CN106754663A - The method for promoting the extracorporeal culturing method and monitoring Adult Mammals cardiomyocyte proliferation of Adult Mammals myocyte survival - Google Patents
The method for promoting the extracorporeal culturing method and monitoring Adult Mammals cardiomyocyte proliferation of Adult Mammals myocyte survival Download PDFInfo
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- CN106754663A CN106754663A CN201611082429.5A CN201611082429A CN106754663A CN 106754663 A CN106754663 A CN 106754663A CN 201611082429 A CN201611082429 A CN 201611082429A CN 106754663 A CN106754663 A CN 106754663A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0657—Cardiomyocytes; Heart cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2503/00—Use of cells in diagnostics
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- G—PHYSICS
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- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
Abstract
Method the present invention relates to promote the extracorporeal culturing method and monitoring Adult Mammals cardiomyocyte proliferation of Adult Mammals myocyte survival, the extracorporeal culturing method is to co-culture the cardiac muscle cell of the cardiac muscle cell of neonatal mammal and Adult Mammals；The method of the monitoring Adult Mammals cardiomyocyte proliferation, comprises the following steps：(1) using described extracorporeal culturing method culture adult cardiac muscle of mammal cell, during the culture, the breeding of the Adult Mammals cardiac muscle cell is monitored using living cells work station；(2) immunostaining treatment is carried out to the newborn adult cardiac muscle of mammal cell obtained in step (1), identifies the property of the newborn adult cardiac muscle of mammal cell.The method can significantly extend the time-to-live of Adult Mammals cardiac muscle cell, using living cells work station, visual record be carried out to cardiomyocyte proliferation, and the method is that research adult cardiomyocytes propagation and related mechanism provide powerful.
Description
Technical field
The invention belongs to biological technical field, and in particular to promote the in vitro culture of Adult Mammals myocyte survival
The method of method and monitoring Adult Mammals cardiomyocyte proliferation.
Background technology
Myocardial infarction and heart failure are the principal diseases that death is caused in world wide.The heart of mammal is only
Only there is power of regeneration in short term after birth, but Adult Mammals cardiac muscle loses power of regeneration, it is difficult to repair the diseases such as myocardial infarction
The cardiac muscle loss that disease is caused.Studying in recent years confirms that adult cardiac muscle is updated with extremely low regeneration rate, but Myocardial Regeneration is next
Source is unclear.The original cardiac muscle cell of Application Hints that pedigree follows the trail of (fate-mapping) technology is the master of adult Myocardial Regeneration
Originate.Based on this, study the mechanism of proliferation of adult cardiomyocytes and stimulate its propagation controlling in myocardial infarction and heart failure
There is important clinical significance in treatment, be also the forward position research direction in Myocardial Regeneration field, and there is no the research mammal heart at present
The external model of muscle cell multiplication.
The content of the invention
In view of this, it is contemplated that overcoming during tradition research cardiomyocyte proliferation method can not differentiate cardiomyocyte proliferation
Cytokinesis this key link, and false positive rate is high and underestimates the double defect of actual value, there is provided one kind promotes adult lactation
The extracorporeal culturing method of animal cardiac muscle cell survival, and a kind of side for monitoring Adult Mammals cardiomyocyte proliferation is provided simultaneously
Method.
To reach above-mentioned purpose, the present invention provides following technical scheme：
1st, a kind of extracorporeal culturing method for promoting Adult Mammals myocyte survival, methods described is：New life is fed
The cardiac muscle cell of newborn animal co-cultures with the cardiac muscle cell of Adult Mammals.
Further, the cardiac muscle cell of the Adult Mammals is by the cell of fluorochrome label.
Further, the cardiac muscle cell of the neonatal mammal and the cardiac muscle cell of Adult Mammals are of the same race or xenogenesis
The cardiac muscle cell of mammal.
Further, the cardiac muscle cell of the neonatal mammal is the cardiac muscle cell of rat；The Adult Mammals
Cardiac muscle cell is the cardiac muscle cell of mouse.
Further, the cardiac muscle cell of the Adult Mammals be the cardiac muscle cell of β-actin-GFP transgenic mices, α-
The cardiac muscle cell of MHC-MerCreMer-GFP pedigree spike mouse, α-MHC-MerCreMer-tdTomato pedigree spike mouse
Cardiac muscle cell or α-MHC-MerCreMer-lacZ pedigree spike mouse cardiac muscle cell in one kind.
Further, the cardiac muscle cell of the neonatal mammal is with the inoculative proportion of the cardiac muscle cell of Adult Mammals
20:1。
2nd, a kind of method for monitoring Adult Mammals cardiomyocyte proliferation, comprises the following steps：
(1) using described extracorporeal culturing method culture adult cardiac muscle of mammal cell, during the culture, using work
Cell work station monitors the breeding of the Adult Mammals cardiac muscle cell；
(2) immunostaining treatment is carried out to the newborn adult cardiac muscle of mammal cell obtained in step (1), identification is described
The property of new life adult cardiac muscle of mammal cell.
