CN104388381B - A kind of method of human marrow mesenchymal stem cell induction differentiation inner ear hair cells - Google Patents
A kind of method of human marrow mesenchymal stem cell induction differentiation inner ear hair cells Download PDFInfo
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Abstract
The invention discloses a kind of method that inducing bone marrow MSCs breaks up inner ear hair cells, using people's marrow as source, bone marrow MSCs are separated, break up inner ear hair cells with the method inducing bone marrow MSCs that feeder cells are combined using combinations of factors；The method of the invention effectively can be divided into inner ear hair cells, differentiation gained cell expression hair cell differential protein by inducing human mesenchymal stem cells, and possess certain hair cell electrophysiological function.
Description
(1) technical field
The present invention relates to the method that a kind of induction differentiation of utilization human marrow mesenchymal stem cell obtains inner ear hair cells.
(2) background technology
The unrepairable damage of mammal inner ear hair cells is to cause one of main reason of hearing loss.Generally
Think, human inner ear's auditory hair cell does not have self-regeneration repair ability, and its damage is irreversible.Recent study is pointed out
Mammal hair cell realizes the possibility of regeneration under certain inductive condition, but for the regeneration of hair cell in pathological process
Reparation still suffers from very big challenge.Therefore finding the cell derived of hair cell regeneration, exploitation damage of hair cell reparation Intervention Strategy is
The key of hearing impairment treatment.
Stem cell with its all can or the focus as regeneration and restoration area research the characteristics of versatility.Numerous researchs
The characteristics of embryonic stem cell, adult stem cell and induced multi-potent stem cell are divided into Various Tissues cell has been illustrated, with
And their application potentials in tissue damage reparation and cell therapy.Therefore stem cell differentiation potential is utilized, is lured with stem cell
Lead based on differentiation, inquiring into hair cell Regeneration and Repair has theoretical foundation.Mescenchymal stem cell (Mesenchymal stem
Cells, MSCs) it is that a class of mesoderma origin has the adult stem cell of height self-renewal capacity and multi-lineage potential.
MSCs is widely present in the tissues such as marrow, fat, muscle, skin, amniotic fluid and umbilical cord or organ, except can be to skeletonization, fat
The mesoderm such as fat and cartilage direction breaks up, and MSCs can also be crossed over and is divided into nerve cell, liver cell, cardiac muscle under certain conditions
Other germinal layer cell types such as cell, islet cells, that is, show the plasticity of height.In addition MSCs is easily isolated, external energy
Enough long-term cultivations expand and keep multi-lineage potential.Therefore MSCs is always the focus in stem-cell research field.Marrow is
One of MSCs the abundantest sources, therefore the induction system that bone marrow MSCs break up inner ear hair cells is set up in research, will be in the future
Hair cell regenerative therapy provides effective cell derived, with important clinical meaning.
(3) content of the invention
It is an object of the present invention to provide a kind of method that inducing bone marrow MSCs breaks up inner ear hair cells, using people's marrow as source,
Bone marrow MSCs are separated, break up inner ear hair cells with the method inducing bone marrow MSCs that feeder cells are combined using combinations of factors.
