CN109266609A - A kind of culture medium and its application in induction amnion mesenchymal stem cell into neural-like cells differentiation - Google Patents
A kind of culture medium and its application in induction amnion mesenchymal stem cell into neural-like cells differentiation Download PDFInfo
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Abstract
A kind of application from induction amnion mesenchymal stem cell to neural-like cells differentiation the present invention relates to stem cell differentiation technique field more particularly to culture medium and its in.It include B27, BDNF, EGF and NGF in culture medium of the invention；Amnion mesenchymal stem cell can be induced to be divided into neuron cell using culture medium provided by the invention, and differentiation efficiency is higher.I.e. visible part cell is adherent after 8 hours and has short and small protrusion to stretch out, visible most cells adherent growth after 2 days, form is irregular, the continuous thickening of protrusion and elongation, different form, the sheet of more protrusion sternzellens of dispersion and neuron cell are divided into after 1 week, protrusion, which is interleaved with each other, to be reticulated；Differentiation rate is up to 60% or more.
Description
Technical field
It is done the present invention relates to stem cell differentiation technique field more particularly to a kind of culture medium and its in induction amnion mesenchymal
Application of the cell into neural-like cells differentiation.
Background technique
The recovery of nervous function is extremely difficult after central lesion, and main cause is the Regenerated energy of intracerebral neuron
Power is very poor.Research at present has shown that intracerebral although with the presence of neural stem cell, and can be divided into neuron and Deiter's cells, but
It is that the position as existing for neural stem cell is limited to very much, quantity is also considerably less, therefore is used for neure damage reparation also right and wrong
Often difficult.As the research of stem cell field deepens continuously, it is found that it is neural thin the stem cell at other positions can also be divided into
Born of the same parents.By embryonic neural or Umbilical Cord Blood Transplant to the mouse intracerebral being damaged, it can moderately improve its nervous function, still
Since the factors such as ethics, immunological rejection limit the extensive use of these stem cells clinically.Medulla mesenchyma is dry thin
Born of the same parents can it is self obtain and can stable amplification in vitro, and can induce differentiation neuroblast in vitro, be expected to become nerveous system
The seed cell of system disease transplantation treatment.But the neural-like cells differentiated are induced effectively can't steadily to express at present
Whether nerve cell characteristic, such neural-like cells have the function of real neuron, however it remains dispute, and from normal human
Interior bone marrow extraction can also cause a degree of actual bodily harm to donor.
For the amnion tissue in Human plactnta source as the waste after pregnant woman childbirth, source is wide, is proliferated fastly and not by ethics road
Moral limitation, is research hotspot in recent years.Amnion be placenta package fetus face one layer of semi-transparent film, surface without nerve,
The tissue such as blood vessel, muscle and lymph, may separate out amniotic epithelial cells and amnion mesenchymal stem cell.Before more than 50 years, section
Scholar and clinicians just have realized that amnion possesses certain biological action.Early stage amnion is once used as covering skin
The dressing of wound, can generation that is anti-infective, mitigating inflammation, and inhibit the immune response of cell, can be with vasostimulant
It generates.These features have obtained relatively broad application it in the adjuvant treatment of a variety of diseases, such as treatment burn, covering
Wound, it is often more important that be used for ocular surface reconstruction.The wherein amnion-derived mescenchymal stem cell (human of Human plactnta
Amnion mesenchymal cells, hAMSCs) from the extraembryonic mesoderm of former phase, have under certain condition to more
A cell line differentiation potential, inside differentiation of germinal layers, such as liver cell, islet-like cells；To mesodermal differentiation, such as osteoblast, soft
Osteocyte, fat cell, cardiac muscle cell；Outside differentiation of germinal layers, such as nerve cell.
HAMSCs not only has the feature of stem cell, and not repellency and immune effect, is cell transplantation and organizational project
Ideal seed cell.HAMSCs equally has the ability for being divided into neural-like cells using suitable inducer in vitro.
This functional characteristic of hAMSCs has greatly attracted from preclinical medicine and clinical medical researcher, to the research of basic science
Persons provide a kind of mode of research and development biology, repair possessed by hAMSCs, substitution and power of regeneration are also clinical
Medicine provides the chance for developing new treatment.But the method efficiency that induction hAMSCs breaks up to neural-like cells at present is still
It is lower, it is not able to satisfy the demand of clinical application.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing a kind of culture medium and its in induction amnion mesenchymal
Application of the stem cell into neural-like cells differentiation.The culture medium can efficiently induce amnion mesenchymal stem cell thin to neural sample
Born of the same parents' differentiation.
