CN105112367A - Mesenchymal stem cell epidermal differentiation inducer and application method thereof - Google Patents
Mesenchymal stem cell epidermal differentiation inducer and application method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of induced differentiation of stem cells, and particularly relates to a mesenchymal stem cell epidermal differentiation inducer. The inducer is composed of KGF, BMP-4, PDGF-AB, VEGF, IL-2 and Forskolin. After a complete culture medium is added, acting concentrations of KGF, BMP-4, PDGF-AB, VEGF, IL-2 and Forskolin are sequentially adjusted to be 20-30ug/L, 5-10ug/L, 5-10ug/L, 10-20ug/L, 5-10ug/L and 1-2mg/L respectively. Cytokines are combined with small molecular compounds to efficiently induce mesenchymal stem cells to be differentiated to be epidermal cells, so that induced differentiation efficiency is greatly improved, and cells after being induced are high in activity; cell transfection is not needed, so that genetic change and risk of getting cancer are avoided, ethical issues are avoided, and the inducer is high in safety.
Description
Technical field
The invention belongs to the differentiation-inducing technical field of stem cell, be specifically related to a kind of mescenchymal stem cell directed differentiation inductor.
Background technology
Skin is the maximum organ of human body, and gross weight accounts for 16% of whose body weight, forms primarily of epidermis, corium and subcutis.Clinically due to the skin injury that severe burn, chronic ulcer, wound etc. cause, need wound healing as early as possible, reduce the dysfunction that paralysed trace causes as far as possible.The development of tissue engineering technique and regenerative medicine makes the exploitation of novel skin reconstituted product become possibility.Organizational project ultimate principle is after being cultivated in vitro by seed cell, increasing, and is integrated in the natural biological stent that designs in advance and forms cell one support complex body, the then corresponding lesion of implanting to human body, repair tissue defect.Three large key elements of organizational project comprise seed cell, timbering material and contribute to the microenvironment of Growth of Cells differentiation.The most basic seed cell built required for organization engineering skin is epidermic cell, and epidermic cell is a kind of cell of the differentiation of end eventually, cultivate a generation in vitro and namely enter ageing state, and its division growth speed is slower, culture condition is strict, and the very difficult short period of time amplifies a large amount of cells to meet the needs of skin tissue engineering.
Mescenchymal stem cell (MSCs) is a kind of stem cell with multi-lineage potential, in fetal development, derive from mesoderm, and the fetal development of mouse starts after 10 days to occur, is almost distributed in all organs of body afterwards with tissue.Adult MSCs is mainly present in marrow, fat, and around the blood vessel in subperiosteum and each Organ and tissue, in addition, neonatal umbilical cord, placenta, amnion etc. are also containing a large amount of MSCs.MSCs has the potential being divided into multiple mesoblastema system, comprises scleroblast, adipocyte, chondrocyte, neurocyte, liver cell, epidermic cell etc.MSCs wide material sources and do not have ethics to limit, be easy to be separated and amplification in vitro, still multi-lineage potential is being kept after repeatedly dividing and going down to posterity, up to now clinical experimental study does not find that MSCs has serious side effects, therefore, MSCs is the seed cell of a kind of ideal cell therapy and regenerative medicine.
The research report of the utilization MSCs external evoked epidermal stem cells differentiation of current bibliographical information is few, and induction method also comparing class seemingly, breaks up pick-up rate also lower.Use and more add Urogastron, Prostatropin (bFGF) for conditioned medium, be aided with vitamin A acid (RA), or applying transgene technique imports EGF, with the cytodifferentiation after bFGF induced transfection.Such as there is investigator to use skin histology homogenate as conditioned medium and add the epidermal differentiation that Urogastron (EGF) carrys out induced dry-cell, prove that MSCs also can break up to epidermic cell in vitro by mark CK19 and CK14 detecting epidermal stem cells, but this method differentiation yield very low (CK19 positive rate is about 10%, CK14 positive rate and is about 12%).Also there are investigator's conbined usage EGF and vitamin A acid to carry out induced lipolysis MSCs and are divided into epidermic cell, induce after 7 days and can find that the form of a small amount of cell in portion changes, become Polygons by fusiformis, part cell forms cobble and to pave the way sample, finds that positive rate is about 12% to the detection of CK19.
