CN105062970B - A kind of derivant and induction differentiation complete medium that mescenchymal stem cell is induced to neuroblast - Google Patents
A kind of derivant and induction differentiation complete medium that mescenchymal stem cell is induced to neuroblast Download PDFInfo
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Abstract
The invention belongs to stem cell induction differentiation technique field, and in particular to a kind of derivant and inducing culture.A kind of derivant that mescenchymal stem cell is induced to neuroblast, includes resveratrol, icariin, hydrocortisone, VEGF, IGF I, EPO.A kind of to induce the induction of neuroblast to break up complete medium mescenchymal stem cell, constituent is：Contain 30~50 μm of ol of resveratrol, 5~10 μm of ol of icariin, 5~10nmol of hydrocortisone, the μ g of VEGF 10~20, the μ g of IGF I 5~10, the μ g of EPO 2~5, surplus behaviour mesenchymal stem cell serum-free complete medium in per inducing culture described in 1000ml.Complete medium is broken up in the induction that mescenchymal stem cell is induced to neuroblast of the present invention, traditional Chinese medicine ingredients resveratrol, icariin is used to combine hydrocortisone and growth factor VEGF, IGF I, EPO co-induction Derived from Mesenchymal Stem Cells directed differentiations for nerve cell, selected inducing component is nontoxic, induced efficiency is high, induction time is short, and induction acquisition neural cell activity is good, without repulsion after cell transplantation, it is safe without ethics problem.
Description
Technical field
The invention belongs to stem cell induction differentiation technique field, and in particular to a kind of derivant and inductive differentiation medium.
Background technology
Since nineteen ninety-five reports that mescenchymal stem cell (MSC) is applied to clinical test first, the MSC that cultivates now by
It is widely used in clinical experimental study, including graft versus host disease(GVH disease)（GVHD）, congestive heart failure, acute myocardial infarction, 2 type glycosurias
Disease, spinal cord injury, cartilage and bone injury, Crohn disease etc..On the other hand, with constantly find new mescenchymal stem cell come
Source, such as fat, amniotic fluid, placenta, umbilical cord, bleeding of the umbilicus, have expanded the available sources of mescenchymal stem cell, thin to be done based on mesenchyma
The various treatment means of born of the same parents provide more preferably seed cell sources.The market-oriented gate in external stem cell drugs field is
Through opening, existing 4 granted listings of stem cell drugs, include 3 that South Korea ratifies：Acute myocardial infarction medicine
Hearticellgram-AMI, articular cartilage defect medicine CartiStem, treat the concurrent anal fistula of complexity clone disease
Autologous stem cells medicine Cuepistem, and the OTC mesenchyma for the treatment of children's graft versus host disease(GVH disease) of Canada's approval
Stem cell drugs Prochymal.New Zealand's medical control office and Drug Administration of Switzerland also have approved Prochymal at this afterwards
2 national list marketing power.But at present, China not yet ratifies any stem cell drugs or launch, and China is 2004
Year, have approved three stem cell drugs altogether within 2005 and 2006 and enter clinical experimental stage.According to Chinese SFDA websites
Query Information, these three medicines be respectively " marrow primary mesenchymal stem cells ", " autologous bone marrow mesenchymal stem cells parenteral solution ",
" mescenchymal stem cell heart infarction parenteral solution ".In addition, not yet start to accept the other any stem cell projects of examination ＆ approval.Therefore, need
Accelerate domestic stem cell basic and clinic studies, strive catching up with and surpassing America and Europe.Nervous system injury and nerve retrograde affection such as pa
Golden Sen Shi diseases, Alzheimer disease, Huntington's disease etc., it is always the problem for perplexing human health, and cell transplantation is to cure
The hope of such disease.Mainly embryonic stem cell and nerve cord currently used for transplantation treatment central lesion is thin
Born of the same parents, but because donor source is difficult, the problems such as ethics objection, immunological rejection and Cell survival difficulty, development is empty
Between it is limited.
Source for mesenchymal stem cells is extensive, materials are easy, is easy to autotransplantation, has powerful multiplication capacity, in vitro
Its multi-lineage potential can be remained during long-term cultivation, specified conditions induction under can be divided into skeletonization, cartilage,
Tendon, myocyte, adipocyte, nerve cell and liver cell etc., it is a kind of preferable tissue engineering seed cell.Mesenchyma is done
Cell has become the focus of stem-cell research, and nerve cell is divided into after induction, available for the nervous system disease and damage
Reparation.