Culture medium provided by the invention is made of basic culture solution, FBS, B27, EGF, BDNF and NGF.
In culture medium provided by the invention, the mass ratio of described EGF, BDNF and NGF are (5~15): (15~25): (10
~20).
In some embodiments, the mass ratio of described EGF, BDNF and NGF are 5:15:10.
In some embodiments, the mass ratio of described EGF, BDNF and NGF are 10:20:15.
In some embodiments, the mass ratio of described EGF, BDNF and NGF are 15:25:20.
B27 is a kind of cell culture additive, for hippocampal neuron and other central nervous system (CNS) neurons
Grow and keep its short-term or long period of activity.
Brain-derived neurotrophic factor (brain derived neurophic factor, BDNF) is synthesized in intracerebral
A kind of protein, it is distributed widely in central nervous system, during development of central nervous system, deposits to neuron
Living, differentiation, growth and development play an important role, and neuronal damage can be prevented to hurt pathological state that is dead, improving neuron, promote
The biological effects such as neuron regeneration and differentiation are damaged, and are also the neuron maintenance of mature maincenter and peripheral nervous system
Existence and normal physiological function institute are required.
Epithelical cell growth factor (cottony-stimulating factor, EGF) can promote the proliferation point of cell
Change, to replace aging and dead cell with newborn cell.
It is sent out around nerve growth factor (nerve growth factor, NGF) is adjustable with the growth of axoneuron
It educates, maintains the survival of neuron.
In culture medium provided by the invention, the volume fraction of the FBS is 10%；The volume fraction of the B27 is 2%.
In culture medium provided by the invention, the concentration of the EGF is 5ng/mL~15ng/mL；The concentration of the BDNF is
15ng/mL~25ng/mL；The concentration of the NGF is 10ng/mL~20ng/mL.
In some embodiments, the concentration of the EGF is 5ng/mL；The concentration of the BDNF is 15ng/mL；The NGF
Concentration be 10ng/mL.
In some embodiments, the concentration of the EGF is 10ng/mL；The concentration of the BDNF is 20ng/mL；The NGF
Concentration be 15ng/mL.
In some embodiments, the concentration of the EGF is 15ng/mL；The concentration of the BDNF is 25ng/mL；The NGF
Concentration be 20ng/mL.
In culture medium provided by the invention, basic culture solution is DMEM/F12 serum-free medium.
The present invention utilizes B27, brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), nerve growth factor
(NGF) induction amnion mesenchymal stem cell is divided into neuron cell, is not only provided with the representative configuration of nerve cell, and table
Up to neuronal marker nestin (Nestin), neuronspecific enolase (neuron specific
Enolase, NSE), Deiter's cells marker glial fibrillary acid protein (glial fibrilamentacidic
Protein, GFAP).Illustrate that culture medium provided by the invention can be such that amnion mesenchymal stem cell fills across differentiation of germinal layers non-
Cell plastid (neural-like cells), and differentiation efficiency is higher.
Application of the culture medium provided by the invention in induction induction amnion mesenchymal stem cell into neural-like cells differentiation.
Experiment shows that hAMSCs is after 8 hours with the culture medium induction containing FBS, B27, EGF, BDNF and NGF
See that part cell is adherent and there is short and small protrusion to stretch out, visible most cells adherent growth after 2 days, form is irregular, and protrusion is not
Disconnected thickening and elongation are divided into different form, the sheet of more protrusion sternzellens of dispersion and neuron cell, dash forward after 1 week
It rises to be interleaved with each other and reticulate.
The present invention also provides a kind of method that induction amnion mesenchymal stem cell breaks up to neural-like cells, filled between amnion
After matter stem cell is with the DMEM/F12 culture medium culture for 24 hours containing 10%FBS, induced 1 week with culture medium of the invention, every 2~3
It replaces fresh culture medium of the present invention.
In the embodiment of the present invention, amnion mesenchymal stem cell is the amnion mesenchymal stem cell in the 3rd~5 generation.
The amnion mesenchymal stem cell in the 3rd~5 generation the preparation method comprises the following steps: successively with trypsase and collagenase type I
After digesting amnion, cell is resuspended with PBS buffer solution, is reached with DMEM/F12 complete medium culture to the degrees of fusion of 10vol%FBS
To 80%~90%, pass on.
