CN105062970A - Inducer and induced differentiation complete medium for inducing mesenchymal stem cells into nerve cells - Google Patents
Inducer and induced differentiation complete medium for inducing mesenchymal stem cells into nerve cells Download PDFInfo
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention belongs to the technical field of stem cell induction differentiation, particularly relates to an inducer and an induction medium, and discloses an inducer and induced differentiation complete medium for inducing mesenchymal stem cells into nerve cells. The inducer for inducing the mesenchymal stem cells into the nerve cells comprises resveratrol, icariin, hydrocortisone, VEGF, IGF-I and EPO. Every one thousand milliliters of the induced differentiation complete medium for inducing the mesenchymal stem cells into nerve cells comprises 30-50 micromoles of resveratrol, 5-10 micromoles of icariin, 5-10 micromoles of hydrocortisone, 10-20 micrograms of VEGF, 5-10 micrograms of IGF-I, 2-5 micrograms of EPO and the balance mesenchymal stem cell serum-free complete media. According to the induced differentiation complete medium for inducing the mesenchymal stem cells into the nerve cells, the traditional Chinese medicine ingredients of resveratrol and icariin are combined with hydrocortisone and the growth factors of VEGF, IGF-I and EPO to synergistically induce the mesenchymal stem cells to be directionally differentiated into the nerve cells, and the selected induction components are all free of poison, high in induction efficiency, short in induction time, good in activity of the nerve cells obtained through induction, free of rejection after cell transplantation, free of ethical issues and 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 inductor and inductive differentiation medium.
Background technology
Since nineteen ninety-five reported first mescenchymal stem cell (MSC) is applied to clinical trial, the MSC of present cultivation is widely used in clinical experimental study, comprise graft versus host disease (GVH disease) (GVHD), congestive heart failure, acute myocardial infarction, diabetes B, Spinal injury, cartilage and bone injury, Crohn's disease etc.On the other hand, along with constantly finding new source for mesenchymal stem cells, as fat, amniotic fluid, placenta, umbilical cord, bleeding of the umbilicus etc., expand the available sources of mescenchymal stem cell, for providing how better seed cell source based on the various treatment meanss of mescenchymal stem cell.The market-oriented gate in external stem cell drugs field is opened, existing 4 granted listings of stem cell drugs, comprise 3: the acute myocardial infarction medicine Hearticellgram-AMI of Korea S's approval, the autologous stem cells medicine Cuepistem of articular cartilage defect medicine CartiStem, treatment complicacy clone disease concurrent anal fistula, and the OTC (over-the-counter) mescenchymal stem cell medicine Prochymal of the treatment children graft versus host disease (GVH disease) of Canada's approval.New Zealand's medical control office and Drug Administration of Switzerland also have approved Prochymal these 2 national list marketing power afterwards.But at present, China not yet ratifies any stem cell drugs or launch, China have approved three stem cell drugs altogether 2004,2005 and 2006 and enters clinical experimental stage.According to the Query Information of Chinese SFDA website, these three medicines are " marrow primary mesenchymal stem cells ", " autologous bone marrow mesenchymal stem cells injection liquid ", " mescenchymal stem cell heart stalk injection liquid " respectively.In addition, not yet start to accept other any stem cell project of examination & approval.Therefore, need to accelerate domestic stem cell basic and clinic studies, strive catching up with and surpassing America and Europe.Nervous system injury and nerve retrograde affection, as Parkinson's disease, alzheimer's disease, Huntington's disease etc., be a difficult problem for puzzlement human health, and Transplanted cells are the hope of curing this type of disease always.At present for mainly embryonic stem cell and the neural stem cell of transplantation treatment central nervous system injury, but due to donor source difficulty, the problems such as ethics objection, immunological rejection and Cell survival difficulty, development space is limited.