The differentiation of stem cell is that portion gene is optionally activated or differential expression, so as to control cell phenotype and egg
The distribution of specific of white matter.Derived from Mesenchymal Stem Cells is the activation that particular cell types depend primarily on gene, and extracellular
An important factor for various factor patterns and concentration are then gene activations in microenvironment.The induction of mesenchymal stem cells into nerve cells
Differentiation at least has both sides meaning：On the one hand, disclosing the gene mechanism of cell has plasticity, and the change of external environment can
The versatility of cell is inspired, so as to surmount the inherent limitations of origin germinal layer, is divided into and carries this germinal layer/other germinal layers mark
Cell.On the other hand, as the new way for producing transplanting nerve cell, treat nervous system injury for clinical transplantation and provide
Chance.
The research report that nerve cell is induced to differentiate into derived mesenchymal stem cells in vitro of document report is very more at present,
Mainly have：（1）Traditional chemical inducer（Antioxidant）Method is such as：β-ME（Beta -mercaptoethanol）、DMSO（Dimethyl sulfoxide (DMSO)）、
BHA（Butylated hydroxy anisole）Combined induction etc.；（2）Growth factor-induced method is such as：Nerve growth factor, epidermal growth factor
Sub- EGF, basic fibroblast growth factor bFGF, vitamin A acid（RA）Induction etc. alone or in combination；（3）Growth factor is with changing
Learn derivant joint；（4）Co-culture or with the conditioned medium close to physiological status；（5）Gene transfects；（6）Salviamiltiorrhizabung, Huang
A kind of reed mentioned in ancient books glycosides etc..
Although Derived from Mesenchymal Stem Cells can be efficiently, rapidly nerve cell by traditional chemical inducer method,
Cell mortality is higher（There is chemical inducer certain toxicity, BHA, DMSO etc. can make cell mutation, exist for cell transplantation
The danger of oncogenicity）；Although cell survival rate is high after the induction of growth factor-induced method, induction time is long, induced efficiency is low.
Gene transfection induction suffers from cancered potential risk because gene alteration is present.
The content of the invention
The purpose of the present invention is the defects of presence for existing derivant and abductive approach, there is provided it is a kind of it is safe and non-toxic,
Induced efficiency is high, short derivant and the induction differentiation culture completely that mescenchymal stem cell is induced to neuroblast of induction time
Base.
To achieve the above object, the technical solution adopted by the present invention is：It is a kind of that mescenchymal stem cell induction is thin into nerve
The derivant of born of the same parents, include resveratrol, icariin, hydrocortisone, VEGF, IGF-I, EPO.
A kind of to induce the induction of neuroblast to break up complete medium mescenchymal stem cell, its constituent is：Often
In inducing culture described in 1000ml containing 30~50 μm of ol of resveratrol, 5~10 μm of ol of icariin, hydrocortisone 5~
The μ g of 10nmol, VEGF 10~20, the μ g of IGF-I 5~10, the μ g of EPO 2~5, surplus behaviour mesenchymal stem cell serum-free culture
Base.
Complete medium is broken up in the described induction that mescenchymal stem cell is induced to neuroblast, and its constituent is：
Contain 40 μm of ol of resveratrol, icariin 8 μm of ol, hydrocortisones in induction differentiation complete medium described in per 1000ml
The μ g of 8nmol, VEGF 15, IGF-I 7 μ g, EPO 4 μ g, surplus behaviour mesenchymal stem cell serum-free culture medium.
It is described to induce the induction of neuroblast to break up complete medium mescenchymal stem cell, by said components by matching somebody with somebody
Than filtration sterilization after mixing.
The derivant and induction differentiation complete medium that mescenchymal stem cell is induced to neuroblast of the present invention, is used
Traditional Chinese medicine ingredients resveratrol, icariin joint hydrocortisone and growth factor VEGF, IGF-I, EPO co-induction mesenchyma
Stem cell is divided into nerve cell, and selected inducing component is nontoxic, and induced efficiency is high, and induction time is short, and induction obtains thin
Cytoactive is good, safe without repulsion, no ethics problem after cell transplantation.