In order to solve problems of the prior art, the present inventor, through repeatedly great many of experiments, surprisingly finds that KGF, BMP-4, PDGF-AB, VEGF, IL-2, Forskolin associating can efficiently induce MSCs to be divided into epidermic cell.Wherein, BMP (Delicious peptide) is one of member of TGF-beta superfamily, plays an important role to the fetal development of bone and Regeneration and Repair.BMP is expressed in numerous embryonic organ and tissue, not only participates in generation and the formation of bone, and closely related with numerous organ, as kidney, lung, eye, neural system, skin, sexual gland etc.BMP is many-sided regulatory factor in vital process, and participate in propagation, differentiation, apoptosis and form generation etc., each member may have different effects to different tissues.Cytokine KGF, PDGF-AB, VEGF, IL-2 can maintain epidermal stem cells microenvironment, can stimulate its propagation simultaneously, grow.Forskolin is a kind of conventional adenylate cyclase activating agent, can permeabilized cells, activated adenyl cyclase (adenylylcyclase), thus the cAMP level in elevate cellular.And they combine, epidermic cell can be divided into by co-induction MSCs.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide mescenchymal stem cell epidermal differentiation inductor, mescenchymal stem cell Induction of committed differentiation can be epidermic cell by this inductor.It is long to there is induction time in current most mescenchymal stem cell epidermal differentiation inductor, and induced efficiency is low, obtains epidermic cell poor activity after induction.
For achieving the above object, the technical solution used in the present invention is: a kind of mescenchymal stem cell epidermal differentiation inductor, be made up of KGF, BMP-4, PDGF-AB, VEGF, IL-2, Forskolin, after adding perfect medium, each composition activity is followed successively by KGF20 ~ 30 μ g/L, BMP-45 ~ 10 μ g/L, PDGF-AB5 ~ 10 μ g/L, VEGF10 ~ 20 μ g/L, IL-25 ~ 10 μ g/L, Forskolin1 ~ 2mg/L.
Preferably, a kind of mescenchymal stem cell epidermal differentiation inductor, after adding perfect medium, each composition activity is followed successively by KGF25 μ g/L, BMP-48 μ g/L, PDGF-AB7 μ g/L, VEGF12 μ g/L, IL-27 μ g/L, Forskolin1.2mg/L.
The application method of mescenchymal stem cell epidermal differentiation inductor of the present invention, required to add respectively in perfect medium according to activity by each composition of mescenchymal stem cell epidermal differentiation inductor, perfect medium is Keratinocyte-SFM, the special serum free medium of human mesenchymal stem cell or DMEM/F12+10%FBS.
Mescenchymal stem cell epidermal differentiation inductor of the present invention, adopt cytokine associating micromolecular compound, efficient inducing mesenchymal stem cell is divided into epidermic cell, differentiation-inducing efficiency greatly improves, after induction, cytoactive is strong, and without the need to cell transfecting, therefore without gene alteration and suffers from cancer risk, without ethics problem, security is high.
Accompanying drawing explanation
Fig. 1 is fat mesenchymal stem cell, P3, (× 200);
The positive epidermic cell of CKl9 after the induction of Fig. 2 mescenchymal stem cell, the fluorescence that takes on a red color (× 200);
The positive epidermic cell of P63 after the induction of Fig. 3 mescenchymal stem cell, the fluorescence that takes on a red color (× 200);
The positive epidermic cell of β 1-integrin after the induction of Fig. 4 mescenchymal stem cell, the fluorescence that takes on a red color (× 200);
Fig. 5 inductor of the present invention compares with traditional method induced lipolysis mescenchymal stem cell induced efficiency;
Fig. 6 inductor of the present invention compares with traditional method inducing umbilical cord mesenchymal stem induced efficiency.
