；
Wherein, cell strainer aperture used is 70 μm；
Wherein, centrifugal speed is 1000r/min, and centrifugation time is 5min.
5) it is inoculated with：Step 3) and step 4) in the cell collected with filling between EGF containing 10ng/mL (epidermal growth factor)
Matter stem cell serum-free culture medium re-suspended cell, is inoculated in six orifice plates, 5%CO237 DEG C of cultures of incubator.
Wherein, basal medium is mesenchymal stem cell serum-free culture medium, can market purchase or self-control.
Wherein, in complete medium, the concentration of EGF (epidermal growth factor) is 10ng/mL；
6) purification：Treat that cell covers with 80-90%, cell is collected, with magnetic bead sorting method purifying cells.By collect cell with
4 DEG C of incubation 1h of STRO-1 monoclonal antibody, with 4 DEG C of incubations of magnetic bead 30min, filter out STRO-1 under magnetic force devices effect+GMSCs, connects
Plant in six new orifice plates, 5%CO237 DEG C of cultures of incubator.
2nd, GMSCs is passed on
Treat that cell covers with 80-90%, inhaled with suction pipe and old culture fluid is abandoned, 0.25% trypsin of 2-3mL is added, digest 1-3
Minute, Microscopic observation cellular contraction becomes bowlder, adds 5-10mL mesenchymal stem cell serum-free culture medium to terminate digestion immediately, receives
Collection cell, 1000r/min is centrifuged 5min, abandons supernatant.Mesenchymal stem cell serum-free of the 5-10mL containing 10ng/mL EGF is added to train
Foster base, carries out cell counting, by 5 × 104Cell/mL density is inoculated in culture dish carries out Secondary Culture, 5%CO2Incubator 37
DEG C culture, changed liquid every 2-3 days once.Observation of cell form in incubation, as a result as shown in Figure 1.After cell inoculation about 4h
Start adherent, 3-4d entrance exponential phase, 6-8d enters plateau.Vitro growth rates are steady, grow in monolayer adherence.Greatly
Part cell is in spindle shape, and form is irregular.
3rd, GMSCs surface marker identification
Treat that cell covers with 80-90%, collect cell (about 1 × 106Individual), add 3g/L Triton to soak 20min, PBS and wash
3 times, 10% lowlenthal serum room temperature closes 2h, respectively Deca 1:CD146, CD105, CD90, CD34, CD45, HLA- of 200 dilutions
The each 50 μ L of DR antibody, while the simple 50 μ L of PBS of matched group Deca, 4 DEG C of process 16h.Next day room temperature places 30min, PBS rinsing 3
Time, each group difference Deca 1:Two anti-igg of FITC labelling of 500 dilutions, room temperature lucifuge incubation 1h, PBS are rinsed 2 times, 10% Fu Er
Malin fixes cell, and flow cytometer determines the positive rate of antigen.Result of the test is shown in Table 1, Fig. 2.
Testing result shows, clone cell surface Antigens CD14 6, CD105, CD90 positive expression, and CD34, CD45,
The negative expression of HLA-DR, illustrates the GMSCs category for obtaining in the present invention from mesoblastic mescenchymal stem cell.
1 GMSCs cell surface marker expression rate result of table
2 GMSCs Osteoblast Differentiation of embodiment
In order to verify the effect of Osteoblast Differentiation of the present invention induction, while 4 groups of matched groups and 3 groups of experimental grouies are set, to experiment
Group and matched group carry out the detection of Alizarin red staining, determination of alkaline phosphatase activity and osteogenesis gene OCN, Col-I.
Matched group 1：For negative control group, it is cellar culture group, the culture fluid for using is：Containing volume fraction 10%FBS
Low sugar DMEM culture fluid.
Matched group 2：For positive controls, it is conventional mescenchymal stem cell osteogenic induction formula, the osteogenic induction liquid for using
For：50 μ g/L vitamin Cs, 1 × 10-8Mol/L dexamethasone, 10mol/L sodium β-glycerophosphate, volume fraction 10%FBS, remaining
Measure as low sugar DMEM culture fluid.
Matched group 3：For positive controls, the osteogenic induction liquid for using is：50 μ g/L vitamin Cs, 1 × 10-8Mol/L ground plug
Meter Song, 10mol/L sodium β-glycerophosphate, 100 μ g/L BMP-2, volume fraction 10%FBS, balance of low sugar DMEM culture fluid.
Matched group 4：For positive controls, the osteogenic induction liquid for using is：50 μ g/L vitamin Cs, 1 × 10-8Mol/L ground plug
Meter Song, 10mol/L sodium β-glycerophosphate, 20 μ g/L VEGF, volume fraction 10%FBS, balance of low sugar DMEM culture fluid.