Brief description of the drawings
Fig. 1 is the adipose-derived mescenchymal stem cell aspect graph of people（×100）；
Fig. 2 is the nerve cell aspect graph occurred after being induced using the culture medium of the present invention（×200）；
Fig. 3 is NeuN positive expression cellular immunity cytochemical staining result figures after the culture medium induction using the present invention
（×400）；
Fig. 4 is the positive expression cellular immunity cytochemical stainings of β-Tubulin III after the culture medium induction using the present invention
Result figure（×400）；
Fig. 5 is using GFAP positive expression cellular immunity cytochemical staining result figures after the culture medium induction of the present invention（×
400）；
Fig. 6 is the positive expression immunofluorescent staining results of β-Tubulin III after the culture medium induction using the present invention
Figure（×200）；In figure：A shows the positive cells of β-Tubulin III, and take on a red color fluorescence；B shows the karyon that Hoechst33258 is redyed, and is in
Blue-fluorescence；C is a and b synthesis.
Embodiment
Experimental method in following embodiments, it is conventional method unless otherwise instructed.Utensil instrument reagent used in experiment
It can all be obtained by commercial sources.
A kind of derivant that mescenchymal stem cell is induced to neuroblast of embodiment 1, includes resveratrol, barrenwort
Glycosides, hydrocortisone, VEGF (VEGF), insulin like growth factor-1（IGF-I）, hematopoietin
（EPO）.
Complete medium is broken up in the induction that mescenchymal stem cell is induced to neuroblast of the present embodiment of embodiment 2, will
By its, each characteristic is dissolved following raw materials according, using human mesenchymal stem cell Serum-free complete medium as matrix（LONZA, goods
Number 00190632）, make each component content is for example following to be settled to 1000ml：Resveratrol（Sigma, R5010-100MG）It is 36g, excessive
Sheep leaves of pulse plants glycosides（Shanghai crystallite biology, 489-32-7,20mg）7 μm of ol, hydrocortisones（Sigma, H3160）6nmol、VEGF
（PeproTech, 96-100-20-2）10μg、IGF-I （PeproTech, 96-100-11-20）6μg、EPO（PeproTech,
CYT-201）3μg.
Complete medium is broken up in the induction that mescenchymal stem cell is induced to neuroblast of the present embodiment of embodiment 3,
By following raw materials according, by its, each characteristic is dissolved, using human mesenchymal stem cell serum free medium as matrix（LONZA, article No.
00190632）, make each component content is for example following to be settled to 1000ml：Resveratrol（Sigma, R5010-100MG）40g, excessive sheep
Leaves of pulse plants glycosides（Shanghai crystallite biology, 489-32-7,20mg）8 μm of ol, hydrocortisones（Sigma, H3160）8nmol、VEGF
（PeproTech, 96-100-20-2）15μg、IGF-I （PeproTech, 96-100-11-20）7μg、EPO（PeproTech,
CYT-201）4μg.
The inducing culture that embodiment 4 is prepared using embodiment 3, to the adipose-derived mescenchymal stem cell of people（AD-MSC）
Nerve cell induction Analytical Chemical Experiment is carried out, step is as follows：
First, prepared by human adipose mesenchymal stem cells：
1st, adipose tissue is received, the container outer wall of adipose tissue is filled with 75% alcohol wipe；
2nd, adipose tissue is dispensed, each T175 blake bottle packing adipose tissue is 50ml.10ml pipettes, remove suction nozzle,
Lower floor's red liquid is first drawn in fat acquisition bottle to discard, remaining upper-layer fat is dispensed after mixing.
3rd, adipose tissue is washed, removes haemocyte.100ml sodium chloride injections are added into T175 blake bottles, tighten lid
Son, 3 minutes are acutely rocked fully to wash adipose tissue, then static 3~5 minutes, make different phase separations, suck lower floor's water
Phase；Operated more than repeating three times, until subnatant is more limpid.
4th, clostridiopetidase A I digests：Add the preheating that equivalent is newly prepared（Half an hour is in 37 DEG C of gas bath shaking table preheating in advance）'s
Clostridiopetidase A I solution（0.1% clostridiopetidase A I compound methods：Weigh 0.1g clostridiopetidase A I powder and be dissolved in 100ml and do not add any factor
In culture medium, with 37 DEG C before preheatings）, sealed membrane sealing, acutely rock blake bottle 5~10 seconds, be placed in vibration gas bath pot, 37
DEG C, 70rpm, digest 60 minutes, acutely blake bottle was rocked 5~10 seconds every 15 minutes, until seeming more smooth.