Source for mesenchymal stem cells extensively, draw materials easily, be convenient to autotransplantation, there is powerful multiplication capacity, its multi-lineage potential can be remained in vitro in long-term cultivation process, can be divided into skeletonization, cartilage, tendon, myocyte, adipocyte, neurocyte and liver cell etc. under specified conditions induction, be a kind of desirable tissue engineering seed cell.Mescenchymal stem cell has become the focus of stem-cell research, is divided into neurocyte, can be used for the reparation of nervous system disorders and damage after induction.
The differentiation of stem cell is that portion gene is optionally activated or differential expression, thus controls the distribution of specific of cell phenotype and protein.Derived from Mesenchymal Stem Cells is the activation that particular cell types depends primarily on gene, and in extracellular microenvironment, various factor pattern and concentration are then the important factors of gene activation.The differentiation-inducing meaning at least with two aspects of mesenchymal stem cells into nerve cells: on the one hand, the gene mechanism disclosing cell has plasticity-, the change of external environment can inspire the versatility of cell, thus surmount the inherent limitations of origin germinal layer, be divided into the cell carrying this germinal layer/other germinal layer mark.On the other hand, as the new way producing transplanting neurocyte, for clinical transplantation treatment nervous system injury offers an opportunity.
The research report that the utilization derived mesenchymal stem cells in vitro of current bibliographical information is induced to differentiate into neurocyte is very many, mainly contains: (1) traditional chemical inducer (antioxidant) method is as β-ME(beta-mercaptoethanol), DMSO(dimethyl sulfoxide (DMSO)), BHA(butylated hydroxy anisole) combined induction etc.; (2) growth factor-induced method is as nerve growth factor, and Urogastron EGF, Prostatropin bFGF, vitamin A acid (RA) are separately or combined induction etc.; (3) somatomedin is combined with chemical inducer; (4) Dual culture or with the conditioned medium close to physiological status; (5) gene transfection; (6) salviamiltiorrhizabung, baicalin etc.
Although Derived from Mesenchymal Stem Cells can be neurocyte by traditional chemical inducer method efficiently, rapidly, but cell mortality is higher, and (chemical inducer has certain toxicity, BHA, DMSO etc. can make cell mutation, there is the danger of tumorigenicity for Transplanted cells); 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 is because there is the potential risk of suffers from cancer in gene alteration.
Summary of the invention
The object of the invention is the defect existed for existing inductor and induction method, provide a kind of safety non-toxic, induced efficiency high, what induction time was short induces the inductor of neuroblast and differentiation-inducing perfect medium by mescenchymal stem cell.
For achieving the above object, the technical solution used in the present invention is: a kind of inductor of mescenchymal stem cell being induced neuroblast, comprises trans-resveratrol, icarin, hydrocortisone, VEGF, IGF-I, EPO.
A kind of differentiation-inducing perfect medium of mescenchymal stem cell being induced neuroblast, its moiety for: containing trans-resveratrol 30 ~ 50 μm of ol, icarin 5 ~ 10 μm of ol, hydrocortisone 5 ~ 10nmol, VEGF10 ~ 20 μ g, IGF-I5 ~ 10 μ g, EPO2 ~ 5 μ g in inducing culture described in every 1000ml, surplus behaviour mesenchymal stem cell serum-free culture medium.
The described differentiation-inducing perfect medium of mescenchymal stem cell being induced neuroblast, its moiety for: containing trans-resveratrol 40 μm of ol, icarin 8 μm of ol, hydrocortisone 8nmol, VEGF15 μ g, IGF-I7 μ g, EPO4 μ g in differentiation-inducing perfect medium described in every 1000ml, surplus behaviour mesenchymal stem cell serum-free culture medium.
The described differentiation-inducing perfect medium of mescenchymal stem cell being induced neuroblast, by said components by filtration sterilization after proportioning mixing.
Of the present inventionly mescenchymal stem cell is induced the inductor of neuroblast and differentiation-inducing perfect medium, employing traditional Chinese medicine ingredients trans-resveratrol, icarin associating hydrocortisone and growth factor VEGF, IGF-I, EPO co-induction Derived from Mesenchymal Stem Cells are neurocyte, selected inducing component is all nontoxic, induced efficiency is high, induction time is short, and it is good that induction obtains cytoactive, without repelling after Transplanted cells, without ethics problem, security is high.