Embodiment
Experimental technique in following embodiment, if no special instructions, is ordinary method.Test utensil instrument reagent used all to obtain by commercial sources.
Embodiment 1 one kinds of mescenchymal stem cell epidermal differentiation inductors, is characterized in that: described inductor is made up of KGF, BMP-4, PDGF-AB, VEGF, IL-2, Forskolin.
Embodiment 2 mescenchymal stem cell epidermal differentiation inductor, by following raw materials according by its separately characteristic dissolve, with human mesenchymal stem cell serum free medium (LONZA, article No. 00190632) be matrix, each component concentration is made to be settled to 1000ml:KGF(Sigma as following, K1751-10 μ g) 20 μ g, BMP-4(Sigma, SRP3016-10 μ g) 5 μ g, PDGF-AB(PeproTech, 96-100-00AB-10 μ g) 5 μ g, VEGF(PeproTech, 96-100-20-10 μ g) 10 μ g, IL-2(PeproTech, 96-200-02-50 μ g) 5 μ g, Forskolin(Abcam, ab120058-100mg) 1mg, filtration sterilization after mixing.
Embodiment 3 mescenchymal stem cell epidermal differentiation inductor, by following raw materials according by its separately characteristic dissolve, take DMEM/F12+10%FBS as matrix, each component concentration is made to be settled to 1000ml:KGF(Sigma as following, K1751-10 μ g) 30 μ g, BMP-4(Sigma, SRP3016-10 μ g) 10 μ g, PDGF-AB(PeproTech, 96-100-00AB-10 μ g) 10 μ g, VEGF(PeproTech, 96-100-20-10 μ g) 20 μ g, IL-2(PeproTech, 96-200-02-50 μ g) 10 μ g, Forskolin(Abcam, ab120058-100mg) 2mg, filtration sterilization after mixing.
Embodiment 4 mescenchymal stem cell epidermal differentiation inductor, by following raw materials according by its separately characteristic dissolve, with Keratinocyte-SFM(Gibco, 170005-42) be matrix, each component concentration is made to be settled to 1000ml:KGF(Sigma as following, K1751-10 μ g) 25 μ g, BMP-4(Sigma, SRP3016-10 μ g) 8 μ g, PDGF-AB(PeproTech, 96-100-00AB-10 μ g) 7 μ g, VEGF(PeproTech, 96-100-20-10 μ g) 12 μ g, IL-2(PeproTech, 96-200-02-50 μ g) 7 μ g, Forskolin(Abcam, ab120058-100mg) 1.2mg, filtration sterilization after mixing.
The inductor that embodiment 5 adopts embodiment 4 to prepare, carries out the differentiation-inducing experiment of epidermic cell to fat mesenchymal stem cell (AD-MSCs)
1, AD-MSCs primary culture in vitro: by the centrifugal 3min of postoperative for suction lipectomy liquid 800r/min, stay the fatty tissue that upper strata is floating, phosphoric acid buffer (PBS) rinsing, centrifugal 5min2 time of 800r/min, stays upper-layer fat; Add equal-volume 0.2% collagenase, 37 DEG C of shaking table digestion 30min; Filtered by pasty state Digestive system 100 order stainless steel filtering nets, the centrifugal 5min of 1500r/min, throw out is resuspended with 1mlDME/F12, PBS rinsing, the centrifugal 8min of 1000r/min, and cell culture medium is resuspended, and 5 × 10
5/ ml inoculates; Put 37 DEG C, 5%CO
2incubator is hatched, and takes the circumstances into consideration to change liquid after leaving standstill 3d.When cell grows to 80% fusion, 0.25% trysinization is gone down to posterity.