In the embodiment of the present invention, the amnion mesenchymal stem cell is inoculated in the coated culture vessel of poly-D-lysine.
In the embodiment of the present invention, the density of the inoculation is 1 × 104cells/mL。
It include B27, BDNF, EGF and NGF in culture medium of the invention；It can be induced using culture medium provided by the invention
Amnion mesenchymal stem cell is divided into neuron cell, and differentiation efficiency is higher.It is that visible part cell is adherent simultaneously after 8 hours
There is short and small protrusion to stretch out, visible most cells adherent growth after 2 days, form is irregular, the continuous thickening of protrusion and elongation, and 1 week
It is divided into different form, the sheet of more protrusion sternzellens of dispersion and neuron cell afterwards, protrusion, which is interleaved with each other, to be reticulated；
Differentiation rate is up to 60% or more.
Detailed description of the invention
Fig. 1 shows hAMSCs cellular morphology figure made from embodiment 1, comprising: Fig. 1-a shows that 40 times of amplifications, Fig. 1-b show 100 times
Amplification；
Fig. 2 shows the surface antigen detection of expression result of hAMSCs, comprising: Fig. 2-a shows that CD90 is shown in the expression of CD105, Fig. 2-b
Expression, Fig. 2-c show that the expression of CD73, Fig. 2-d show that the expression of HLA-DR, Fig. 2-e show that the expression of CD34, Fig. 2-f show CD45's
Expression.
Specific embodiment
The present invention provides a kind of culture medium and its in induction amnion mesenchymal stem cell into neural-like cells differentiation
Using those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that institute
There are similar replacement and change apparent to those skilled in the art, they are considered as being included in the present invention.
Method and application of the invention is described by preferred embodiment, and related personnel can obviously not depart from the present invention
Hold, in spirit and scope to methods herein and application is modified or appropriate changes and combinations, carrys out the implementation and application present invention
Technology.
The instrument that the present invention uses is all common commercially available product, can all be bought in market.
Below with reference to embodiment, the present invention is further explained:
1 hAMSCs of embodiment is primary to be separately cultured, identify and passes on
30~50cm is made with by amnion2The tissue of size is dispensed into 50mL centrifuge tube (volume 10ml~15ml).
Pancreatin digestion: being added 0.25% trypsase of three times volume, sets 37 DEG C, digests in 200R constant temperature oscillator
30min, every 10min taking-up are acutely rocked with hand.
Secondary rinsing: after digestion, the tissue block digested is transferred to the 250mL equipped with PBS buffer solution with tweezers
It in liquid storage bottle, covers tightly lid and is acutely rocked with hand, PBS buffer solution rinses 2-3 times.10~30cm is made in amnion again2Size
Tissue.
Type I collagen enzymic digestion: being added the 0.5% Type I collagen enzyme of 20mL, and adding DMEM/F12 culture medium to final volume is
100mL.It sets 37 DEG C, digest in 200R constant temperature oscillator, every 10min taking-up is acutely rocked with hand, until tissue block digests completely.
Sieving: after the completion of digestion, the tissue fluid digested is dispensed to 50mL centrifuge tube, every 20~25mL of pipe, the bodies such as addition
Long-pending PBS buffer solution, 2000rpm are centrifuged 5min.10mLPBS buffer resuspension cell precipitation, 200 μm of cells are added after abandoning supernatant
The screen to filtrate.2000rpm is centrifuged 5min.Supernatant is abandoned, DMEM/F12 complete medium weight of the 5~10mL containing 10wt%FBS is added
Outstanding cell.
Cell count and inoculation: carrying out cell count to cell suspension, adjusts cell suspension density according to count results and arrives
1.0×105Cell/mL~1.5 × 105Cell/mL is inoculated in culture dish, 5%CO237 DEG C of incubator cultures.
Originally culture: after cell inoculation 48h, primary cell is carried out to change liquid, is replaced fresh containing 10wt%FBS's
DMEM/F12 complete medium places 5%CO237 DEG C of incubator cultures.
Passage: covering with 80%~90% to cell, is inhaled with suction pipe and abandons old culture solution, and 2~3mL0.25wt% tryptose is added
Enzyme digests 1~3 minute, when microscopic observation cellular contraction is rounded, the DMEM/F12 culture solution containing 10%FBS in right amount is added immediately
Digestion is terminated, cell is collected, 1500rpm is centrifuged 5min, abandons supernatant.The DMEM/F12 containing 10%FBS in right amount is added to cultivate completely
Base carries out cell count, by 1 × 105Cell/mL density, which is inoculated in culture dish, carries out secondary culture, 5%CO237 DEG C of incubator
Culture, it is primary every passage in 3-4 days.