Accompanying drawing explanation
Fig. 1 is the adipose-derived mescenchymal stem cell aspect graph (× 100) of people;
Fig. 2 is the neurocyte aspect graph (× 200) occurred after adopting substratum of the present invention induction;
Fig. 3 adopts rear NeuN positive expression cellular immunization cytochemical staining result figure (× 400) of substratum of the present invention induction;
Fig. 4 adopts rear β-Tubulin III positive expression cellular immunization cytochemical staining result figure (× 400) of substratum of the present invention induction;
GFAP positive expression cellular immunization cytochemical staining result figure (× 400) after Fig. 5 adopts substratum of the present invention to induce;
Fig. 6 adopts rear β-Tubulin III positive expression immunofluorescent staining result figure (× 200) of substratum of the present invention induction; In figure: a shows β-Tubulin III positive cell, and take on a red color fluorescence; B shows the karyon that Hoechst33258 redyes, in blue-fluorescence; C is the synthesis of a and b.
Embodiment
Experimental technique in following embodiment, if no special instructions, is ordinary method.Test utensil instrument reagent used all to obtain by commercial sources.
Mescenchymal stem cell is induced the inductor of neuroblast by embodiment 1 one kinds, comprise trans-resveratrol, icarin, hydrocortisone, vascular endothelial growth factor (VEGF), insulin like growth factor-1 (IGF-I), erythropoietin (EPO).
The differentiation-inducing perfect medium of mescenchymal stem cell being induced neuroblast of embodiment 2 the present embodiment, by following raw materials according by its separately characteristic dissolve, with human mesenchymal stem cell Serum-free complete medium for matrix (LONZA, article No. 00190632), each component concentration is made to be settled to 1000ml as following: trans-resveratrol (Sigma, R5010-100MG) 36g, (Shanghai crystallite is biological for icarin, 489-32-7, 20mg) 7 μm of ol, hydrocortisone (Sigma, H3160) 6nmol, VEGF(PeproTech, 96-100-20-2) 10 μ g, IGF-I(PeproTech, 96-100-11-20) 6 μ g, EPO(PeproTech, CYT-201) 3 μ g.
The differentiation-inducing perfect medium of mescenchymal stem cell being induced neuroblast of embodiment 3 the present embodiment, by following raw materials according by its separately characteristic dissolve, with human mesenchymal stem cell serum free medium for matrix (LONZA, article No. 00190632), each component concentration is made to be settled to 1000ml as following: trans-resveratrol (Sigma, R5010-100MG) 40g, (Shanghai crystallite is biological for icarin, 489-32-7, 20mg) 8 μm of ol, hydrocortisone (Sigma, H3160) 8nmol, VEGF(PeproTech, 96-100-20-2) 15 μ g, IGF-I(PeproTech, 96-100-11-20) 7 μ g, EPO(PeproTech, CYT-201) 4 μ g.
The inducing culture that embodiment 4 adopts embodiment 3 to prepare, carry out the differentiation-inducing experiment of neurocyte to the adipose-derived mescenchymal stem cell of people (AD-MSC), step is as follows:
One, human adipose mesenchymal stem cells preparation:
1, fatty tissue is received, with the container outer wall of the alcohol wipe dress fatty tissue of 75%;
2, packing fatty tissue, each T175 culturing bottle packing fatty tissue is 50ml.10ml transfer pipet, removes suction nozzle, first draws lower floor's red liquid discard in fat acquisition bottle, carries out packing after the mixing of residue upper-layer fat.
3, fatty tissue is washed, removing hemocyte.In T175 culturing bottle, add 100ml sodium chloride injection, tighten lid, acutely rock 3 minutes fully to wash fatty tissue, then static 3 ~ 5 minutes, difference is separated, sucks lower floor's aqueous phase; Repeat above operation three times, until subnatant is comparatively limpid.