Osteogenic induction liquid used in experimental group 1 is：50 μ g/L vitamin Cs, 1 × 10-8Mol/L dexamethasone, 10mol/L
Sodium β-glycerophosphate, 50 μ g/L BMP-2,10 μ g/L VEGF, volume fraction 10%FBS, balance of low sugar DMEM culture fluid.
Osteogenic induction liquid used in experimental group 2 is：50 μ g/L vitamin Cs, 1 × 10-8Mol/L dexamethasone, 10mol/L
Sodium β-glycerophosphate, 100 μ g/L BMP-2,20 μ g/L VEGF, volume fraction 10%FBS, balance of low sugar DMEM culture fluid.
Osteogenic induction liquid used in experimental group 3 is：50 μ g/L vitamin Cs, 1 × 10-8Mol/L dexamethasone, 10mol/L
Sodium β-glycerophosphate, 150 μ g/L BMP-2,30 μ g/L VEGF, volume fraction 10%FBS, balance of low sugar DMEM culture fluid.
The formula of each group culture fluid such as table 2：
The formula of 2 each group culture fluid of table
Test method and result：
(1) Alizarin red staining
3rd generation GMSCs prepared by Example 1, by 1 × 104Cell/mL density is inoculated in 6 well culture plates, cultivate to
Cell reach 60%～70% fusion above when, each group changes osteogenic induction liquid shown in table 2 respectively.Induction differentiation culture 28d pair
Cell carries out Alizarin red staining.The concrete grammar of Alizarin red staining is：
Culture fluid is abandoned, with PBS cell 2 to 3 times；4% paraformaldehyde is added to fix 10-15min；Poly first is abandoned in suction
Aldehyde, PBS 3 times；Add 2% alizarin red, 37 DEG C of incubation 30min.Incubation terminate after inhale abandon residual liquid, with 37 DEG C preheating go from
Sub- water is cleaned 2 to 3 times, each 20s；Observe under inverted microscope after drying and gather image.Each group cell dyeing effect is such as
Fig. 3.
Multiple to each group 3 using image analysis software Image-Pro-Plus 5.0 (Media Cybernetics, the U.S.)
Cell in hole carries out Mineral nodules areal calculation.Mineral nodules area is as shown in table 3 and fig. 4.
3 mineralising area result of calculation of table
Note：*, * * represents P 0.01.
From table 3 and Fig. 3, Fig. 4, matched group 1 does not contain inducible factor, does not carry out Osteoblast Differentiation induction to cell, therefore,
Do not colour, mineralising area is zero.There was no significant difference with matched group 2 for matched group 3,4, and the base in conventional inducing culture is described
Add single BMP-2 or VEGF on plinth and all do not significantly improve GMSCs Osteoblast Differentiation ability；Each experimental group and each matched group it
Between all have pole significant difference (*, p 0.01), illustrate on the basis of conventional inducing culture while adding BMP-2 and VEGF
Two kinds of factors can effectively improve GMSCs Osteoblast Differentiation ability；Experimental group 2 has pole significant difference (* *, p with experimental group 1,3
0.01), illustrate under BMP-2 and VEGF synergy, the too high or too low concentration collocation of rational concentration Burden can more have
The raising GMSCs Osteoblast Differentiation ability of effect；Mineralising area average in experimental group 2 is 82.63 ± 1.48%, is 2 ore deposit of matched group
Change area 1.96 times, illustrate that the induction liquid of the Osteoblast Differentiation shown in experimental group of the present invention 2 carries out the effect of Osteoblast Differentiation induction most
Good.
(2) alkaline phosphatase activitieses (ALP) are determined
To cell, alkaline phosphatase activitieses are carried out respectively at induction differentiation culture 0d, 14d, 21d and 28d to each group GMSCs
Quantitative determination.Specifically assay method is：
Original fluid is abandoned, PBS is washed 3 times, add 200 μ L 0.2%TritonX-100,37 DEG C of incubation 1h per hole, visible under mirror
Cell lysis is complete, suctions out lysate to new centrifuge tube, takes supernatant, give over to standby after centrifugation.
Determining the protein quantity is carried out to above-mentioned lysate, is carried out in 96 orifice plates.Say by BCA determination of protein concentration test kit
Bright book is required to arrange and determines hole, gauge orifice and blank well, sets 3 multiple holes per group, per 20 μ L of boreliquid, after 37 DEG C of incubation 30min
Detection 562nmOD value, blank well returns to zero.Protein concentration standard curve is drawn according to gauge orifice OD value, each hole OD value is substituted into, is calculated
The protein concentration of each group.
