CN105462917B - Periodontal ligament stem cell adipogenic differentiation inducing liquid and method - Google Patents
Periodontal ligament stem cell adipogenic differentiation inducing liquid and method Download PDFInfo
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Abstract
The invention relates to an inducing liquid and a method for inducing periodontal ligament stem cell adipogenic differentiation. The adipogenic differentiation induction liquid comprises a basal medium, IBMX, insulin, indomethacin, dexamethasone, PRP and FBS; preferably consists of the following components in the following content: 0.1mmoL/L IBMX, 10mg/L insulin, 0.1mmoL/L indomethacin, 1 mu moL L dexamethasone, 5 v/v% PRP, 10 v/v% FBS and the balance of basal medium, wherein the basal medium is DMEM/F12 medium. The adipogenic differentiation induction method comprises inducing using the adipogenic differentiation induction liquid. According to the method, the PRP is added on the basis of the conventional induction culture solution, so that the adipogenesis efficiency of the PDLSCs is effectively improved, and the adipogenesis induction of the PDLSCs can be successfully realized.
Description
Technical field
The present invention relates to stem cell induction differentiation fields more particularly to a kind of periodontal ligament stem cell into rouge induction liquid and
Method.
Background technique
Parodontium is one layer of connective tissue between root of the tooth and alveolar bone, is important Periodontal Supporting Tissue, mainly by
Fibrocyte, cementoblast and some undifferentiated mesenchymal cell compositions.Under normal circumstances, periodontium has itself
Update and repair ability, repair action be to be realized by periodontal ligament cell self-renewing.In disease or by the external world
When stimulation, regeneration is made by the continuous proliferation and differentiation of cell in parodontium.Seo in 2004 etc. is from the third molar pulled out
Isolated periodontal ligament stem cell (Periodontal Ligament Sem Cells, PDLSCs) in periodontal membrane tissue.In addition,
The inner surface of tooth socket is also with the presence of PDLSCs after extraction.Recently, someone has obtained highly purified from human adult periodontal tissue
PDLSCs clone.
PDLSCs has the stem cell properties of potential Multidirectional Differentiation, it can not only be divided into Ya Ti periodontal tissue,
It can be the cell of other pedigrees with transdifferentiationof.There is no adipose tissues in parodontium under physiological status, but have been reported
It is shown in stem cell under the inductions such as dexamethasone and insulin, isobutyl methylxanthine and is divided into fat cell.Fat cell
Repairing soft tissue defects as beauty has the advantage that plasticity is strong, is suitable for different defect shapes.Existing foreign scholar at present
Research has shown that PDLSCs can be adjusted by cAMP and is successfully divided into fat cell.He Huixia etc. passes through STRO-1 immunomagnetic beads point
Screening system PDLSCs is selected, the 2nd generation PDLSCs of Beads enrichment is taken, 3 days is cultivated after cell grows to 90% convergence degree, changes rouge into
Induce liquid A (basal medium A, 100mmolL-1Indomethacin, 250mmolL-1IBMX、5mmg·L-1Insulin,
1mmol·L-1Dexamethasone, 20%FBS) induction 3 days, then change adipogenic induction maintaining liquid B (basal medium B, 5mmgL-1
Insulin) induction 1 day, according to 3:1 scheme continuous induction 21 days, in experiment 96.54% PDLSCs can induction at fat
Cell, it was demonstrated that PDLSCs efficient adipogenic induction potential is expected to become the source of human stem cell that soft tissue defects are repaired in beauty
(He Huixia, Liu Hongchen, Wang Dongsheng wait periodontal ligament stem cell to test [J] to fat cell direction Induction of committed differentiation, Hua Xikou
Chamber medical journal, 2010,28 (2): 203-207).
But periodontal ligament stem cell need to be further improved at the induction liquid and method of rouge induction in the prior art.
Summary of the invention
In view of this, the present invention provides a kind of periodontal ligament stem cell into rouge induction liquid and method.
To achieve the goals above, it adopts the following technical scheme that
A kind of periodontal ligament stem cell includes basal medium, 3-isobutyl-1-methylxanthine at rouge induction liquid
(IBMX), insulin, Indomethacin, dexamethasone, platelet rich plasma (PRP) and fetal calf serum (FBS).