4, collagenase I digests: collagenase I solution (the 0.1% collagenase I compound method: take 0.1g collagenase I powder dissolution and do not add in the substratum of any factor in 100ml adding the preheating (half an hour is in the gas bath shaking table preheating of 37 DEG C in advance) that equivalent is newly prepared, with 37 DEG C of preheatings before), sealed membrane seals, acutely rock culturing bottle 5 ~ 10 seconds, be placed in vibration gas bath pot, 37 DEG C, 70rpm, digest 60 minutes, culturing bottle is acutely rocked 5 ~ 10 seconds, until seem comparatively level and smooth every 15 minutes.
5, isolation medium vascular component (SVF): be dispensed in the core barrel of 50ml by aseptic 40 mesh filter screens of postdigestive tissue, centrifugal 10 minutes of room temperature 400g, the precipitation obtained is SVF.
6, purification precipitation: after centrifugal, SVF is deposited on bottom centrifuge tube, carefully removes the collagenase solution of upper strata grease and lower floor from top to bottom with transfer pipet.Attention: leave a small amount of solution, in order to avoid disturbance sedimentation cell above SVF precipitation.Appropriate physiological saline re-suspended cell, dispels, room temperature 400g, and 10 minutes centrifugal.Centrifugal complete, carefully suck supernatant liquor, can not directly outwell.During absorption, head of pipette should be placed in the top of centrifuge tube so that go out to deoil thoroughly.10ml substratum suspension cell, is then aggregated into cell in 50ml centrifuge tube, and cross 100 mesh sieves, again room temperature 300g, 10 minutes centrifugal.
7, cell seeding: add 20ml substratum after centrifugal and fully mix.Based on tissue block method: the area according to culturing bottle carries out cell seeding.The fat quantity obtained according to every square centimeter of inoculation 0.16ml liposuction is inoculated, and namely inoculates 12ml liposuction fat quantity in each T75 culturing bottle.I.e. every 100ml fatty tissue, finally can inoculate 8 T75 culturing bottles.The cell suspension conversion carried out untreated fat quantity and finally obtain, and then inoculating cell.
8, primary cell culture: horizontal culturing bottle, is positioned over carbonic acid gas fixed temperature and humidity incubator by culturing bottle.Culture condition: (37 ± 0.5) DEG C, carbonic acid gas volume fraction is (5 ± 0.2) %.Substratum: human mesenchymal stem cell Serum-free complete medium (LONZA, 00190632).
9, change liquid: original cuiture 24h, carry out full dose and change liquid.After this change liquid every 3 days full doses, place carbonic acid gas fixed temperature and humidity incubator and cultivate.
10, primary cell results: about 7d, when the area percentage of the cell clone group of original cuiture arrives 70% ~ 80%, digestion results.In culturing bottle, adding digestive ferment, (digestive ferment is 0.125%Trypsin-EDTA solution, uses front room temperature (20 ~ 25 DEG C) to place 15 ~ 25min, every 75cm
2add 2ml digestive ferment solution), digestion time is 5 ~ 8min, add at the bottom of substratum 2 ~ 3ml blows and beats bottle repeatedly and come off to cell major part, move in 50ml centrifuge tube, in former culturing bottle, add 4 ~ 5ml sodium chloride injection washing bottle wall, add in centrifuge tube and be settled to 50ml, after transfer pipet piping and druming suspends, the aseptic strainer filtering of 100um, filtered liquid is collected in 50ml centrifuge tube, 1000rpm, 10min centrifuge washing.