5th, isolation medium vascular component（SVF）：Postdigestive tissue is dispensed into 50ml heart pipe with sterile 40 mesh filter screen
In, room temperature 400g is centrifuged 10 minutes, and obtained precipitation is SVF.
6th, purification precipitation：After centrifugation, SVF is deposited on centrifugation bottom of the tube, and upper strata oil is carefully removed from top to bottom with pipette
Fat and the collagenase solution of lower floor.Pay attention to：A small amount of solution is left above SVF precipitations, in order to avoid disturbance sedimentation cell.It is appropriate raw
Manage salt solution and cell is resuspended, dispel, room temperature 400g, centrifuge within 10 minutes.Centrifugation finishes, and carefully sucks supernatant, it is impossible to directly outwells.
Head of pipette should be placed in the top of centrifuge tube in order to thoroughly go out to deoil during absorption.10ml culture medium suspension cells, then
Cell is aggregated into 50ml centrifuge tubes, crosses 100 mesh sieves, again room temperature 300g, is centrifuged within 10 minutes.
7th, cell seeding：After centrifugation plus 20ml culture mediums fully mix.Based on tissue block method：Entered according to the area of blake bottle
Row cell seeding.The fat mass obtained according to inoculation 0.16ml liposuctions every square centimeter is inoculated with, i.e., in each T75 blake bottles
It is inoculated with 12ml liposuction fat masses.I.e. per 100ml adipose tissues, 8 T75 blake bottles may finally be inoculated with.Carry out untreated fat
The cell suspension conversion measured and finally given, and then inoculating cell.
8th, primitive cell culture：Horizontal blake bottle, blake bottle is positioned over carbon dioxide constant temperature and humidity incubator.Cultivate bar
Part：（37±0.5）DEG C, carbon dioxide volume fraction is（5±0.2）% .Culture medium：Human mesenchymal stem cell serum-free is trained completely
Support base（LONZA, 00190632）.
9th, liquid is changed：Original cuiture 24h, carry out full dose and change liquid.Hereafter liquid is changed every 3 days full doses, it is permanent places carbon dioxide
Constant temperature and humidity incubator is cultivated.
10th, primary cell harvests：7d or so, the area percentage of the cell clone group of original cuiture reach 70%~80%
When, digestion harvest.Digestive ferment is added in blake bottle（Digestive ferment is 0.125%Trypsin-EDTA solution, uses preceding room temperature（20
~25 DEG C）Place 15~25min, every 75 cm2Add 2ml digestion enzyme solutions）, digestion time is 5~8min, adds culture medium 2
~3ml blows and beats bottom of bottle and largely come off to cell repeatedly, moves into 50ml centrifuge tubes, and 4~5ml sodium chloride is added in original culture bottle
Parenteral solution rinses bottle wall, adds in centrifuge tube and is settled to 50ml, after pipette piping and druming suspends, the sterile strainer filterings of 100um, and filtering
Liquid is collected into 50ml centrifuge tubes, 1000rpm, 10min centrifuge washings.
11st, primary cell passes on：Remaining cell precipitation amount in single centrifuge tube is observed, is suitably merged thin in several centrifuge tubes
Born of the same parents are precipitated in 1 centrifuge tube, add appropriate culture medium, gently blow and beat resuspension cell, are settled to 30ml, and piping and druming is mixed, taken
Sample counts.1000rpm after counting, 10min secondary centrifuging.Supernatant is removed, addition culture medium is appropriate in centrifuge tube, gently blows
Resuspension cell is beaten, is seeded to after constant volume in new culture vessel, passage cell density is 5000~6000/cm2, i.e.,
（3.75~4.5）×105Individual cells/T75, according to 4.5 × 105Individual cells/T75 is passed on.Indicated on culture vessel thin
The information such as born of the same parents' algebraically and incubation time.Culture vessel is positioned over into carbon dioxide constant temperature and humidity incubator to start to cultivate, cultivates bar
Part：Carbon dioxide constant temperature and humidity incubator,（37±0.5）DEG C, carbon dioxide volume fraction is（5±0.2）%.Cultivate to cell
Merge up to 85%~90%.
2nd, fatty MSC identifications
1st, P3 fat subsitutes MSC are taken, flow cytometer detection cell surface marker, the results are shown in Table 1.
