CN109666633A - A method of improving brown fat stem cell myocardiac differentiation efficiency - Google Patents
A method of improving brown fat stem cell myocardiac differentiation efficiency Download PDFInfo
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Abstract
The invention discloses a kind of methods for the raising brown fat stem cell myocardiac differentiation efficiency for belonging to cell engineering field.The cardiac stem cells abundance in brown adipose tissue source, it is big to collect cell concentration, patient is easy to receive, the present invention improves brown fat stem cell myocardiac differentiation using extracellular matrix derived from Cardiac Fibroblasts, solves the problems, such as that current isolated culture method is lower for the efficiency of brown fat stem cell Cardiomyocytes differentiation.
Description
Technical field
The invention belongs to cell engineering fields, and in particular to a kind of raising brown fat stem cell cells into cardiomyocytes point
Change the method for efficiency.
Background technique
Seed cell is the basis of cardiac muscle tissue engineering strategy and cell therapy strategy.Currently used for damaged myocardium reparation
Seed cell includes the cardiac muscle cell of terminal differentiation, tissue stem cell (such as fat of various separate sources and different developmental phases
Mescenchymal stem cell (ADSCs), mesenchymal stem cell etc.) and myeloid-lymphoid stem cell (such as embryonic stem cell (ESCs) and lure
Lead multipotential stem cell (iPSC)) etc..These cells can improve cardiac function to a certain extent, but there is also such as sources
Limited, the disadvantages of amplification efficiency is low, Myocardium Differentiation ability is limited, certain oncogenicity.Simultaneously because the severe microenvironment in heart infarction area
Feature also makes the cell viability measurement of transplanting lower, so that it be caused to receive certain limit to the repairing effect of damaged myocardium
System.For these problems, recent domestic scholar is had made intensive studies, and obtains some significant results of study.Mesh
For the purpose of preceding research is mainly transported to improve stem cell, survived, being proliferated, break up, function, pass through drug, the factor or micro-
Environment is pre-processed to improve the ability that stem cell breaks up to functional cell.Tissue-derived stem cell is cardiac regeneration and cardiac muscle
Another kind of important seed cell source in Tissue Engineering Study mainly includes derived from bone marrow mescenchymal stem cell (Bone
Marrow derived Mesenchymal Stem Cells, BMSCs), adipose-derived mescenchymal stem cell (Adipose
Derived Stem Cells, ADSCs) etc..Existing research shows that mescenchymal stem cell can effectively be divided into cardiac muscle cell,
And the clinical experimental study of existing heart infarction treatment, it was demonstrated that it is effectively improved effect to the systolic and diastolic functions of infarcted hearts.
Wherein, the tissue that is easily obtained as human body of fat, containing a large amount of mescenchymal stem cell, 2004, the head such as Planat-Benard
The secondary stem cell for demonstrating mouse adipose source have 0.02% to 0.07% can Spontaneous Differentiation be cardiac muscle cell.2006,
Yamada etc. has developed a kind of method for separating BADSCs, and demonstrates BADSCs with efficient Myocardium Differentiation ability, differentiation potency
Power can reach 20%, and internal heart infarction promotes the heart function of heart infarction rat researches show that can effectively be differentiated to form cardiac muscle cell
Energy.Liu et al. improves in BADSCs isolation technics, improves the purity and quantity of BADSCs, meets cardiac muscular tissue's structure
Build the quantity of required seed cell.Our study groups utilize the brackets such as BADSCs and aquagel, PNIPAAm/SWCNTs
Myocardial Regeneration research is carried out, it was demonstrated that Cardiomyocytes break up BADSCs in vivo, and have to the function of heart infarction heart apparent
Facilitation.The cardiac stem cells (ADSC) in brown adipose tissue source have abundance, and collection cell concentration is big, and patient is easy to
The advantages such as receiving.But current isolated culture method is lower for the efficiency of brown fat stem cell Cardiomyocytes differentiation, compels to be essential
Find the method for improving its myocardiac differentiation.
Summary of the invention
It is an object of the invention to propose a kind of method for improving brown fat stem cell myocardiac differentiation efficiency.
To realize the above-mentioned technical purpose, this invention takes the following technical solutions:
A method of brown fat stem cell myocardiac differentiation efficiency is improved, is spread out using Cardiac Fibroblasts
Raw extracellular matrix culture brown fat stem cell, is divided into cardiac muscle cell.