11, primary cell goes down to posterity: observe remaining cell precipitation amount in single centrifuge tube, and suitably merge in several centrifuge tube in cell precipitation to 1 centrifuge tube, add appropriate substratum, blow and beat resuspension cell gently, be settled to 30ml, piping and druming mixes, sampling counting.1000rpm, 10min secondary centrifuging after counting.Remove supernatant liquor, in centrifuge tube, add substratum in right amount, blow and beat resuspension cell gently, be seeded in new culture vessel after constant volume, passage cell density is 5000 ~ 6000/cm
2, i.e. (3.75 ~ 4.5) × 10
5individual cells/T75, according to 4.5 × 10
5individual cells/T75 goes down to posterity.Culture vessel indicates the information such as cell algebraically and incubation time.Culture vessel is positioned over carbonic acid gas fixed temperature and humidity incubator to start to cultivate, culture condition: carbonic acid gas fixed temperature and humidity incubator, (37 ± 0.5) DEG C, carbonic acid gas volume fraction is (5 ± 0.2) %.Be cultured to cytogamy and reach 85% ~ 90%.
Two, fatty MSC qualification
1, get P3 fat subsitutes MSC, flow cytometer detection cell surface marker, the results are shown in Table 1.
Table 1 flow cytomery P3 fat subsitutes MSC 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%, prove that this cell is fatty MSC.
Three, induced lipolysis MSC is divided into neurocyte: the MSC passing for 3 generations, 0.125%Trypsin-EDTA solution digestion collecting cell, prepares cell suspension, cell counting count board living cell counting density, and adjusts density 1 × 10
4/ cm
2, be inoculated in and be placed with in advance in 24 orifice plates of the disinfection cap slide of poly-lysine process, prepare cell climbing sheet.Treat that cell merges close to 80%, carry out differentiation-inducing when growing vigorous again, grouping is as table 2.
Table 2 induced lipolysis MSC is divided into the experiment grouping of neurocyte
Four, neurocyte qualification after induction
1, morphological observation: under inverted microscope, dynamically observes morphological change before and after fatty MSC induction.
2, immunocytochemical stain method (SABC method) detects:
(1) the rear cell climbing sheet of induction is collected, 0.01MPBS(PH=7.2) rinse, 5min × 3 time, cold acetone fixes 10min, and PBS rinses 5min × 3 time;
(2) 3%H
2o
2solution hatches 30min, and to eliminate the activity of endogenous peroxydase, 0.01MPBS rinses, 5min × 3 time;
(3) drip closed sheep blood serum (reagent A), hatch 30min for 37 DEG C, absorb unnecessary serum, do not wash;
(4) drip little mouse-anti NeuN, the anti-β of rabbit-Tubulin III primary antibodie, little mouse-anti GFAP respectively, 4 DEG C of refrigerator overnight, PBS rinses, 5min × 3 time;
(5) drip biotin labeled corresponding anti-mouse or anti-rabbit two anti-(reagent B), hatch 1h for 37 DEG C, 0.01MPBS rinses, 5min × 3 time;
(6) drip the chain enzyme avidin (reagent C) of horseradish peroxidase-labeled, hatch 30min for 37 DEG C, 0.01MPBS rinses, 5min × 3 time;
(7) DAB colour developing: get distilled water 1ml, add each one of A, B, C composition in test kit, after mixing, drop on the cell specimen of above-mentioned process; Develop the color under room temperature, under mirror, control the reaction times.(general 2 ~ 10min) adding distil water termination reaction when being satisfied with to colour developing;
(8) the transparent mounting of conventional dehydration: 50% alcohol 1min, 70% alcohol 1min, 80% alcohol 1min, 95% alcohol 1min, 100% alcohol I 5min, 100% alcohol II 5min, dimethylbenzene I 5min, dimethylbenzene II 5min, neutral gum mounting.
Control experiment: replace first antibody with 0.01MPBS, all the other steps are identical.
Immunocytochemical method detects the expression of NeuN, β-Tubulin III, GFAP before and after induction, and randomly draw 4 slides, under 200 times of visuals field, random selecting 5 visuals field, calculate the ratio of positive cell respectively.Positive cell account for total cell proportion with mean ± standard deviation (
) represent.Adopt SPSS11.0 software to carry out statistical study, experimental data with (
) represent, multiple-group analysis adopts variance analysis, and between two groups, the comparison of rate adopts χ
2inspection, P<0.05 thinks statistical significance.