The flow cytomery P3 fat subsitutes MSC surface markers of table 1
Wherein, positive mark's thing CD29, CD73, CD90, CD49d are expressed more than 95%, negative marker thing CD14, CD34,
CD45, HLA-DR expression are less than 2%, it was demonstrated that the cell is fatty MSC.
3rd, induced lipolysis MSC is divided into nerve cell：The MSC in 3 generations is passed, 0.125%Trypsin-EDTA solution digestions are received
Collect cell, prepare cell suspension, cell counting count board living cell counting density, and adjust density 1 × 104/cm2, it is inoculated in and puts in advance
It is equipped with 24 orifice plates of the sterilization cover glass handled through poly-D-lysine, prepares cell climbing sheet.Cell is treated close to 80% fusion, it is raw
Induction differentiation, packet such as table 2 are carried out when long vigorous again.
The induced lipolysis MSC of table 2 is divided into the experiment packet of nerve cell
4th, nerve cell is identified after inducing
1st, morphological observation：Under inverted microscope, morphological change before and after fatty MSC inductions is dynamically observed.
2nd, immunocytochemical stain method（SABC methods）Detection：
（1）Collect cell climbing sheet after inducing, 0.01M PBS（PH=7.2）Rinse, 5min × 3 time, cold acetone is fixed
10min, PBS rinse 5min × 3 time；
（2）3%H2O2Solution is incubated 30min, and to eliminate the activity of endogenous peroxydase, 0.01M PBS are rinsed, 5min
× 3 times；
（3）Closing sheep blood serum is added dropwise（Reagent A）, 37 DEG C of incubation 30min, unnecessary serum is absorbed, is not washed；
（4）The anti-NeuN of mouse, the primary antibodies of rabbit-anti β-Tubulin III, the anti-GFAP of mouse, 4 DEG C of refrigerator overnights, PBS are added dropwise respectively
Rinse, 5min × 3 time；
（5）Corresponding anti-mouse or the anti-rabbit secondary antibody of biotin labeling is added dropwise（Reagent B）, 37 DEG C are incubated 1h, 0.01M PBS
Rinse, 5min × 3 time；
（6）The chain enzyme avidin of horseradish peroxidase-labeled is added dropwise（Reagent C）, 37 DEG C are incubated 30min, 0.01M PBS
Rinse, 5min × 3 time；
（7）DAB develops the color：Distilled water 1ml is taken, each drop of A, B, C composition in kit is added, after mixing, is added dropwise to above-mentioned
On the cell specimen of processing；Develop the color at room temperature, the reaction time is controlled under mirror.When being satisfied with to colour developing（General 2~10 min）Add steaming
Distilled water terminating reaction；
（8）It is conventional to be dehydrated transparent mounting：50% alcohol 1min, 70% alcohol 1min, 80% alcohol 1min, 95% alcohol 1min,
The 5min of 100% alcohol I, the 5min of 100% alcohol II, the 5min of dimethylbenzene I, the 5min of dimethylbenzene II, neutral gum mounting.
Control experiment：First antibody is replaced with 0.01M PBS, remaining step is identical.
NeuN, β-Tubulin III, GFAP expression before and after immunocytochemical method detection induction, randomly select 4
Slide, 5 visuals field are randomly selected under 200 times of visuals field, calculate the ratio of positive cell respectively.Positive cell account for total cell ratio with
Mean ± standard deviation（）Represent.Using SPSS11.0 softwares carry out statistical analysis, experimental data with（）Represent, it is more
Group compares using variance analysis, between two groups the comparison of rate use χ2Examine, P<0.05 thinks statistically significant.
3rd, immunofluorescence staining detects
Immunofluorescence dyeing step is same as above, and primary antibody is rabbit-anti β-Tubulin III, and fluorescence secondary antibody is Cy3- goat anti-rabbit iggs, cloudy
Property control with PBS replace first antibody.
Three groups of β-Tubulin III expression before and after immunofluorescence staining detection induction, and randomly select 4 glass
Piece, 5 visuals field are randomly selected under 200 times of visuals field of every slide, it is total to count the cell that positive cell sum and Hoechst are redyed
Number, calculate the ratio of the positive cells of β-Tubulin III.Positive cell account for total cell ratio with（）Represent.Using
SPSS11.0 softwares carry out statistical analysis, experimental data with（）Represent, multiple-group analysis uses variance analysis, rate between two groups
Comparison use χ2Examine, P<0.05 thinks statistically significant.