Preferably, the periodontal ligament stem cell is 1-10v/v% at the content of PRP in rouge induction liquid.
Preferably, the periodontal ligament stem cell is 5v/v% at the content of PRP in rouge induction liquid.
Preferably, the periodontal ligament stem cell is grouped as at rouge induction liquid by following groups of following contents:
0.1mmoL/L IBMX, 10mg/L insulin, 0.1mmoL/L Indomethacin, 1 μm of oL/L dexamethasone, 5v/v%PRP, 10v/
V%FBS, surplus are basic culture medium, and the basal medium is DMEM/F12 culture medium.
The platelet rich plasma (Platelet-rich plasma, PRP) that the present invention uses is obtained by secondary centrifuging method
The platelet concentrate arrived comes from autoblood, obtains and is easy, small to injury of human, and immunological rejection is not present, rich in more
Kind growth factor such as platelet derived growth factor, transforming growth factor β, vascular endothelial growth factor etc., the PRP of various concentration has
The effects of promoting the proliferation of stem cell, skeletonization and breaking up at rouge, clinically has a good application prospect.
Preferably, the PRP is prepared by the following method: 10mL autologous peripheral blood is centrifuged 10min under the conditions of 400g, from
Blood sample is divided into supernatant layer, white cellular layer and red cell layer from top to bottom after the heart, takes red cell layer top 1-
The supernatant layer and white cellular layer of 2mm and whole, are centrifuged 5min under the conditions of 400g, after centrifugation blood sample from top to bottom according to
It is secondary to be divided into supernatant layer, white cellular layer and red cell layer, take supernatant layer and white cellular layer to be centrifuged under the conditions of 1200g
5min, it is PRP that bottom 2.5mL is retained after centrifugation.
It is highly preferred that PRP is activated before use, the activation method are as follows: PRP is moved into the rush of chloride containing calcium
Solidifying pipe, 4 DEG C overnight after be centrifuged 12min under the conditions of 800g, take 0.22 μm of whole supernatants filtering to get the PRP after activation.
A kind of periodontal ligament stem cell is induced using above-mentioned at rouge induction liquid at rouge method of inducing differentiation.
Preferably, the periodontal ligament stem cell is at rouge method of inducing differentiation, comprising the following steps: after parodontium tissue digestion
Cell culture is carried out, STRO-1 is sub-elected+PDLSCs is cultivated, using it is above-mentioned at rouge induction liquid to 2-5 generation
PDLSCs is induced.
Preferably, STRO-1 is sub-elected using magnetic bead sorting method+ PDLSCs。
It is further preferred that magnetic bead sorting method are as follows: the cell cultivated after digestion and 4 DEG C of STRO-1 monoclonal antibody incubations will be collected
1h filters out STRO-1 under magnetic force devices effect with magnetic bead in 4 DEG C of incubation 30min+ PDLSCs。
Preferably, the periodontal ligament stem cell is at rouge method of inducing differentiation, comprising the following steps:
1) the primary separation of PDLSCs and passage:
The periodontal membrane tissue for scraping lower 1/3 section in tooth root, is cut into about 1mm3Size；Appropriate clostridiopetidase A-is added
Dispase enzyme 1:1 mixture slaking liquid, after 37 DEG C of digestion 30-35min, 1000rpm is centrifuged 5min, abandons supernatant, appropriate PBS is added
It is resuspended, 1000rpm is centrifuged 5min, abandons supernatant, and cell is resuspended in the DMEM/F12 culture medium that the 10%FBS containing volume fraction is added, and adjusts
Whole cell density is to 1 × 104The every hole cells/mL, 2mL is inoculated in six orifice plates, in 5%CO2, cultivate under the conditions of 37 DEG C；
It is long to convergence degree 80-90% to cell, cell is collected, sub-elects STRO-1 with magnetic bead sorting method+PDLSCs is used
Cell, adjustment cell density to 1 × 10 is resuspended in the DMEM/F12 culture medium of the 10%FBS containing volume fraction4Cells/mL, 2mL are every
Hole is inoculated in six orifice plates, in 5%CO2, cultivate under the conditions of 37 DEG C；
It is long to convergence degree 80-90% to cell, culture solution is abandoned, appropriate 0.25m/v% trypsin solution is added, digests 1-
3min, when microscopic observation cellular contraction is rounded, the DMEM/F12 culture medium that the 10%FBS containing volume fraction in right amount is added immediately is terminated
Cell is collected in digestion, and 1000rpm is centrifuged 5min, abandons supernatant；It is resuspended with the DMEM/F12 culture medium of the 10%FBS containing volume fraction
Cell, adjustment cell density to 5 × 104Cells/mL is inoculated in culture dish in 5%CO2, carry out passage training under the conditions of 37 DEG C
It supports, was changed the liquid once every 2-3 days；
2) PDLSCs breaks up at rouge:
The 3rd generation PDLSCs is taken, by 1 × 104Cells/mL density is inoculated in 12 well culture plates, and culture to convergence degree reaches
It when 70-80% or more, is changed to above-mentioned at rouge induction liquid, changed liquid every 3 days, continuous induction 21 days.