3, immunofluorescence staining detects
Immunofluorescence dyeing step is the same, and primary antibodie is the anti-β of rabbit-Tubulin III, and fluorescence two resists for Cy3-goat anti-rabbit igg, and negative control PBS replaces first antibody.
Immunofluorescence staining detects the expression of three groups of β-Tubulin III before and after induction, and randomly draw 4 slides, often open random selecting 5 visuals field under slide 200 times of visuals field, the total cellular score that counting positive cell sum and Hoechst redye, calculate the ratio of β-Tubulin III positive cell.Positive cell account for total cell proportion with (
) represent.Adopt SPSS11.0 software to carry out statistical study, experimental data with (
) represent, multiple-group analysis adopts variance analysis, and between two groups, the comparison of rate adopts χ
2inspection, P<0.05 thinks statistical significance.
Five, result
1, morphological observation
Chemical induction group: after going down to posterity, fatty MSC is soon adherent, and cell proliferation is vigorous, cell is homogeneous inoblast sample form, as shown in Figure 1.After pre-induced 12h, originally the fat mesenchymal stem cell cell space of fusiformis shrinks, and cell edges becomes irregularity, has many thin projections.At the end of pre-induced, a part of cell body is rounded.After formal induction, cell body shrinks further, rounded, trilateral, visible multiple projection, and sends branch, forms coniform whole end; Some projections are extended gradually.After 5h no longer there is considerable change in cellular form, and most cells presents typical neuron form.After induction, cellular portions is dead, come off.The floating death of most cells after continuation cultivation 5d.
Somatomedin group: after differentiation-inducing cultivation, in 3d, fatty MSC is substantially unchanged.5 ~ 10d part cell body increases gradually, and cytoplasmic process attenuates elongation.About 2 weeks, part cell body was rounded, and more greatly, cell space stretches out one, two or more projection, and projection is longer, and refractivity is good, and regional area flanking cell projection is linked to be netted, and about the cell of half presents typical neurocyte form.Cell survival is good, continues cultivation and does not see obvious cells float, obscission in 2 weeks.
Inducing culture group of the present invention: inducing culture of the present invention, after induction 1 ~ 2d, cellular form change obviously, cell cytosol, tenuigenin shrink centered by nucleus, cell shortens, smaller volume, cell forms cell paste that is bipolar or multipole, and the Cytoplasmic shrinkage of part cell forms cell process, cellular form is little ellipse, star-like, tadpole type, elongated strip etc., the reflective enhancing of cell.3 ~ 5d induces, and cell process extends further and attenuates, and part cell stretches out many projections, and refractivity is good, and regional area flanking cell projection is linked to be netted, and most cells presents typical neurocyte form, as shown in Figure 2.After 7d, cell state is good, and the survival time is long, does not see obvious cells float, obscission.
2, immunocytochemical stain result
Immunocytochemical stain result shows, and NeuN positive cell karyon is painted, and endochylema is painted shallow, as shown in Figure 3.Induction first three groups NeuN is negative expression, and after chemical inducer induction, NeuN positive cell rate is (72.3 ± 2.1) %; After growth factor-induced, NeuN positive cell rate is (39.6 ± 2.8) %; After inducing culture induction of the present invention, NeuN positive cell rate is (86.6 ± 4.5) %.The differentiation-inducing rate of inducing culture of the present invention is higher than chemical inducer and somatomedin (P<0.05), and difference has statistical significance.
Immunocytochemical stain result shows, and β-Tubulin III positive cell endochylema is painted, and in brown yellow granule, nucleus is not painted, as shown in Figure 4.Induction first three groups β-Tubulin III is negative expression, and after chemical induction, β-Tubulin III positive cell rate is (73.2 ± 1.3) %; After growth factor-induced, β-Tubulin III positive cell rate is (49.6 ± 3.1) %; β-Tubulin III positive cell rate (92.6 ± 2.3) % after inductor induction of the present invention.The differentiation-inducing rate of inducing culture of the present invention is higher than chemical inducer and somatomedin (P<0.05), and difference has statistical significance.