5th, result
1st, morphological observation
Chemical induction group：Fatty MSC is adherent fast after passage, and cell molecular marker for increased proliferation, cell is in homogeneous fibroblast sample
Form, as shown in Figure 1.After pre-induced 12h, the fat mesenchymal stem cell cell space of originally fusiformis is shunk, and cell edges become
Must be irregular, there are many thin projections.At the end of pre-induced, a part of cell body is rounded.After formal induction, cell body
Further shrink, rounded, triangle, it is seen that multiple projections, and branch is sent, form coniform whole end；Some projections
Gradually elongation.Significant change no longer occurs for cellular morphology after 5h, and typical neuron form is presented in most cells.Induction
Afterwards, cellular portions are dead, come off.It is dead to continue most cells floating after cultivating 5d.
Growth factor group：After induction differentiation culture, fatty MSC is substantially unchanged in 3d.5~10d parts cell body by
Cumulative big, cytoplasmic process attenuates elongation.2 weeks or so, part cell body was rounded, larger, cell space stretch out one, it is two or more prominent
Rise, projection is longer, and refractivity is good, and regional area flanking cell projection is linked to be netted, the typical nerve of cell presentation of about half
Cellular morphology.Cell survival is good, continues culture and has not seen obvious cells float, obscission in 2 weeks.
The inducing culture group of the present invention：Inducing culture of the present invention, cellular morphology change is obvious after inducing 1~2d, carefully
Born of the same parents' endochylema, cytoplasm are shunk centered on nucleus, and cell shortens, and volume diminishes, and cell forms bipolar or multipole cell body,
The Cytoplasmic shrinkage of part cell forms cell process, and cellular morphology is in small oval, star-like, tadpole type, elongated strip etc., carefully
The reflective enhancing of born of the same parents.3~5d is induced, and cell process further extends and attenuated, and part cell stretches out more projections, and refractivity is good,
Regional area flanking cell projection is linked to be netted, the typical nerve cell form of most cells presentation, as shown in Figure 2.After 7d,
Cell state is good, time-to-live length, has not seen obvious cells float, obscission.
2nd, immunocytochemical stain result
Immunocytochemical stain result shows that the coloring of NeuN positive cells karyon, endochylema coloring is shallow, as shown in Figure 3.Lure
It is that radiolucent table reaches to lead first three groups NeuN, and NeuN positive cell rates are after chemical inducer induction（72.3±2.1）%；Growth because
NeuN positive cell rates are after son induction（39.6±2.8）%；NeuN positive cell rates are after the inducing culture induction of the present invention
（86.6±4.5）%.The inducing culture induction differentiation rate of the present invention is higher than chemical inducer and growth factor（P<0.05）, it is poor
It is different statistically significant.
Immunocytochemical stain result shows, the coloring of the positive cell endochylemas of β-Tubulin III, in brown yellow granule, carefully
Karyon is not colored, as shown in Figure 4.It is that radiolucent table reaches to induce first three groups β-Tubulin III, β-Tubulin III after chemical induction
Positive cell rate is（73.2±1.3）%；The positive cell rates of β-Tubulin III are after growth factor-induced（49.6±3.1）%；This
The positive cell rates of β-Tubulin III after the induction of invention derivant（92.6±2.3）%.The inducing culture induction differentiation of the present invention
Rate is higher than chemical inducer and growth factor（P<0.05）, difference is statistically significant.
Immunocytochemical stain result shows, the coloring of GFAP positive cells endochylema, and in brown yellow granule, nucleus is not
Color, as shown in Figure 5.It is that radiolucent table reaches to induce first three groups GFAP, and GFAP positive cell rates are after chemical induction（13.3±
2.1）%；GFAP positive cell rates are after growth factor-induced（32.1±1.3）%；GFAP positive cell rates after present invention induction
（16.6±3.6）%.The inducing culture induced lipolysis mescenchymal stem cell of the present invention is to Astrocyte differentiation rate less than life
The long factor（P<0.05）, difference is statistically significant, slightly above chemical inducer.
3rd, immunofluorescence dyeing result
Immunofluorescence technique coloration result shows, the endochylemas of the positive cells of β-Tubulin III takes on a red color fluorescence, such as a institutes in Fig. 6
Show, the karyon that Hoechst33258 is redyed is in blue-fluorescence, as shown in b in Fig. 6.It is the moon to induce first three groups β-Tubulin III
Property expression, the positive cell rates of β-Tubulin III are after chemical induction（73.4±2.1）%；β-Tubulin after growth factor-induced
III positive cell rate is（48.5±1.8）%；The positive cell rates of β-Tubulin III are after the inducing culture induction of the present invention
（91.8±1.3）%.The inducing culture induction differentiation rate of the present invention is higher than chemical inducer and growth factor（P<0.05）, it is poor
It is different statistically significant.