The technical solution adopted by the present invention is：
The present invention provides a kind of method of human marrow mesenchymal stem cell induction differentiation inner ear hair cells, and methods described is：
Human marrow mesenchymal stem cell is taken, is inoculated with (preferably with 105Individual/cm2Density inoculation) to induction liquid I, in 37 DEG C, 5%CO2Training
Fiber differentiation 7 days in case are supported, induction liquid II are then changed, in 37 DEG C, 5%CO2Incubator continues to cultivate 7-10 days, 0.25% pancreatin
After EDTA digestion, it is seeded on chicken embryo utricle interstitial cell (i.e. feeder cells), induction liquid III is added, in 37 DEG C, 5%
CO2Incubator continues to induce 14-21 days, obtains ripe inner ear hair cells；
Induce the final concentration of liquid I composition：Volumetric concentration 5%FBS, 45-55ng/ml IGF-1 (insulin-like growth factor-Ⅱs
1), 20-25ng/ml EGF (epithelical cell growth factor), 10 μM of RA (ATRA) and 20-30ng/ml FGF2 (into
Fibroblast growth factor 2), solvent is DMEM/F12 basic culture solutions；
Induce the final concentration of liquid II composition：Volumetric concentration 5%FBS, 5-15ng/ml BMP4 (BMP 4) and 20-
30ng/ml FGF2, solvent is DMEM/F12 basic culture solutions；
Induce the final concentration of liquid III composition：Volumetric concentration 5%FBS and 20-30ng/ml FGF2, solvent is DMEM/F12 bases
Nutrient solution；
The method of chicken embryo utricle interstitial cell pretreatment is：By chicken embryo utricle interstitial cell, (preferred growth is extremely
The chicken embryo utricle interstitial cell of 90% degree of converging) soaked with the DMEM nutrient solutions of the μ g/ml mitomycin Cs containing final concentration 2,37
DEG C, 5%CO2Incubator culture 3 hours, discards suspension, obtains pretreated chicken embryo utricle interstitial cell.
Further, preferably described induction liquid I final concentration composition：Volumetric concentration 5%FBS, 50ng/ml IGF-1,25ng/ml
EGF、10-6M RA and 25ng/ml FGF2, solvent is DMEM/F12 basic culture solutions.
Further, preferably described induction liquid II final concentration composition：Volumetric concentration 5%FBS, 10ng/ml BMP4 and 25ng/
Ml FGF2, solvent is DMEM/F12 basic culture solutions.
Further, preferably described induction liquid III final concentration composition：Volumetric concentration 5%FBS and 25ng/ml FGF2, solvent is
DMEM/F12 basic culture solutions.
Further, the feeder cells are chicken embryo utricle interstitial cell, and preparation method is mainly by digesting and passing on
Method is separately cultured utricle interstitial cell for 18 days from fertilization in chicken embryo, 3-5 is for cell, after mitomycin C processing, as
Feeder cells, preferably described chicken embryo utricle interstitial cell is prepared as follows：Separated from 18 days chicken embryos of fertilization oval
Capsule, 0.25% pancreatin after 37 DEG C digest 15-20 minutes, is stopped, mistake with addition volume final concentration 10%FBS DMEM nutrient solutions
200 eye mesh screens, collect cell suspension, and 1000rpm/min is centrifuged 6-8 minutes, and cell is with addition volume final concentration 10%FBS's
DMEM nutrient solutions suspend, and are inoculated in blake bottle, put 37 DEG C, 5%CO2Incubator culture, changes liquid after 48 hours, discards not first
Attached cell, adherent continuation is cultivated to 90% degree of converging, and with 0.25% pancreatin/EDTA digestion and inoculated and cultured, is designated as P1 generations thin
Born of the same parents, by such Secondary Culture, to P3 generations, that is, obtain chicken embryo utricle interstitial cell.
The factor of the present invention can be obtained by mode purchased in market, and 18 days chicken embryos of fertilization have purchased from Hangzhou Tian Yuan cultivation
Limit company.
Compared with prior art, the beneficial effects are mainly as follows：The method of the invention can be induced effectively
Human marrow mesenchymal stem cell is divided into inner ear hair cells, and differentiation gained cell is not only expressed hair cell differential protein, more attached most importance to
What is wanted is that differentiation and maturation cell possesses certain hair cell electrophysiological function.
(4) illustrate
Fig. 1 is the cellular morphology micrograph (100 times of amplification) for inducing liquid II to induce the 7th day；A figures are not start induction
MSCs, B figure are the cell for inducing liquid II to induce the 7th day.