2, the qualification of AD-MSCs
(1) get P3 for AD-MSCs, flow cytometer detection cell surface marker, streaming result is as table 1.
The detection of table 1AD-MSCs surface marker
wherein, positive mark's thing CD29, CD73, CD90, CD49d express and are greater than 95%, and negative marker thing CD14, CD34, CD45, HLA-DR express lower than 2%, and proving this cell is MSCs.
3, AD-MSCs is differentiation-inducing to epidermic cell: get the AD-MSCs of P3 with 5*10
5/ hole is inoculated in 6 orifice plates, spend the night adherent after change inducing culture continuous induction 8d of the present invention into, blank group and positive controls are set simultaneously.Divide into groups as shown in table 2.
The differentiation-inducing grouping of table 2
4, immunocyte fluorescent dye detects the expression of CKl9, P63, β 1-integrin: the cell number often counting CKl9, P63, β 1-integrin stained positive under the group equal random selecting of cell climbing sheet 5 light microscopics under (× 200) visual field.Test repetition 3 times.
Result: immunocyte fluorescent dye finds, blank group is without the cell of CKl9, P63, β 1-integrin stained positive.Inductor CKl9, P63, β 1-integrin staining positive cells number of the present invention is all obvious more than experimental group two (positive control), and illustrate that inductor of the present invention is obviously better than traditional inductor, as accompanying drawing 5 shows, result has statistical significance.Accompanying drawing 2,3,4 shows the cell of CKl9, P63, β 1-integrin stained positive respectively.
The inductor that embodiment 6 adopts embodiment 4 to prepare, carries out the differentiation-inducing experiment of epidermic cell to umbilical cord mesenchymal stem cells (UC-MSCs)
1, the separation and Culture (tissue mass cell culture) of UC-MSCs
(1) in umbilical cord acquisition bottle, 30ml physiological saline is added, primary wash umbilical cord.
(2) aseptic nipper transfer umbilical cord is in 10cm sterile petri dish, and aseptic operation to be cut umbilical cord scissors into about 2cm number section, adds brine blood clot, removes bloodstain washings limpider until basic.
(3) blood vessel is rejected: walk formula by blood vessel spiral and reject two arteries, a vein.
(4) magnificent Tong Shi glue is separated: the white connective tissue between amnion and blood vessel is Wal Tong Shi glue, is torn, put into sterilized petri dishes with long handle pincers.
(5) Washed Red Blood Cells: add 10ml water for injection in sterilized petri dishes, washing colloids 1min, wash 2 times altogether.
(6) colloid is weighed: be moved in 50ml centrifuge tube by the magnificent Tong Shi glue after washing, record of weighing.
(7) tissue homogenate: with aseptic long handle operating scissors, in centrifuge tube, tissue shear is cut into 1mm
3the tissue homogenate block of left and right, shear time 15 ~ 20min.
(8) constant volume kind bottle: according to gel weight, add appropriate umbilical cord MSC special culture media, constant volume tissue homogenate concentration is 0.4g/ml, after electric pipettor piping and druming homogenate evenly, every 75cm
2inoculation 1ml tissue homogenate (i.e. 0.4g/75cm
2), add 15ml umbilical cord MSC serum free medium (LONZA, 00190632) in T75 culturing bottle, cultivate after rocking culturing bottle mixing.
(9) culture condition: carbonic acid gas fixed temperature and humidity incubator, parameter: (37 ± 0.5) DEG C, carbonic acid gas volume fraction is (5 ± 0.2) %.
(10) original cuiture: primary 4 ~ 5d half amount changes liquid once, (intend adopting plate centrifuge, in culturing bottle after initial centrifugation, remove half amount substratum, add half amount fresh culture, resuspension uterus tissue pieces), after original cuiture 14 ~ 16d, P0 reaches 80% ~ 90% for cell density, had digestive transfer culture.