3 three groups of induction results contrasts of table（N=20,）
Note P*<0.05 relative to growth factor group and chemical induction group, P*△<0.05 relative to growth factor group.
To sum up, as shown in table 3, it is high with the positive cell rates of NeuN, β-Tubulin III after the inducing culture induction of the present invention
In chemical induction and growth factor-induced（P<0.05）, GFAP positive cell rates are less than growth factor-induced（P<0.05）, explanation
The inducing culture induced efficiency of the present invention is higher than chemical inducer and growth factor, and cell primary differentiation is nerve after induction
Cell, rather than Deiter's cells.
The inducing culture that embodiment 5 is prepared using embodiment 3, to Mesenchymal Stem Cells from Umbilical Cord（UC-MSC）Carry out
Nerve cell induction Analytical Chemical Experiment, step are as follows：
First, umbilical cord MSC is separately cultured（Tissue mass cell culture）
1st, 30ml physiological saline, primary wash umbilical cord are added into umbilical cord acquisition bottle
2nd, aseptic nipper shifts umbilical cord into 10cm sterile petri dish, and aseptic operation is cut is cut into about 2cm numbers section by umbilical cord, adds
Enter brine blood clot, until basic removal bloodstain cleaning solution is limpider.
3rd, blood vessel is rejected：Formula, which is walked, by blood vessel spiral rejects two arteries, a vein.
4th, magnificent Tong Shi glue is separated：White connective tissue between amnion and blood vessel is Wal Tong Shi glue, uses long handle
Pincers is torn, and is put into sterilized petri dishes.
5th, Washed Red Blood Cells：10ml waters for injection are added into sterilized petri dishes, washing colloids 1min, are washed 2 times altogether.
6th, colloid is weighed：Magnificent Tong Shi glue after washing is moved into 50ml centrifuge tubes, record of weighing.
7th, tissue homogenate：With sterile long handle operating scissors, tissue is cut into 1mm in centrifuge tube3The tissue homogenate of left and right
Block, shear time 15~20 minutes.
8th, constant volume kind bottle：According to gel weight, appropriate umbilical cord MSC special culture medias are added, constant volume tissue homogenate concentrations are
0.4g/ml, after electric pipettor piping and druming homogenate uniformly, per 75cm2It is inoculated with 1ml tissue homogenate（That is 0.4g/75cm2）, T75 cultures
15ml umbilical cord MSC serum free mediums are added in bottle（LONZA, 00190632）, rock after blake bottle mixes and cultivate.
9th, condition of culture：Carbon dioxide constant temperature and humidity incubator, parameter：（37±0.5）DEG C, carbon dioxide volume fraction is
（5±0.2）%.
10th, original cuiture：Primary 4~5 days half amounts change liquid once,（Plan use plate centrifuge, in blake bottle tentatively from
After the heart, half amount culture medium is removed, adds half amount fresh culture, resuspension uterus tissue pieces）, original cuiture 14~16 days it
Afterwards, P0 reaches 80%~90% for cell density, had digestive transfer culture.
11st, P0 is for passage：Digestive ferment is 0.125%Trypsin-0.01% EDTA solution, every 75 cm2Addition 1~
1.5ml digests enzyme solutions, and digestion time is 40~60sec, adds 2~3ml of serum free medium and blows and beats bottom of bottle repeatedly, moves into
In 50ml centrifuge tubes, 5ml normal saline flushing bottle walls are added in original culture bottle, adds in centrifuge tube and is settled to 50ml, statistics is thin
Born of the same parents' total amount.
12nd, washing inoculation passage：Parameter of noncentricity, 250g/min, 15min, after removing supernatant, add fresh medium and determine
It is inoculated with after appearance in new blake bottle, passage cell density is（6~8）x105/75cm2。
13rd, cell culture and passage：After P1 generations, liquid being changed weekly 2 times, being passed within every 3~4 days, cell fusion should be during passage
80%~90%, passage cell density is（6~8）*105/75cm2。
2nd, umbilical cord MSC identification
（1）P3 is taken for umbilical cord MSC, flow cytometer detection cell surface marker, streaming result such as table 4.