Fig. 2 is the electrophoretogram for inducing liquid II to induce the 7th day cell expression hair cell marker gene Atoh1；A is induction liquid II
7 days cell Atoh1 gene expression detection gel electrophoresis figures are induced, band β-actin are as internal reference, and swimming lane 0D represents not start to lure
The MSCs led the, -7D of swimming lane II represent the cell for inducing liquid II to induce the 7th day；The egg that B1 is Atoh1 in the MSCs for do not start induction
Atoh1 expression in white expression, cells of the B2 to induce the induction the 7th day of liquid II.
Fig. 3 is the cellular morphology micrograph (100 times of amplification) of the chicken embryo utricle interstitial cell in P3 generations.
Fig. 4 is the Immunofluorescence test figure for inducing cell expression hair cell specific proteins after the induction of liquid III；A is Atoh1
With Myosin 7a expression；B is that Myosin 7a and nucleus C23 are expressed.
Fig. 5 is the Immunofluorescence test figure for inducing cell expression hair cell specific proteins after the induction of liquid III；A is Espin
Expressed with C23 in nucleus, B is C23 expression in Myosin 7a and nucleus, C is the superposition of A and the width figures of B two.
Fig. 6 is cell hair cell electrophysiological function detection figure after induction liquid III is induced；A is that FM1-43FX is dyed and nucleus
Interior C23 expression, it is A and the superposition of the width figures of B two that B, which is that C23 expresses C in Myosin 7a and nucleus,.
(5) embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：The separation of human marrow mesenchymal stem cell
The marrow of Adult Healthy Volunteers, pH value is washed for 7.4 phosphate buffers (PBS, purchased from Sheng Gong biotech firms)
After twice, and marrow is diluted to 10 with PBS7Individual/ml cell suspension, gently adds to human lymphocyte separating liquid upper strata
(Ficoll-Hypaque, D=1.077 ± 0.002g/ml, biological purchased from raw work), both volume ratios are 3：2；Room temperature (25 DEG C),
After 2200rpm/min is centrifuged 25 minutes, middle white cellular layer (mononuclearcell layer) is drawn；Add addition volume final concentration
The IMDM basic culture solutions (being purchased from Corning companies) of 10% hyclone (FBS, purchased from Gibco companies), wash cell, so
1000rpm/min is centrifuged 6-8 minutes afterwards, is removed nutrient solution, with same nutrient solution suspension cell, is inoculated in blake bottle, puts
37 DEG C, 5%CO2Incubator culture, liquid is changed after 48 hours first, discards non-attached cell, and adherent continuation, which is cultivated to 90%, to be converged
Degree, with 0.25% pancreatin/EDTA (being purchased from Corning companies) digestion and inoculated and cultured, is designated as P1 for cell, by so passing on
Culture, to P3-P5 generations, obtains the single fiber-like human marrow mesenchymal stem cell (MSCs) of form.
Embodiment 2：The separation of chicken embryo utricle interstitial cell
From 18 days chicken embryo (cultivating Co., Ltd purchased from Hangzhou Tian Yuan) middle separation utricles of fertilization, 0.25% pancreatin, 37 DEG C
After digestion 15-20 minutes, stopped, mistake with addition volume final concentration 10%FBS DMEM nutrient solutions (being purchased from Corning companies)
200 eye mesh screens, collect cell suspension, and 1000rpm/min is centrifuged 6-8 minutes, and cell is with addition volume final concentration 10%FBS's
DMEM nutrient solutions suspend, and are inoculated in blake bottle, put 37 DEG C, 5%CO2Incubator culture, changes liquid after 48 hours, discards not first
Attached cell, adherent continuation is cultivated to 90% degree of converging, and is digested with 0.25% pancreatin/EDTA and is seeded to Tissue Culture Flask training
Support, be designated as P1 for cell, by such Secondary Culture, to P3 generations, that is, obtain the single chicken embryo utricle interstitial cell of form.
Embodiment 3：The induction of human marrow mesenchymal stem cell vitro differentiation inner ear hair cells
The chicken embryo utricle interstitial cell for growing to 90% degree of converging prepared by Example 2, with containing the μ g/ml of final concentration 2
The DMEM nutrient solutions immersion of mitomycin C (being purchased from Sigma companies), 37 DEG C, 5%CO2Incubator culture 3 hours, discards suspension
Liquid, after being embathed with PBS, obtains pretreated chicken embryo utricle interstitial cell.