HAMSCs identification: 2nd generation logarithmic growth phase cell (form such as Fig. 1), Flow cytometry surface antigen are taken
CD105, CD90, CD73, CD45, CD34, HLA-DR expression, Cell-Quest software analyze result.Each sample analysis
8000~10000 cells.
Flow cytometry the results show that cell surface antigen CD105, CD90, CD73 expression are positive, and CD45,
CD34, HLA-DR expression are negative, illustrate hAMSCs obtained in the present invention belong to from mesoblastic mescenchymal stem cell (table 1,
Fig. 2).
1 hAMSCs surface antigen positive expression rate of table
| Cell phenotype | CD105 | CD90 | CD73 | HLA-DR | CD34 | CD45 |
| Positive expression rate (%) | 99.60 | 99.50 | 100.00 | 0.00 | 0.00 | 0.20 |
2 hAMSCs of embodiment differentiation
HAMSCs made from embodiment 1 is induced with different culture medium and is broken up, while 4 groups of control groups and 3 groups of experiments are set
Group chooses the 3rd~5 generation hAMSCs, by 1 × 104Cells/mL density is inoculated in the sterility cover glass for being placed with poly-D-lysine processing
In six orifice plates of piece, DMEM/F12 culture medium of the 2mL containing 10vol%FBS is added in every hole.Replace 1~control group of control group afterwards for 24 hours
6 and 1~experimental group of experimental group 3 in the corresponding culture solution of each group, in 37 DEG C, 5%CO2, saturated humidity stationary culture, every 2
~3 days culture solutions more renewed.
Control group 1 is negative control group, is routine culture group, the culture solution used are as follows: the 10%FBS's containing volume fraction
DMEM/F12 culture solution.
Control group 2 is positive controls, the induction liquid used are as follows: the DMEM/ of 10%FBS, 2%B27,10ng/mL EGF
F12 culture solution.
Control group 3 is positive controls, the induction liquid used are as follows: the DMEM/ of 10%FBS, 2%B27,20ng/mL BDNF
F12 culture solution.
Control group 4 is positive controls, the induction liquid used are as follows: the DMEM/ of 10%FBS, 2%B27,15ng/mL NGF
F12 culture solution.
Control group 5 is positive controls, the induction liquid used are as follows: 10%FBS, 5%B27,20ng/mL bFGF, 20ng/
The DMEM/F12 culture solution of mL BDNF, 20ng/mL NGF.
Control group 6 is positive controls, the induction liquid used are as follows: 10%FBS, 5%B27,10ng/mL EGF, 20ng/mL
The DMEM/F12 culture solution of bFGF, 35ng/mL NGF.
Liquid is induced used in experimental group 1 are as follows: 10%FBS, 2%B27,5ng/mL EGF, 15ng/mL BDNF, 10ng/
The DMEM/F12 culture solution of mL NGF.
Liquid is induced used in experimental group 2 are as follows: 10%FBS, 2%B27,10ng/mL EGF, 20ng/mL BDNF, 15ng/
The DMEM/F12 culture solution of mL NGF.
Liquid is induced used in experimental group 3 are as follows: 10%FBS, 2%B27,15ng/mL EGF, 25ng/mL BDNF, 20ng/
The DMEM/F12 culture solution of mL NGF.
HAMSCs breaks up morphologic observation:
I.e. visible part cell is adherent after 8 hours and has short and small protrusion to stretch out, the visible adherent life of most cells after 2 days
Long, form is irregular, the continuous thickening of protrusion and elongation, is divided into that form is different, the sheet of more protrusions of dispersion are starlike thin after 1 week
Born of the same parents and neuron cell, protrusion, which is interleaved with each other, to be reticulated.
Immunohistochemistry:
Culture 7 days after take out cell climbing sheet, according to immunohistochemical staining kit specification carry out Nestin, NSE,
The immunohistochemical staining of GFAP, DAB colour developing.
Culture solution in 6 well culture plate of coverslip will be placed with to be sucked out, PBS buffer solution rinses cell tile 5 minutes, is repeated 3 times.