The method of above-mentioned raising brown fat stem cell myocardiac differentiation efficiency, is operated in accordance with the following steps:
S1. it takes 1 day age SD rat heart to be placed in the PBS liquid of pre-cooling, shreds, utilize 0.05% pancreatin digestion group completely
Block is knitted, is centrifuged within 1000rpm/7 minutes, is obtained cell precipitation, be resuspended in DMEM in high glucose plus 15% fetal calf serum and be seeded in training
It supports in ware, not adherent cardiac muscle cell is discarded after 1 hour, obtains primary Cardiac Fibroblasts.
S2. it is digested after adding 10% fetal calf serum culture Cardiac Fibroblasts to 85-90% fusion using high sugar-DMEM,
And be inoculated in 24 culture orifice plates, it cultivates 10 days, discards culture medium, PBS is rinsed 3 times, and it is 0.3% that volume fraction, which is added,
The NH of Triton X-100 and 20mM4OH PBS handles 5 minutes under the conditions of 37 DEG C, discards digestive juice, PBS is washed twice, obtains the heart
The extracellular matrix of myofibroblast secretion.
S3. 3 week old SD rats are taken, the neck that breaks is put to death, and the brown around back and shoulder blade is taken out after the immersion of 75% ethyl alcohol
Adipose tissue is washed three times with the PBS of pre-cooling, is shredded, and 10mL mixture slaking enzyme is added, and 37 DEG C of Stirrings digest 40min, sufficiently
The screen to filtrate after pressure-vaccum discards remnant tissue's block, collects digestive juice, and 600g is centrifuged 8min, is washed with the α-MEM culture medium of serum-free
It washs precipitating 1 time, cell inoculation is cultivated on the extracellular matrix derived from Cardiac Fibroblasts, finally obtains differentiation
Cardiac muscle cell.
In the step S3 ingredient of mixture slaking enzyme be α-MEM culture medium, 0.25% pancreatin, type Ⅳ collagenase,
Dispase enzyme, four weight ratio are 4:4:1:1.
Compared with prior art, the invention has the following beneficial effects: the cardiac stem cells in brown adipose tissue source to come
Source is abundant, and collection cell concentration is big, and patient is easy to receive, and the present invention is improved using extracellular matrix derived from Cardiac Fibroblasts
The method of brown fat stem cell myocardiac differentiation improves the efficiency of brown fat stem cell Cardiomyocytes differentiation.
Detailed description of the invention
A-a1 is that phase contrast microscope is caused to observe derivative matrix morphology figure in Fig. 1；A-a2 is that sirius red detects collagen point
Butut；B is that SEM detects derivative matrix figure；C is total protein measurement result figure；D is that immunofluorescence dyeing detects matrix components figure.
Be in Fig. 2 A be CCK-8 measurement cell proliferative conditions figure；B is that immunofluorescence dyeing detects PH3 expression figure.
Fig. 3 is that immunofluorescence dyeing detects Gata4 expression figure.
Fig. 4 is the expression figure that immunofluorescence dyeing detects cardiomyocyte markers object.
Fig. 5 is the differentiation figure of Flow cytometry BADSC cells into cardiomyocytes.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples, but implementation of the invention is not limited only to this.
1 brown fat stem cell myocardiac differentiation of embodiment
1. taking newborn 1 day age SD rat, chest is opened, coring is dirty, in the PBS liquid of pre-cooling, shreds, and utilize 0.05% pancreas
Enzyme digests repeatedly until tissue block digestion completely, is centrifuged, cell precipitation is obtained, in DMEM in high glucose plus 15% for 1000rpm/7 minutes
It is resuspended and is seeded on culture dish in fetal calf serum (gibco), not adherent cardiac muscle cell is discarded after 1 hour, obtains the primary heart
Myofibroblast.
2. it is digested after adding 10% fetal calf serum culture Cardiac Fibroblasts to 85-90% fusion using high sugar-DMEM, and
It is inoculated in 24 culture orifice plates, cultivates 10 days.Culture medium is discarded, and is rinsed 3 times using PBS, 0.3% (volume/volume) is added
The NH of Triton X-100 and 20mM4OH PBS is handled 5 minutes under the conditions of 37 DEG C, discards digestive juice, and PBS is washed twice, obtained
The extracellular matrix of cardiac fibroblasts.As shown in Figure 1A-a1, light is under the microscope, it can be seen that orifice surface is myocardium
The extracellular matrix of fibroblasts to secrete covers, and forms the porous matrix network of an interconnection.