Immunocytochemical stain result shows, and GFAP positive cell endochylema is painted, and in brown yellow granule, nucleus is not painted, as shown in Figure 5.Induction first three groups GFAP is negative expression, and after chemical induction, GFAP positive cell rate is (13.3 ± 2.1) %; After growth factor-induced, GFAP positive cell rate is (32.1 ± 1.3) %; The present invention induces rear GFAP positive cell rate (16.6 ± 3.6) %.Inducing culture induced lipolysis mescenchymal stem cell of the present invention is to Astrocyte differentiation rate lower than somatomedin (P<0.05), and difference has statistical significance, a little more than chemical inducer.
3, immunofluorescence dyeing result
Immunofluorescence technique coloration result shows, and the endochylema of β-Tubulin III positive cell takes on a red color fluorescence, and as shown in a in Fig. 6, the karyon that Hoechst33258 redyes is blue-fluorescence, as shown in b in Fig. 6.Induction first three groups β-Tubulin III is negative expression, and after chemical induction, β-Tubulin III positive cell rate is (73.4 ± 2.1) %; After growth factor-induced, β-Tubulin III positive cell rate is (48.5 ± 1.8) %; After inducing culture induction of the present invention, β-Tubulin III positive cell rate is (91.8 ± 1.3) %.The differentiation-inducing rate of inducing culture of the present invention is higher than chemical inducer and somatomedin (P<0.05), and difference has statistical significance.
Three groups, table 3 induction results contrast (n=20,
)
Note P*<0.05 relative to somatomedin group and chemical induction group, P*
△<0.05 is relative to somatomedin group.
To sum up, as shown in table 3, by NeuN, β-Tubulin III positive cell rate after inducing culture induction of the present invention higher than chemical induction and growth factor-induced (P<0.05), GFAP positive cell rate is lower than growth factor-induced (P<0.05), illustrate that inducing culture induced efficiency of the present invention is higher than chemical inducer and somatomedin, and after induction, cell primary differentiation is neurocyte, but not neurogliocyte.
The inducing culture that embodiment 5 adopts embodiment 3 to prepare, carry out the differentiation-inducing experiment of neurocyte to Mesenchymal Stem Cells from Umbilical Cord (UC-MSC), step is as follows:
One, the separation and Culture (tissue mass cell culture) of umbilical cord MSC
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 ~ 20 minutes.
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: half amount changed liquid once in primary 4 ~ 5 days, (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 ~ 16 days, 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, 15min, after removing supernatant liquor, inoculate after adding fresh medium constant volume in new culturing bottle, passage cell density is (6 ~ 8) x10
5/ 75cm
2.
13, cell cultures and going down to posterity: after P1 generation, change weekly liquid 2 times, go down to posterity for every 3 ~ 4 days, when going down to posterity, cytogamy should 80% ~ 90%, and passage cell density is (6 ~ 8) * 10
5/ 75cm
2.
Two, the qualification of umbilical cord MSC
(1) get P3 for umbilical cord MSC, flow cytometer detection cell surface marker, streaming result is as table 4.
The detection of table 4 umbilical cord MSC 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 umbilical cord MSC.
Three, inducing umbilical cord mesenchymal stem is divided into neurocyte: the umbilical cord MSC passing for 3 generations, and 0.125%Trypsin-EDTA solution digestion collecting cell, prepares cell suspension, cell counting count board living cell counting density, and adjusts density 1 × 10
4/ cm
2, be inoculated in and be placed with in advance in 24 orifice plates of the disinfection cap slide of poly-lysine process, prepare cell climbing sheet.Treat that cell merges close to 80%, carry out differentiation-inducing when growing vigorous again, grouping is as table 5.