Compared with prior art, the invention has the following beneficial effects:
1) periodontal ligament stem cell of the present invention is added on the basis of conventional induction broth at rouge induction liquid and method
The PRP that human peripheral extracts, effectively increases the fatization efficiency of PDLSCs, can successfully realize luring at rouge to PDLSCs
It leads, it was demonstrated that PDLSCs efficient adipogenic induction potential repairs soft tissue defects application seed cell for beauty and provides mostly one kind
Selection.
2) raw material that the present invention uses are the thirds that 12-18 years old teenager needs to pull out when carrying out orthodontic treatments
It grinds one's teeth in sleep, is thrown away at present essentially as Biohazard Waste；And the present invention carries out adipogenic induction side for being separately cultured PDLSCs
Method effectively realizes the recycling of Biohazard Waste.
Detailed description of the invention
Fig. 1 is the flow cytometer detection figure of PDLSCs cell surface marker expression.Figure A- figure F be respectively CD146, CD105,
The flow cytometer detection figure of CD73, HLA-DR, CD34 and CD45.
Fig. 2 is cellular morphology figure.Wherein, Fig. 2A, Fig. 2 B and Fig. 2 C are respectively P0 generation, P1 generation and P3 100 times of enlarged drawings of generation.
Fig. 3 is the result figure of negative control group oil red O stain.Wherein Fig. 3 A and Fig. 3 B is respectively 200 times and 400 times amplifications
Figure.
Fig. 4 is the result figure of positive controls oil red O stain.Wherein Fig. 4 A and Fig. 4 B is respectively 200 times and 400 times amplifications
Figure.
Fig. 5 is the result figure of 1 oil red O stain of experimental group.Wherein Fig. 5 A and Fig. 5 B is respectively 200 times and 400 times of enlarged drawings.
Fig. 6 is the result figure of 2 oil red O stain of experimental group.Wherein Fig. 6 A and Fig. 6 B is respectively 200 times and 400 times of enlarged drawings.
Fig. 7 is the result figure of 3 oil red O stain of experimental group.Wherein Fig. 7 A and Fig. 7 B is respectively 200 times and 400 times of enlarged drawings.
Fig. 8 is the induction differentiation rate result figure in effect example 1.
Fig. 9 is the detection of expression result figure of PPAR γ -2 in effect example 2.
Figure 10 is the detection of expression result figure of LPL in effect example 2.
Specific embodiment
In order to better illustrate the present invention, it is described further in the following with reference to the drawings and specific embodiments.Make in the present invention
Detection method etc. be all it is known in the art, details are not described herein.
The portion of reagent or instrument source that the present invention uses are as shown in table 1, do not indicate the reagent in source or instrument is this
Field conventional reagent or instrument can be commercially available from market.
The portion of reagent and instrument source that table 1, the present invention use
Embodiment 1
A kind of periodontal ligament stem cell is at rouge induction liquid, following components comprising following contents: 0.1mmoL/L IBMX,
10mg/L insulin, 0.1mmoL/L Indomethacin, 1 μm of oL/L dexamethasone, 1v/v%PRP, 10v/v%FBS, surplus are
DMEM/F12 culture medium.