Carry out sirius red dyeing to it, detect the content and distribution of collagen in matrix, as shown in Figure 1A-a2, cardiac muscle at
Contain a large amount of collagen in extracellular matrix derived from fibrocyte and is evenly distributed.Scanning electron microscope (SEM) detection shows ECM
Ultra microstructure is by sheet and granular material structure composition.In order to further detect cell-derived extracellular matrix after de- cell
Reservation (Figure 1B), the Tot Prot before and after de- cell is measured, takes off Tot Prot and de- cell after cell as the result is shown
It is preceding to reduce by 17% or so, but no difference of science of statistics, illustrate in taking off cell processes i.e. will loss section divide albumen, but still can be with
Retain the composition of albumen derived from most cells (Fig. 1 C).Immunofluorescence test matrix composition is utilized simultaneously, as the result is shown
Contain collagen (I, III, IV type) in extracellular matrix component derived from Cardiac Fibroblasts, fine laminins
(fibronection), Laminin lens (laminin), tubulin (Fig. 1 D).
3. preparing brown fat stem cell (BADSCs) mixture slaking enzyme solution, 37 DEG C of pre- stand-by heats, in mixture slaking enzyme solution
α-MEM culture medium (Gibco): 0.25% pancreatin (sigma): type Ⅳ collagenase (sigma): Dispase enzyme (sigma)=4:4:
1:1.3 week old SD rats are taken, the neck that breaks is put to death, and is transferred in superclean bench after 75% ethyl alcohol soaking disinfection, is cut off rat back
Skin carefully takes out the brown adipose tissue around back and shoulder blade with scissors, is washed three times with the PBS of pre-cooling.The group of collection
It knits and shreds as far as possible, 10mL mixture slaking enzyme is then added, 37 DEG C of Stirrings digest 40min, until being visible by naked eyes bulk rouge
Fat tissue, the culture medium containing serum stop digestion, and the screen to filtrate after abundant pressure-vaccum discards remnant tissue's block, collect digestive juice,
600g is centrifuged 8min, wash and is precipitated 1 time with the α-MEM culture medium of serum-free, with suitable density by cell inoculation in culture plate,
And it is inoculated on extracellular matrix derived from Cardiac Fibroblasts and is cultivated.
The detection of 2 conversion ratio of embodiment
1. the cell of culture is rinsed with PBS using the proliferative conditions of 1/2/3/4/5/7 day BADSC of CCK-8 detection culture
3 times, the CCK-8 solution containing 10% is added, and be incubated for 1.5 hours at 37 DEG C, supernatant is drawn, using microplate reader in 450nm condition
Lower detection OD value, as shown in Figure 2 A, the BADSC being inoculated on derivative host material have stronger proliferative capacity.Using immune
The expression of fluorescence detection cell cycle coherent detection point PH3, it is as shown in Figure 2 B, as a result same to confirm that extracellular derivative matrix promotes
The cell cycle of BADSC is activated, and the cell into the S phase dramatically increases.
2. the further differentiation situation of detection BADSC Cardiomyocytes pedigree on host material.It collects culture 3 days and 7 days
BADSC cell, PBS are washed three times, after 4% paraformaldehyde fixes 30 minutes, carry out immunofluorescence dyeing, addition increases with Myocardium Differentiation
It grows and survives relevant myocardium earlier markers Gata4 antibody, and utilize laser co-focusing progress Image Acquisition.As shown in figure 3,
Compared with culture BADSC merely, BADSC cell expression control group expression Gata4 in the derivative matrix of Cardiac Fibroblasts compared with
It is more, and with the extension of incubation time, the expression of the Gata4 of experimental group and control group is significantly lowered, simple BADSC culture group
Substantially without the expression of Gata4, and derive the cell on host material still and can detecte the table of the myocardium earlier markers in part
It reaches, this proves that derivative matrix can promote BADSC continuous expression Gata4, promotes the differentiation and survival of BASC cells into cardiomyocytes.
14 days feeding, the differentiation of collection sample detection BADSC cells into cardiomyocytes of 3.BADSC training.As shown in figure 4, passing through ratio
Expression discovery compared with cardiomyocyte markers object α-actinin, c-TnT, Tnnt2, it is centripetal to derive the BADSC cultivated on host material
Myocyte's differentiation is more.