Table 5 induction of cord MSC is divided into the experiment grouping of neurocyte
Four, neurocyte qualification after induction
Method and step, with embodiment 4, are identified neurocyte after induction, from morphological observation, and immunocytochemistry, three groups of inductor effects in Immunofluorescence test tripartite face contrast table 5.
Five, result
1, morphological observation: result is with embodiment 4, three groups of cellular form equal neuralward cell direction changes after induction, but chemical inducer group has more cells float dead, and inducing culture group cell state of the present invention is good, survival time is long, does not see obvious cells float, obscission.
2, immunocytochemical stain result, in table 6.
3, immunofluorescence dyeing result, in table 6.
Three groups, table 6 induction results contrast (n=20,
)
Note P*<0.05 relative to somatomedin group and chemical induction group, P*
△<0.05 is relative to somatomedin group
To sum up, as shown in table 6, by NeuN, β-Tubulin III positive cell rate after inducing culture induction of cord MSC of the present invention higher than chemical induction and growth factor-induced (P<0.05), GFAP positive cell rate is lower than growth factor-induced (P<0.05), illustrate that inducing culture induced efficiency of the present invention is higher than chemical inducer and somatomedin, and after induction, cell primary differentiation is neurocyte, but not neurogliocyte.
Claims (3)
1. mescenchymal stem cell is induced an inductor for neuroblast, comprise trans-resveratrol, icarin, hydrocortisone, VEGF, IGF-I, EPO.
2. mescenchymal stem cell is induced the differentiation-inducing perfect medium of neuroblast for one kind, its moiety for: containing trans-resveratrol 30 ~ 50 μm of ol, icarin 5 ~ 10 μm of ol, hydrocortisone 5 ~ 10nmol, VEGF10 ~ 20 μ g, IGF-I5 ~ 10 μ g, EPO2 ~ 5 μ g in differentiation-inducing perfect medium described in every 1000ml, surplus behaviour mesenchymal stem cell serum-free perfect medium.
3. inducing culture according to claim 2, it is characterized in that: containing trans-resveratrol 40 μm of ol, icarin 8 μm of ol, hydrocortisone 8nmol, VEGF15 μ g, IGF-I7 μ g, EPO4 μ g in differentiation-inducing perfect medium described in every 1000ml, surplus behaviour mesenchymal stem cell serum-free culture medium.
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| 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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| CN107058225A (en) * | 2017-02-10 | 2017-08-18 | 郑州大学 | A kind of co-induction culture medium and using the culture medium inducing umbilical cord mesenchymal stem into neuron cell method |
| 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 |
| CN109852654A (en) * | 2018-12-28 | 2019-06-07 | 广州润虹医药科技股份有限公司 | Composition and its application of stem cell secretion cell factor can be induced |
| 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 |
| CN109694851A (en) * | 2019-01-16 | 2019-04-30 | 吉林省拓华生物科技有限公司 | A kind of inducing composition of human mesenchymal stem cell, induction differentiation culture solution and its external evoked methods and applications |
| EP4092106A4 (en) * | 2020-01-13 | 2024-03-06 | Qingdao Restore Biotechnology Co Ltd | Inducer for inducing mesenchymal stem cells to differentiate into islet cells |
| CN111394311A (en) * | 2020-04-20 | 2020-07-10 | 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 |
| WO2021212592A1 (en) * | 2020-04-20 | 2021-10-28 | 青岛瑞思德生物科技有限公司 | Serum-free complete culture medium capable of inducing differentiation of mesenchymal stem cells from corneal epithelial 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 |
| JP2022534332A (en) * | 2020-04-20 | 2022-07-29 | 青島瑞思徳生物科技有限公司 | Serum-free complete medium that induces differentiation of mesenchymal stem cells into corneal epithelial cells |
| JP7239686B2 (en) | 2020-04-20 | 2023-03-14 | 青島瑞思徳生物科技有限公司 | Serum-free complete medium that induces differentiation of mesenchymal stem cells into corneal epithelial cells |
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