Determination of alkaline phosphatase activity is carried out to above-mentioned lysate, is carried out in 96 orifice plates.Try by alkaline phosphatase assay
Agent box description is required to arrange and determines hole, gauge orifice and blank well, and 37 DEG C are incubated 15min, set 3 multiple holes per group, add 150 per hole
μ L nitrite ion, gently concussion orifice plate mixing.Detection 520nmOD value, blank well returns to zero.Alkali phosphatase is calculated according to below equation
Activity：
Alkaline phosphatase activitieses (U/gprot)=(determining hole absorbance-blank well absorbance) ÷ (gauge orifice absorbance-
Blank well absorbance) × phenol standard concentration ÷ sample to be tested protein concentration (gprot/mL).
Experimental result is as shown in table 4 and fig. 5.
4 different time points each group Cellular alkaline phosphatase of table activity
Test result indicate that, after cell culture 14d, 21d, 28d, within the same time, each induction group is with not induce group (right
According to group 1) activity of alkali phosphatase all has pole significant difference (7.44 ± 0.06, p 0.01), experimental group 1,3 and each matched group
Have pole significant difference (※, p 0.01), experimental group 2 and experimental group 1,3 have pole significant difference (※※, p 0.01), experimental group 2
All there is pole significant difference (* *, p 0.01) with other each groups.Alkaline phosphatase activitieses increase with the cell culture time and raise,
But it is unobvious not induce group (matched group 1) to rise, and experimental group 2 is significantly raised.
(3) detection of osteogenesis gene OCN, Col-I
Each group GMSCs after induction differentiation culture 28d, the RT-PCR method detection significant gene OCN of osteoblast,
The expression of Col-I, Runx2.Cell total rna extraction is carried out by TRIzol kit specification, M-MLV reverse transcription reagent box is obtained
cDNA.Water is expressed by SYBR Green quantitative PCR kit description gene OCN, Col-I, Runx2 significant to osteoblast
Put down and detected.β-actin is used as reference gene.2- △ △ Ct quantitative analysis.
5 primer sequence table of table
Experimental result is as shown in Figure 6.It will be appreciated from fig. 6 that after cell culture 28d, each induction group with do not induce group (matched group 1)
Relatively, significant gene OCN, Col-I expression of osteoblast all has pole significant difference (*, p 0.01), experimental group 1,3
Compare with each matched group, all have pole significant difference (※, p 0.01), experimental group 2 all has pole significance with other each induction groups
Difference (* *, p 0.01).
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of Osteogenic Induction Medium, it is characterised in that including vitamin C, dexamethasone, sodium β-glycerophosphate, bone formation
Albumen -2, VEGF and basal medium.
2. Osteogenic Induction Medium according to claim 1, it is characterised in that each component in the Osteogenic Induction Medium
Consumption be：
Vitamin C：10～100 μ g/L；
Dexamethasone：(1～10) × 10-8mol/L；
Sodium β-glycerophosphate：1～20mol/L；
BMP-2：50～150 μ g/L；
VEGF：10～30 μ g/L；
Basal medium：Supply.
3. Osteogenic Induction Medium according to claim 1 and 2, it is characterised in that each in the Osteogenic Induction Medium
The consumption of component is：
Vitamin C：50μg/L；
Dexamethasone：1×10-8mol/L；
Sodium β-glycerophosphate：10mol/L；
BMP-2：50～150 μ g/L；
VEGF：10～30 μ g/L；
Basal medium：Supply.
4. Osteogenic Induction Medium according to any one of claim 1 to 3, it is characterised in that the osteogenic induction training
In foster base, the consumption of each component is：
Vitamin C：50μg/L；
Dexamethasone：1×10-8mol/L；
Sodium β-glycerophosphate：10mol/L；
BMP-2：100μg/L；
VEGF：20μg/L；
Basal medium：Supply.
5. Osteogenic Induction Medium according to any one of claim 1 to 4, it is characterised in that the basal medium
It is the low sugar DMEM culture medium containing 10% volume fraction FBS.
6. a kind of inducing mesenchymal stem cell bone is to the method for differentiation, it is characterised in that using any one of claim 1 to 5
Osteogenic Induction Medium culture mescenchymal stem cell.
7. method according to claim 6, it is characterised in that the mescenchymal stem cell be.
8. the method according to claim 6 or 7, it is characterised in that the mescenchymal stem cell be
Stem cell.
9. the method according to any one of claim 6 to 8, it is characterised in that the inoculation of the mescenchymal stem cell is close
Spend for (1～10) × 104cell/mL.
10. the method according to any one of claim 6 to 9, it is characterised in that the culture temperature of the mescenchymal stem cell
Spend for 37 DEG C, incubation time is not less than 14 days.
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