4. utilizing the ratio of 14 days BADSC myocardiac differentiations of Flow cytometry culture.4% poly first will be utilized
The fixed BADSC cell of aldehyde carries out streaming antibody label, and antibody diluted concentration is 1:200, utilizes flow cytomery c-TnT
Positive ratio.As shown in figure 5, the ratio without BADSC expression c-TnT in derivative matrix group is 15.33%, and derive matrix group
The ratio of middle BADSC expression c-TnT is 42.57%.Extracellular matrix derived from further confirmed myocardial fibroblast can promote
Into the differentiation of BADSC cells into cardiomyocytes.
Disclosed above is only a specific embodiment of the invention, and still, the present invention is not limited to this, any ability
What the technical staff in domain can think variation should all fall into protection scope of the present invention.
Claims (3)
1. a kind of method for improving brown fat stem cell myocardiac differentiation efficiency, which is characterized in that using cardiac muscle at fibre
Cell-derived extracellular matrix culture brown fat stem cell is tieed up, cardiac muscle cell is divided into.
2. improving the method for brown fat stem cell myocardiac differentiation efficiency according to claim 1, which is characterized in that
It is operated in accordance with the following steps:
S1. it takes 1 day age SD rat heart to be placed in the PBS liquid of pre-cooling, shreds, digest tissue block completely using 0.05% pancreatin,
It is centrifuged within 1000rpm/7 minutes, obtains cell precipitation, be resuspended and be seeded in culture dish in DMEM in high glucose plus 15% fetal calf serum,
Not adherent cardiac muscle cell is discarded after 1 hour, obtains primary Cardiac Fibroblasts；
S2. it digests, and is inoculated with after adding 10% fetal calf serum culture Cardiac Fibroblasts to 85-90% fusion using high sugar-DMEM
It in 24 culture orifice plates, cultivates 10 days, discards culture medium, PBS is rinsed 3 times, and the Triton X- that volume fraction is 0.3% is added
The NH of 100 and 20mM4OH PBS handles 5 minutes under the conditions of 37 DEG C, discards digestive juice, PBS is washed twice, obtains cardiac muscle into fiber
The extracellular matrix of cell secretion；
S3. 3 week old SD rats are taken, the neck that breaks is put to death, and the brown fat around back and shoulder blade is taken out after the immersion of 75% ethyl alcohol
Tissue, is washed three times with the PBS of pre-cooling, is shredded, and 10 mL mixture slaking enzymes are added, and 37 DEG C of Stirrings digest 40min, sufficiently
The screen to filtrate after pressure-vaccum discards remnant tissue's block, collects digestive juice, and 600g is centrifuged 8min, is washed with the α-MEM culture medium of serum-free
It washs precipitating 1 time, cell inoculation is cultivated on the extracellular matrix derived from Cardiac Fibroblasts, finally obtains differentiation
Cardiac muscle cell.
3. improving the method for brown fat stem cell myocardiac differentiation efficiency according to claim 2, which is characterized in that
In the step S3 ingredient of mixture slaking enzyme be α-MEM culture medium, 0.25% pancreatin, type Ⅳ collagenase, Dispase enzyme, four
The mass ratio of person is 4:4:1:1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110499281A (en) * | 2019-08-26 | 2019-11-26 | 首都医科大学附属北京世纪坛医院 | A method of quickly establishing H9c2 cardiac muscle cell's fat model |
| CN111793609A (en) * | 2020-09-08 | 2020-10-20 | 北京达熙生物科技有限公司 | Method for promoting proliferation and differentiation of adipose-derived stem cells |
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| CN104940997A (en) * | 2014-03-27 | 2015-09-30 | 复旦大学 | Human tissue-engineered cardiac muscle tissue |
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| CN107735113A (en) * | 2015-06-03 | 2018-02-23 | 威斯康星校友研究基金会 | Extracellular matrix and its injectable formulation derived from cardiac fibroblast for treating ischemic disease or damage |
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| CN104940997A (en) * | 2014-03-27 | 2015-09-30 | 复旦大学 | Human tissue-engineered cardiac muscle tissue |
| CN107735113A (en) * | 2015-06-03 | 2018-02-23 | 威斯康星校友研究基金会 | Extracellular matrix and its injectable formulation derived from cardiac fibroblast for treating ischemic disease or damage |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110499281A (en) * | 2019-08-26 | 2019-11-26 | 首都医科大学附属北京世纪坛医院 | A method of quickly establishing H9c2 cardiac muscle cell's fat model |
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Application publication date: 20190423 |