The P3-P5 generation human marrow MSCs that the method for Example 1 is obtained, are seeded to induction liquid I, 37 DEG C, 5%CO2Incubator
Middle Fiber differentiation 7 days, wherein per liquid is changed for 2-3 days, then changing induction liquid II, 37 DEG C, 5%CO2Incubator continues to cultivate 7-10 days,
Wherein per liquid is changed within 2-3 days, after 0.25% pancreatin EDTA digestion, with 105Individual/cm2Density to be seeded to pretreated chicken embryo oval
On capsule interstitial cell, induction liquid III, 37 DEG C, 5%CO are added2Incubator continues to induce 14-21 days, collects inducing cell and is reflected
It is fixed, obtain ripe inner ear hair cells.
Induce the final concentration of liquid I composition：Volumetric concentration 5%FBS, 50ng/ml IGF-1,25ng/ml EGF, 10-6M RA and
25ng/ml FGF2, solvent is DMEM/F12 basic culture solutions (being purchased from Corning companies).
Induce the final concentration of liquid II composition：Volumetric concentration 5%FBS, 10ng/ml BMP4 and 25ng/ml FGF2, solvent is base
Plinth nutrient solution (DMEM/F12)；
Induce the final concentration of liquid III composition：Volumetric concentration 5%FBS and 25ng/ml FGF2, solvent is the training of DMEM/F12 bases
Nutrient solution.
IGF-1, EGF and FGF2 are purchased from Peprotech companies, and BMP4 is purchased from RD companies, and RA is purchased from Sigma companies.
Embodiment 4：The identification of noble cells
(1) morphological observation：The cell of the 7th day is induced to utilize micro- sem observation without the MSCs and induction liquid II of induction
Cellular morphology, and photograph to record, as a result as shown in Figure 1, picture amplifies 100 times.
(2) hair cell specific gene is detected：Induce the cell after the induction of liquid II (public purchased from Invitrogen with TRizol
Department) total serum IgE is extracted, extracting method by specification is carried out, then with Reverse Transcriptase kit RNA PCR kit (AMV) Ver3.0 (purchases
From TaKaRa companies) carry out reverse transcription.The cDNA obtained using reverse transcription is template, in primer Atoh1 (primer β-actin conducts
Internal reference) in the presence of enter performing PCR reaction, detect Atoh1 expression conditions, and internal reference be used as using β-actin.PCR programs are：
94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 50 DEG C~65 DEG C annealing 30s, 72 DEG C of extension 30s, totally 30~40 circulations, 72 DEG C
Extend 10min, annealing temperature and period are selected according to the concrete condition of primer.PCR primer is subjected to Ago-Gel
Electrophoretic analysis, as a result as shown in A in Fig. 2.
The primer is as follows:Atoh1 (5'-ATCTACATCAACGCCTTGTCCGAGC-3', 5'-
CGAAAGTCGAGAAGTGCAGAGCGTC-3')；β-actin (5'-ACACCTTCTACAATGAGCTG-3', 5'-
CTGCTTGCTGATCCACATCT-3'), synthesized by Shanghai JaRa biotech firm.