PRP is prepared by the following method in the present embodiment: by with the peripheral blood of the same donor of tooth in super-clean bench blood
Sample is transferred to 15mL centrifuge tube, the every pipe of 10mL, and 400g is centrifuged 10min.Blood sample is divided into three layers from top to bottom after centrifugation: yellowish
Color supernatant layer, white cellular layer and red cell layer.Draw whole supernatant layers, white cellular layer and red cell layer top 1-
2mm, moves into new 15mL centrifuge tube, and 400g is centrifuged 5min.Supernatant layer and white cellular layer are drawn after centrifugation, move into new 15mL
Centrifuge tube, 1200g are centrifuged 5min.Supernatant is abandoned after centrifugation, retains nethermost 2.5mL, i.e. platelet rich plasma.Immigration contains
The solidifying pipe of the rush of calcium chloride stays overnight rear 800g for 4 DEG C and is centrifuged 12min, draws out the whole supernatants promoted in solidifying pipe, and 0.22 μm is filtered, and is filtered
- 20 DEG C of liquid save for use.After PRP is mixed with calcium chloride and fibrin ferment, growth factor is that the α particle from blood platelet releases
Come, then can be obtained by the PRP of activation through centrifugal filtration.
Use the above-mentioned method broken up at rouge induction liquid induction periodontal ligament stem cell at rouge, comprising the following steps:
1) the primary separation of PDLSCs and passage
Materials: it by the third molar for needing to pull out because of correction of 12-18 years old health donors, quickly immerses and contains 3 times dual anti-4
DEG C pre-cooling PBS in it is spare.Tooth body is rinsed with the direction from root of the tooth to corona containing 3 times of dual anti-PBS, thoroughly removes blood stains, weight
It is 3 times multiple.It unidirectionally scrapes lower 1/3 section in root of periodontal membrane tissue with knife blade crown root, moves into centrifuge tube, and with eye scissors by group
It knits block and is cut into about 1mm3Size.
Digestion: appropriate mixture slaking liquid being added into tissue pieces, sets in 37 DEG C of constant-temperature tables and digests 30-35min, digests
After, 1000rpm is centrifuged 5min, abandons supernatant, and appropriate PBS is added and is resuspended, and 1000rpm is centrifuged 5min, abandons supernatant；Culture is added
Cell is resuspended in base (the DMEM/F12 culture medium of the 10%FBS containing volume fraction), makes its density 1 × 104Cells/mL is inoculated in
Six orifice plates, every hole 2mL, 5%CO237 DEG C of incubator cultures.The mixture slaking liquid is by 3g/L collagenase solution and 4g/L
The ratio of Dispase enzyme solutions 1:1 by volume mixes.
Purifying: it is long to convergence degree 80-90% to cell, cell is collected, with magnetic bead sorting method purifying cells；Specific method
Are as follows: the cell of collection and 4 DEG C of incubation 1h of STRO-1 monoclonal antibody are sieved under magnetic force devices effect with magnetic bead in 4 DEG C of incubation 30min
Select STRO-1+PDLSCs is added culture medium (the DMEM/F12 culture medium of the 10%FBS containing volume fraction) and cell, adjustment is resuspended
Cell density is 1 × 104The every hole cells/mL, 2mL is inoculated in six new orifice plates, 5%CO237 DEG C of incubator cultures.
PDLSCs identification: it is long to convergence degree 80-90% to cell, collect cell (about 1 × 106It is a), 3g/L is added
TritonX-100 impregnates 20min, and PBS is washed 3 times, and 10% lowlenthal serum room temperature closes 2h, and it is diluted that 1:200 is added dropwise respectively
Each 50 μ l of CD146, CD105, CD73, HLA-DR, CD34, CD45 antibody, while control group is added dropwise 50 μ l of simple PBS, at 4 DEG C
Manage 16h.Next day is placed at room temperature for 30min, and PBS is rinsed 3 times, and diluted two anti-igg of FITC label of 1:500 is added dropwise in each group respectively, often
Temperature, which is protected from light, is incubated for 1h, and PBS is rinsed 2 times, the fixed cell of 10% formalin, the positive rate of upper machine measurement antigen.