15 minutes are fixed with 4% paraformaldehyde, is dried after PBS buffer solution rinsing, neutral gum is adhered on glass slide, 4 DEG C of refrigerator mistakes
Night.3% hydrogen peroxide is added dropwise to be incubated for 15 minutes, eliminates the activity of endogenous peroxydase, PBS buffer solution rinses 5 minutes, weight
It is 3 times multiple.It is added dropwise appropriate primary antibody (Nestin, NSE, GFAP), is incubated in 37 DEG C of wet box 1 hour, 4 DEG C overnight respectively.Next day takes out
PBS buffer solution rinses 5 minutes afterwards, and secondary antibody is added dropwise, is incubated for 40 minutes in 37 DEG C of wet box, PBS buffer solution rinses 5 minutes, repeats 3
Secondary, the DAB solution that Fresh is added dropwise develops the color 5-10 minutes, in light microscopic observation, is rinsed color development stopping with tap water.It will
After drying piece, haematoxylin is redyed 1 minute, and indigo plant is returned in differentiation, and gradient alcohol dehydration, dimethylbenzene is transparent, neutral gum mounting.
To cell obtained by each group after immunohistochemistry detects, formula is pressed to the cell of Nestin, NSE, GFAP dyeing:
Induce differentiation rate=staining positive cells number/total number of cells × 100%
It is counted, the differentiation rate for calculating each group is compared.Every group sampling 3 times, respectively detect after statistical data.System
Count result such as table 2.
2 group of cells differentiation rate of table
Table 2 is the results show that control group 1 cell Nestin, NSE, GFAP are showed no coloring, differentiation rate 0, control group 2~6
Cell Nestin, NSE, GFAP show coloring, but tinctorial yield is lower；And the coloring of experimental group 1~3 is then all very deep.Statistics
Induce differentiation rate, the results showed that, control group 1 is cell undifferentiated, differentiation rate 0；1~6 cell of control group all occurs to a certain degree
Differentiation, wherein the significant effect of control group 4~6 be better than control group 1~3；And the differentiation rate of experimental group 1~3 is then significantly higher than
Control group 1~6.Through statistical analysis, any one the differentiation rate of experimental group 1~3 is all significantly better than control group 1~6 (p < 0.05).
Wherein the differentiation effect of experimental group 2 is best, and differentiation rate and control group are deposited there are extremely significant difference (p < 0.01) with embodiment 1 or 3
In significant difference (p < 0.05).
These results suggest that illustrating that differential medium used in the present invention effectively can induce hAMSCs to nerve in 7 days
First like cell differentiation, divergaence time is short, and differentiation rate is high, therefore differentiation efficiency is higher.
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a kind of culture medium, which is characterized in that be made of basic culture solution, FBS, B27, EGF, BDNF and NGF.
2. culture medium according to claim 1, which is characterized in that the mass ratio of described EGF, BDNF and NGF be (5~
15): (15~25): (10~20).
3. according to culture medium described in claim 1, which is characterized in that the volume fraction of the FBS is 10%；The body of the B27
Fraction is 2%.
4. according to culture medium described in claim 1, which is characterized in that
The concentration of the EGF is 5ng/mL~15ng/mL；
The concentration of the BDNF is 15ng/mL~25ng/mL；
The concentration of the NGF is 10ng/mL~20ng/mL.
5. culture medium according to claim 1, the basic culture solution is DMEM/F12 serum-free medium.
6. the described in any item culture mediums of Claims 1 to 5 divide in induction induction amnion mesenchymal stem cell to neural-like cells
Application in change.
7. a kind of method that induction amnion mesenchymal stem cell breaks up to neural-like cells, which is characterized in that amnion mesenchymal is dry
After cell is with the DMEM/F12 culture medium culture for 24 hours containing 10%FBS, with the described in any item culture medium inductions of Claims 1 to 5
1 week, every the described in any item culture mediums of Claims 1 to 5 that replacement in 2~3 days is fresh.
8. the method according to the description of claim 7 is characterized in that the amnion mesenchymal stem cell is the amnion in the 3rd~5 generation
Mescenchymal stem cell.
9. the method according to the description of claim 7 is characterized in that the amnion mesenchymal stem cell is inoculated in poly-D-lysine
Coated culture vessel.
10. according to the method described in claim 9, it is characterized in that, the density of the inoculation is 1 × 104cells/mL。
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| CN114990065A (en) * | 2022-05-16 | 2022-09-02 | 康妍葆(北京)干细胞科技有限公司 | Directional inducer of neural stem cells, induction method and application |
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