(3) hair cell differential protein Immunofluorescence test：The cell after liquid II and the induction induction of liquid III is induced through 4% poly
After the fixed 15min of (25 DEG C) of formaldehyde room temperature, paraformaldehyde is removed with PBS (pH value is 7.4) rinsings, then use 0.05%Triton
X-100 (being purchased from Sigma companies) soaking at room temperature is handled 10 minutes, then with 5% lowlenthal serum (biological purchased from raw work) in room temperature
1h is closed, twice, cell is divided into 4 groups to PBS, be separately added into primary antibody, group 1 is anti-Atoh1, and group 2 is anti-Myosin7a and anti-
C23, group 3 is that anti-Espin, group 4 are anti-Myosin7a and anti-C23, and primary antibody is purchased from Santa cruz companies, and 4 DEG C of wet box are incubated
Overnight；PBS embathes three times, each 10min；Sequentially add FITC mark secondary antibodies, group 1 is that Alexa 488, group 2 are Alexa 647
Secondary antibody is marked with Dylight 405, group 3 is Alexa 488, group 4 is Dylight 649 and Dylight405 marks secondary antibody (two
It is anti-to be purchased from Lian Ke biotech firms), 37 DEG C are incubated 1h, and PBS embathes three times；Its moderate resistance Atoh1 primary antibody group cells DAPI (is purchased from
Sigma companies) core is contaminated, laser confocal microscope (Carl Zeiss) observation is taken pictures.As a result see that B1 and B2 (amplify in Fig. 2
200 times), Fig. 4 and Fig. 5 (amplifying 630 times).
(4) hair cell electrophysiological function is detected：Utilize FM1-43FX (being purchased from Invitrogen companies) Coloration experiment detection
Noble cells electrophysiological function, after cell is cleaned with HBSS (being purchased from Sheng Gong biotech firms) after induction liquid III is induced, adds 5 μM
FM1-43FX is incubated at room temperature 30s, is cleaned immediately with HBSS, then is embathed with HBSS after 20min, carries out immunofluorescence experiment detection
Myosin7a is expressed, and step and antibody are with hair cell differential protein Immunofluorescence test.As a result as shown in Figure 6,630 times are amplified.
As a result show, induction gained noble cells can express Hair Cell Progenitors and (induce the induction gained of liquid II thin
Born of the same parents) specific gene and albumen, ripe hair cell specific proteins, what is more important differentiation and maturation cell has certain capillary
Born of the same parents' electrophysiological function, shows that this abductive approach effectively can break up inner ear hair cells by inducing bone mesenchymal stem cell.
Finally, in addition it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (6)
1. a kind of method of human marrow mesenchymal stem cell induction differentiation inner ear hair cells, it is characterised in that methods described is：Take
Human marrow mesenchymal stem cell, is seeded to induction liquid I, in 37 DEG C, 5%CO2Fiber differentiation 7 days in incubator, then change induction
Liquid II, in 37 DEG C, 5%CO2Incubator continues to cultivate 7-10 days, after 0.25% pancreatin EDTA digestion, is seeded to pretreated
On chicken embryo utricle interstitial cell, induction liquid III is added, in 37 DEG C, 5%CO2Incubator continues to induce 14-21 days, obtains ripe
Inner ear hair cells；
Induce the final concentration of liquid I composition：Volumetric concentration 5%FBS, 45-55ng/ml IGF-1,20-30ng/ml EGF, 10 μM of RA
With 20-30ng/ml FGF2, solvent is DMEM/F12 basic culture solutions；
Induce the final concentration of liquid II composition：Volumetric concentration 5%FBS, 5-15ng/ml BMP4 and 20-30ng/ml FGF2, solvent is
DMEM/F12 basic culture solutions；
Induce the final concentration of liquid III composition：Volumetric concentration 5%FBS and 20-30ng/ml FGF2, solvent is the culture of DMEM/F12 bases
Liquid；
The chicken embryo utricle interstitial cell preprocess method is：Chicken embryo utricle interstitial cell is used and contains 2 μ of final concentration g/ml
Rimocidin C DMEM nutrient solutions immersion, in 37 DEG C, 5%CO2Incubator culture 3 hours, discards suspension, after being pre-processed
Chicken embryo utricle interstitial cell.
2. the method for human marrow mesenchymal stem cell induction differentiation inner ear hair cells as claimed in claim 1, it is characterised in that institute
State induction liquid I final concentration composition：Volumetric concentration 5%FBS, 50ng/ml IGF-1,25ng/ml EGF, 10-6M RA and 25ng/ml
FGF2, solvent is DMEM/F12 basic culture solutions.