FCM analysis result is as shown in Figure 1, PDLSCs cell surface marker expression rate is as shown in table 2.By 2 He of table
Fig. 1 is it is found that clone cell surface Antigens CD14 6, CD105, CD73 express the positive, and HLA-DR, CD34, CD45 expression are negative,
Illustrate that PDLSCs obtained in the present invention belongs to from mesoblastic mescenchymal stem cell.
Table 2, PDLSCs cell surface marker expression rate
| Cell phenotype | CD146 | CD105 | CD73 | HLA-DR | CD34 | CD45 |
| Positive expression rate (%) | 99.8 | 99.8 | 99.8 | 0.2 | 0.2 | 0.3 |
Passage: will identify that correct PDLSCs carries out secondary culture, inhaled with suction pipe and abandon old culture solution, be added appropriate
0.25m/v% trypsase digests 1-3min, when microscopic observation cellular contraction is rounded, is added contains volume fraction in right amount immediately
The DMEM/F12 culture medium of 10%FBS terminates digestion, collects cell, and 1000rpm is centrifuged 5min, abandons supernatant.It is added and contains body in right amount
The DMEM/F12 culture medium of fraction 10%FBS carries out cell count, by 5 × 104Cells/mL density is inoculated in culture dish
Carry out secondary culture, 5%CO237 DEG C of incubator cultures, changed the liquid once every 2-3 days.Cellular morphology is observed in incubation, is tied
Fruit is as shown in Figure 2.Start adherent after cell inoculation about 4h, 2-3d enters exponential phase of growth, and cell proliferation rate is very fast, 4-6d into
Enter plateau.Vitro growth rates are steady, grow in monolayer adherence.Most cells are in class shuttle shape, and form is irregular.
2) at rouge induction:
The 3rd generation PDLSCs is taken, by 1 × 104Cells/mL density is inoculated in 12 well culture plates, culture to cell confluency degree
When reaching 70-80% or more, adipogenic induction liquid culture 3d is changed, changes liquid, continuous induction 7-21d every 3d.
Embodiment 2
The present embodiment is substantially the same manner as Example 1, and what is be used only is different at rouge induction culture medium.Make in the present embodiment
Contain following components of following contents at rouge induction culture medium: 0.1mmoL/LIBMX, 10mg/L insulin,
0.1mmoL/L Indomethacin, 1 μm of oL/L dexamethasone, 5v/v%PRP, 10v/v%FBS, surplus are DMEM/F12 culture medium.
Embodiment 3
The present embodiment is substantially the same manner as Example 1, and what is be used only is different at rouge induction culture medium.Make in the present embodiment
Contain following components of following contents at rouge induction culture medium: 0.1mmoL/L IBMX, 10mg/L insulin,
0.1mmoL/L Indomethacin, 1 μm of oL/L dexamethasone, 10v/v%PRP, 10v/v%FBS, surplus are DMEM/F12 culture medium.
In order to verify the effect of Osteoblast Differentiation induction of the present invention, to induce liquid and method to carry out into rouge point in embodiment 1-3
Change induction and be used as experimental group 1-3, while negative control group and positive controls are set, oil red O is carried out to experimental group and control group
It dyes, the detection of expression of peroxisome proliferators activated receptor γ -2 (PPAR γ -2) and lipoprotein lipase (LPL).Each
Control group is all substantially the same manner as Example 1, and what is be used only is different at rouge induction liquid (or culture solution).
The culture solution of negative control group is the DMEM/F12 culture medium of the 10%FBS containing volume fraction.
Being grouped as at rouge induction liquid by following groups of following contents in positive controls: 0.1mmoL/L IBMX,
10mg/L insulin, 0.1mmoL/L Indomethacin, 1 μm of oL/L dexamethasone, 10v/v%FBS, surplus are DMEM/F12 culture
Base.