The detection of the umbilical cord MSC surface markers of table 4
Wherein, positive mark's thing CD29, CD73, CD90, CD105 are expressed more than 95%, negative marker thing CD14, CD34,
CD45, HLA-DR expression are less than 2%, it was demonstrated that the cell is umbilical cord MSC.
3rd, inducing umbilical cord mesenchymal stem is divided into nerve cell：Pass the umbilical cord MSC, 0.125%Trypsin- in 3 generations
EDTA solution digestions collect cell, prepare cell suspension, cell counting count board living cell counting density, and adjust density 1 × 104/
cm2, it is inoculated in and is placed with advance in 24 orifice plates of the sterilization cover glass handled through poly-D-lysine, prepares cell climbing sheet.Treat thin
Born of the same parents carry out induction differentiation, packet such as table 5 again close to 80% fusion when growing vigorous.
The induction of cord MSC of table 5 is divided into the experiment packet of nerve cell
4th, nerve cell is identified after inducing
Method and steps is identified nerve cell after induction, from morphological observation, Tri-labeling method with embodiment 4
Learn, three groups of derivant effects in Immunofluorescence test tripartite face contrast table 5.
5th, result
1st, morphological observation：As a result with embodiment 4, after induction three groups of cellular morphologies to nerve cell direction change, but
Chemical inducer group has more cells float death, and the inducing culture group cell state of the present invention is good, time-to-live length, not
See obvious cells float, obscission.
2nd, immunocytochemical stain result, 6 are shown in Table.
3rd, immunofluorescence dyeing result, 6 are shown in Table.
6 three groups of induction results contrasts of table（N=20,）
Note P*<0.05 relative to growth factor group and chemical induction group, P*△<0.05 relative to growth factor group
To sum up, as shown in table 6, it is positive with NeuN, β-Tubulin III after the inducing culture induction of cord MSC of the present invention
Cell rate is higher than chemical induction and growth factor-induced（P<0.05）, GFAP positive cell rates are less than growth factor-induced（P<
0.05）, illustrate that the inducing culture induced efficiency of the present invention is higher than chemical inducer and growth factor, and cell is main after induction
It is divided into nerve cell, rather than Deiter's cells.
Claims (2)
1. a kind of induce the induction of neuroblast to break up complete medium mescenchymal stem cell, its constituent is：Often
Described in 1000ml induction differentiation complete medium in containing 30~50 μm of ol of resveratrol, 5~10 μm of ol of icariin,
5~10nmol of hydrocortisone, the μ g of VEGF 10~20, the μ g of IGF-I 5~10, the μ g of EPO 2~5, surplus is the human world
Mesenchymal stem cells Serum-free complete medium.
2. the inducing culture according to claim 1, it is characterised in that：Induction differentiation culture completely described in per 1000ml
Contain 40 μm of ol of resveratrol, 8 μm of ol of icariin, the μ g of hydrocortisone 8nmol, VEGF 15, IGF-I 7 μ g, EPO in base
4 μ g, surplus behaviour mesenchymal stem cell serum-free culture medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510525294.4A CN105062970B (en) | 2015-08-25 | 2015-08-25 | A kind of derivant and induction differentiation complete medium that mescenchymal stem cell is induced to neuroblast |
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| CN107058225B (en) * | 2017-02-10 | 2020-06-23 | 郑州大学 | Compound induction culture medium and method for inducing umbilical cord mesenchymal stem cells into neuron-like cells by adopting culture medium |
| CN109852654B (en) * | 2018-12-28 | 2021-02-26 | 广州润虹医药科技股份有限公司 | Composition capable of inducing stem cells to secrete cytokines and application thereof |
| CN109694851B (en) * | 2019-01-16 | 2021-03-16 | 吉林省拓华生物科技有限公司 | Inducing composition and inducing differentiation culture solution of human mesenchymal stem cells, and in-vitro inducing method and application thereof |
| CN113106054B (en) * | 2020-01-13 | 2022-10-14 | 青岛瑞思德生物科技有限公司 | Inducer for inducing and differentiating mesenchymal stem cells into islet cells |
| CN111394311B (en) * | 2020-04-20 | 2022-07-01 | 青岛瑞思德生物科技有限公司 | Serum-free complete culture medium for inducing mesenchymal stem cells to differentiate into corneal epithelial cells |
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