3. the method for human marrow mesenchymal stem cell induction differentiation inner ear hair cells as claimed in claim 1, it is characterised in that institute
State induction liquid II final concentration composition：Volumetric concentration 5%FBS, 10ng/ml BMP4 and 25ng/ml FGF2, solvent is DMEM/F12
Basic culture solution.
4. the method for human marrow mesenchymal stem cell induction differentiation inner ear hair cells as claimed in claim 1, it is characterised in that institute
State induction liquid III final concentration composition：Volumetric concentration 5%FBS and 25ng/ml FGF2, solvent is DMEM/F12 basic culture solutions.
5. the method for human marrow mesenchymal stem cell induction differentiation inner ear hair cells as claimed in claim 1, it is characterised in that institute
Chicken embryo utricle interstitial cell is stated to be prepared as follows：Utricle, 0.25% pancreatin, 37 DEG C are separated within 18 days in chicken embryo from fertilization
After digestion 15-20 minutes, stopped with addition volume final concentration 10%FBS DMEM nutrient solutions, cross 200 eye mesh screens, collect cell
Suspension, 1000rpm/min is centrifuged 6-8 minutes, and cell is suspended with addition volume final concentration 10%FBS DMEM nutrient solutions, inoculation
In blake bottle, 37 DEG C, 5%CO are put2Incubator culture, liquid is changed after 48 hours first, discards non-attached cell, adherent continuation training
Support to 90% degree of converging, digested and be seeded in Tissue Culture Flask with 0.25% pancreatin/EDTA and cultivated, be designated as P1 for cell, lead to
Such Secondary Culture is crossed, to P3 generations, that is, chicken embryo utricle interstitial cell is obtained.
6. the method for human marrow mesenchymal stem cell induction differentiation inner ear hair cells as claimed in claim 1, it is characterised in that institute
State induction liquid II and cultivate postdigestive cell with 105Individual/cm2Density be seeded to pretreated chicken embryo utricle interstitial cell
On.
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| CN105112367B (en) * | 2015-08-28 | 2018-07-27 | 青岛瑞思科生物科技有限公司 | A kind of mescenchymal stem cell epidermal differentiation derivant and its application process |
| WO2017146035A1 (en) * | 2016-02-22 | 2017-08-31 | 学校法人順天堂 | Method for producing inner ear cells |
| CN107384852A (en) * | 2017-07-27 | 2017-11-24 | 山东兴瑞生物科技有限公司 | A kind of sertoli cell is divided into method and the application of inner ear hair cells |
| CN112852715B (en) * | 2021-03-08 | 2023-03-28 | 浙江大学 | Method for directionally differentiating induced pluripotent stem cells into inner ear hair cell-like cells |
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| WO2008076556A2 (en) * | 2006-11-15 | 2008-06-26 | Massachusetts Eye & Ear Infirmary | Generation of inner ear cells |
| CN101215546A (en) * | 2007-12-31 | 2008-07-09 | 浙江大学 | Method for obtaining inner ear hair cell precursor induced by bone marrow mesenchymal stem cells |
| CN101215545A (en) * | 2007-12-31 | 2008-07-09 | 浙江大学 | Method for obtaining neural progenitor cells induced by bone marrow mesenchymal stem cells |
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| WO2008076556A2 (en) * | 2006-11-15 | 2008-06-26 | Massachusetts Eye & Ear Infirmary | Generation of inner ear cells |
| CN101215546A (en) * | 2007-12-31 | 2008-07-09 | 浙江大学 | Method for obtaining inner ear hair cell precursor induced by bone marrow mesenchymal stem cells |
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Denomination of invention: A method for inducing differentiation of inner ear hair cells by human bone marrow mesenchymal stem cells Granted publication date: 20170822 Pledgee: Zhejiang Hangzhou Yuhang Rural Commercial Bank Co.,Ltd. Science and Technology City Branch Pledgor: HANGZHOU S-EVANS BIOSCIENCES, Ltd. Registration number: Y2024980003656 |