Effect example 1, oil red O stain
Culture is carried out to periodontal ligament stem cell according to experimental group or control group method or at rouge induction, respectively the
7d, 14d and 21d carry out oil red O stain to cell.
The cell of oil red O stain is counted, the differentiation rate of each group is calculated, as a result as shown in table 3 and Fig. 3-Fig. 8.
Induce the calculation method of differentiation rate are as follows: under 100 times of magnification fields of inverted microscope, every hole counts 3 visuals field at random, calculates point
Change the ratio that cell accounts for total number of cells, count 3 holes in parallel every time, calculate average).
Induce differentiation rate=staining positive cells number/total number of cells × 100% ... ... ... (1)
Table 3, each group induce differentiation rate
After cultivating 21d as the result is shown, negative control group is free of inducible factor, does not carry out cell at rouge induction, because
This, does not colour, and is zero at rouge differentiation rate.Experimental group 2 and other each groups have significant difference (P ﹤ 0.01, * *)；Experimental group 1
There is significant difference (P ﹤ 0.01, *) with positive controls, experimental group 3；There was no significant difference with positive controls for experimental group 3.It is real
Testing in group 2 is 96.25 ± 1.04% at rouge differentiation rate mean value, is nearly 2 times of positive controls.Illustrate the embodiment of the present invention 2
Shown at rouge induction liquid carry out it is best at the effect of rouge induction.
The detection of expression of effect example 2, PPAR γ -2 and LPL
Culture is carried out to periodontal ligament stem cell according to experimental group or control group method or at rouge induction, continuous induction training
The detection of expression of PPAR γ -2 and LPL is carried out after feeding 7d, 14d and 21d to cell.
Cell total rna extraction is carried out by TRIzol kit specification；M-MLV reverse transcription reagent box obtains cDNA.It presses
SYBR Green quantitative PCR kit specification detects PPAR γ -2, LPL expression.Using GAPDH as internal reference base
Cause.With 2-△△CtMethod carries out quantitative analysis.The primer used is as shown in table 4.As a result as shown in Figure 9 and Figure 10.
Table 4, primer sequence table
The experimental results showed that after cell Fiber differentiation 7d, 14d, 21d, at same time point, each induction group and group is not induced
(negative control group) compares, and the significant gene PPAR γ -2 of lipoblast, LPL expression have significant difference (P ﹤
0.01), experimental group 1 and positive controls, experimental group 3 have significant difference (P ﹤ 0.01, *)；Experimental group 2 and other each induction groups
There is significant difference (P ﹤ 0.01, * *).
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (5)
1. a kind of periodontal ligament stem cell is at rouge induction liquid, which is characterized in that include basal medium, IBMX, insulin, Yin
Diindyl U.S. is pungent, dexamethasone, PRP and FBS；
The PRP is prepared by the following method: 10mL autologous peripheral blood is centrifuged 10min, blood after centrifugation under the conditions of 400g
Sample is divided into supernatant layer, white cellular layer and red cell layer from top to bottom, takes red cell layer top 1-2mm and whole
Supernatant layer and white cellular layer, 5min is centrifuged under the conditions of 400g, blood sample is divided into supernatant from top to bottom after centrifugation
Layer, white cellular layer and red cell layer take supernatant layer and white cellular layer to be centrifuged 5min under the conditions of 1200g, and centrifugation terminates
Retaining bottom 2.5mL afterwards is PRP；
PRP is activated before use, the activation method are as follows: the solidifying pipe of the rush that PRP is moved into chloride containing calcium, 4 DEG C overnight
It is centrifuged 12min under the conditions of 800g afterwards, takes 0.22 μm of whole supernatants filtering to get the PRP after activation；
The periodontal ligament stem cell is grouped as at rouge induction liquid by following groups of following contents: 0.1mmoL/L IBMX,
10mg/L insulin, 0.1mmoL/L Indomethacin, 1 μm of oL/L dexamethasone, 5v/v%PRP, 10v/v%FBS, surplus is base
Basal culture medium, the basal medium are DMEM/F12 culture medium.
2. a kind of periodontal ligament stem cell is at rouge method of inducing differentiation, which is characterized in that lured using described in claim 1 at rouge differentiation
Drain is induced.