(11) P0 is for passage: digestive ferment is 0.125%Trypsin-0.01%EDTA solution, every 75cm
2add 1 ~ 1.5ml digestive ferment solution, digestion time is 40 ~ 60sec, adds at the bottom of serum free medium 2 ~ 3ml blows and beats bottle repeatedly, move in 50ml centrifuge tube, add 5ml normal saline flushing bottle wall in former culturing bottle, add in centrifuge tube and be settled to 50ml, statistics cell total amount.
(12) wash inoculation to go down to posterity: parameter of noncentricity, 250g/min, 10min, after removing supernatant liquor, inoculate after adding fresh medium constant volume in new culturing bottle, passage cell density is (6 ~ 8) × 10
5/ 75cm
2.
(13) cell cultures with go down to posterity: P1 generation after, change weekly liquid 2 times, within every 3 ~ 4 days, go down to posterity, when going down to posterity, cytogamy should 80% ~ 90%, and passage cell density is (6 ~ 8) × 10
5/ 75cm
2.
2, the qualification of UC-MSCs
(1) get P3 for umbilical cord mesenchymal stem cells, flow cytometer detection cell surface marker, streaming result is as table 3.
The detection of table 3UC-MSCs surface marker
Wherein, positive mark's thing CD29, CD73, CD90, CD105 express and are greater than 95%, and negative marker thing CD14, CD34, CD45, HLA-DR express lower than 2%, prove that this cell is MSCs.
3, UC-MSCs is differentiation-inducing to epidermic cell: get the UC-MSCs of P3 with 5*10
5/ hole is inoculated in 6 orifice plates, spend the night adherent after change inducing culture continuous induction 8d of the present invention into.Blank group and positive controls are set simultaneously.Divide into groups as shown in table 4.
The differentiation-inducing grouping of table 4
4, immunocyte fluorescent dye detects the expression of each group of CKl9, P63, β 1-integrin: the cell number often counting CKl9, P63, β 1-integrin stained positive under the group equal random selecting of cell climbing sheet 5 light microscopics under (× 200) visual field respectively, test repetition 3 times.
Result: immunocyte fluorescent dye finds, blank group is without the cell of CKl9, P63, β 1-integrin stained positive, and inductor CKl9, P63, β 1-integrin staining positive cells number of the present invention is all obvious more than experimental group two (positive control), indicate that inductor of the present invention is obviously better than traditional inductor, as accompanying drawing 6 shows, result has statistical significance.
Claims (4)
1. a mescenchymal stem cell epidermal differentiation inductor, it is characterized in that: be made up of KGF, BMP-4, PDGF-AB, VEGF, IL-2, Forskolin, after adding perfect medium, each composition activity is followed successively by KGF20 ~ 30 μ g/L, BMP-45 ~ 10 μ g/L, PDGF-AB5 ~ 10 μ g/L, VEGF10 ~ 20 μ g/L, IL-25 ~ 10 μ g/L, Forskolin1 ~ 2mg/L.
2. mescenchymal stem cell epidermal differentiation inductor according to claim 1, it is characterized in that: after adding perfect medium, each composition activity is followed successively by KGF25 μ g/L, BMP-48 μ g/L, PDGF-AB7 μ g/L, VEGF12 μ g/L, IL-27 μ g/L, Forskolin1.2mg/L.
3. mescenchymal stem cell epidermal differentiation inductor according to claim 1 and 2, is characterized in that: described perfect medium is Keratinocyte-SFM, the special serum free medium of human mesenchymal stem cell or DMEM/F12+10%FBS.
4. the application method of a mescenchymal stem cell epidermal differentiation inductor, it is characterized in that: required to add respectively in perfect medium according to activity by each composition of mescenchymal stem cell epidermal differentiation inductor, perfect medium is Keratinocyte-SFM, the special serum free medium of human mesenchymal stem cell or DMEM/F12+10%FBS.
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