3. periodontal ligament stem cell according to claim 2 is at rouge method of inducing differentiation, which is characterized in that the parodontium is dry
Cell is at rouge method of inducing differentiation, comprising the following steps: carries out cell culture after parodontium tissue digestion, sub-elects STRO-1+
PDLSCs is cultivated, and is induced for PDLSCs at rouge induction liquid 2-5 using above-mentioned.
4. periodontal ligament stem cell according to claim 3 is at rouge method of inducing differentiation, which is characterized in that use magnetic bead sorting
Method sub-elects STRO-1+PDLSCs, magnetic bead sorting method are as follows: the cell cultivated after digestion and 4 DEG C of STRO-1 monoclonal antibody incubations will be collected
1h filters out STRO-1 under magnetic force devices effect with magnetic bead in 4 DEG C of incubation 30min+PDLSCs。
5. periodontal ligament stem cell according to claim 4 is at rouge method of inducing differentiation, which is characterized in that the parodontium is dry
Cell is at rouge method of inducing differentiation, comprising the following steps:
1) the primary separation of PDLSCs and passage:
The periodontal membrane tissue for scraping lower 1/3 section in tooth root, is cut into about 1mm3Size；Appropriate clostridiopetidase A-Dispase enzyme is added
1:1 mixture slaking liquid, after 37 DEG C of digestion 30-35min, 1000rpm is centrifuged 5min, abandons supernatant, and appropriate PBS is added and is resuspended,
1000rpm is centrifuged 5min, abandons supernatant, and cell is resuspended in the DMEM/F12 culture medium that the 10%FBS containing volume fraction is added, and adjusts cell
Density is to 1 × 104The every hole cells/mL, 2mL is inoculated in six orifice plates, in 5%CO2, cultivate under the conditions of 37 DEG C；
It is long to convergence degree 80-90% to cell, cell is collected, sub-elects STRO-1 with magnetic bead sorting method+PDLSCs, with containing volume
Cell, adjustment cell density to 1 × 10 is resuspended in the DMEM/F12 culture medium of score 10%FBS4The every hole inoculation of cells/mL, 2mL
In six orifice plates, in 5%CO2, cultivate under the conditions of 37 DEG C；
It is long to convergence degree 80-90% to cell, culture solution is abandoned, appropriate 0.25m/v% trypsin solution is added, digests 1-
3min, when microscopic observation cellular contraction is rounded, the DMEM/F12 culture medium that the 10%FBS containing volume fraction in right amount is added immediately is terminated
Cell is collected in digestion, and 1000rpm is centrifuged 5min, abandons supernatant；It is resuspended with the DMEM/F12 culture medium of the 10%FBS containing volume fraction
Cell, adjustment cell density to 5 × 104Cells/mL is inoculated in culture dish in 5%CO2, carry out passage training under the conditions of 37 DEG C
It supports, was changed the liquid once every 2-3 days；
2) PDLSCs breaks up at rouge:
The 3rd generation PDLSCs is taken, by 1 × 104Cells/mL density is inoculated in 12 well culture plates, and culture to convergence degree reaches 70-
It when 80% or more, is changed to above-mentioned at rouge induction liquid, changed liquid every 3 days, continuous induction 7-21 days.
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| Jung-Yeon Lee et al..The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells.《In Vitro Cell.Dev.Biol.-Animal》.2010,第47卷摘要. * |
| Ui-Lyong Lee et al..Effect of platelet-rich plasma on dental stem cells derived from human impacted third molars.《Regen. Med.》.2011,第6卷(第1期),摘要. * |
| 乔静 等.富血小板血浆对人牙周膜细胞增殖及分化的影响.《上海口腔医学》.2008,第17卷(第1期),第2.3，2.4节. * |
| 贺慧霞 等.牙周膜干细胞向脂肪细胞方向定向诱导分化实验.《华西口腔医学杂志》.2010,第28卷(第2期),第1节. * |
| 韩劼 等.人牙周膜细胞体外定向诱导分化为脂肪细胞的研究.《口腔医学研究》.2006,第22卷(第2期),第1.2，1.2.2，1.3节. * |
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