Further, in step (1), it is described culture be humidity be 95%, temperature be 37 DEG C, CO2Concentration is 5% condition
Lower culture 7d.
Further, in step (1), when the utilization living cells work station is monitored, the imaging interval time is 10min-1h.
Further, in step (2), the property of the newborn adult cardiac muscle of mammal cell includes Cardiac-specific albumen
The expression of label cTnI and sarcomere structure are formed.
The beneficial effects of the present invention are：The invention provides the external training for promoting Adult Mammals myocyte survival
The method of the method for supporting and monitoring Adult Mammals cardiomyocyte proliferation, it is thin that this method can significantly extend Adult Mammals cardiac muscle
The time-to-live of born of the same parents, using living cells work station, visual record is carried out to cardiomyocyte proliferation, the method is research adult cardiac muscle
Cell is bred and related mechanism provides powerful.
Brief description of the drawings
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below and carries out
Explanation：
Fig. 1 is the operational flowchart of embodiment 1；
Under the conditions of Fig. 2 is to co-culture, Adult Mammals cardiomyocyte cell death, survival and proliferative conditions figure；
Fig. 3 is Adult Mammals cardiomyocyte proliferation rate and cell nuclei graph of a relation；
Fig. 4 is Adult Mammals cardiac muscle cell size and proliferation rate graph of a relation；
Fig. 5 changes with time figure for Ki67 (A) positive rates and PH3 (B) positive rate of Adult Mammals cardiac muscle cell.
Specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
Embodiment 1
Tested by operational flowchart in Fig. 1
First, neonatal mammal cardiac muscle cell (NRVM) extracts with Adult Mammals cardiac muscle cell (ACM) and separates
1st, NRVM is extracted and separated：
1-2 age in days SD rat suckling mouse isolated hearts are taken, is put into the PBS of precooling rapidly, rinsed 3-5 times and cut off sustainer
Deng tissue, heart is then cut into 1mm3Size, adds the mass fraction of 1ml to be 0.15% pancreatin digestive juice, and blow repeatedly
1min is beaten, digestive juice is slowly sucked, continuous 2 additions 1ml pancreatin digestive juice carries out digestion process, each digestive juice quality point
Number terminates for 10% hyclone, is placed on ice；The clostridiopetidase A that 3-5ml mass fractions are 0.08% is added, is blown and beaten repeatedly
Scattered to tissue block, be put into incubator and digest 30min；The tissue that taking-up has digested is blown and beaten repeatedly, until tissue is substantially complete
Totally disappeared, 200 mesh filters filtering, filtrate is centrifuged 5min, abandons supernatant with 1000r/min, and DMEM high glucose mediums are resuspended, are put into training
Differential velocity adherent 1h in ware is supported, cardiac muscle cell is separated with non-myocardial infarction；Finally it is seeded in 24 orifice plates.
2nd, ACM is extracted and separated：
(1) reagent prepares
A) tyrode is prepared：500ml common desktop liquid is prepared, is 7.3 with NaOH regulations pH, formula is shown in Table 1.
| Molar concentration (mM/L) | Content (g/L) | |
| NaCl | 140 | 6.89 |
| KCl | 4 | 0.3 |
| Hepes | 10 | 5(ml) |
| MgCl2.6H2O | 1.0 | 0.2 |
| Glucose | 10 | 2 |
B) working solution is prepared
No. 1 liquid：A) tyrode 350ml is taken, 437.5mg thiosulfonic acids, 438mg biacetyl monoximes (BDM) is separately added into；
No. 2 liquid：No. 1 liquid of 50ml is taken, 50mg clostridiopetidase As, 6mg pancreatin, 0.11mg CaCl is separately added into2；
No. 3 liquid：No. 1 liquid of 50ml is taken, 250mg BSA, 0.70mg CaCl is separately added into2；
No. 4 liquid：No. 1 liquid of 50ml is taken, 250mg BSA, 1.39mg CaCl is separately added into2；
Above-mentioned working solution is used after being both needed to filtration sterilization.
(2) Adult Mammals cardiac muscle cell extracts and separates：
Water bath temperature is adjusted to 37 DEG C, using the pipeline 30min of 75% ethanol disinfection Langendorff perfusion systems,
Again with aseptic water washing 3-5 times, then No. 1 liquid and No. 2 liquid are separately added into the long tube and short tube of Langendorff perfusion systems
In, flow velocity is adjusted, remove bubble.
β-actin-GFP transgenosis adult mice isolated hearts are taken, is placed in 4 DEG C of PBS, moved under microscope；With eye
Section's tweezer removes paraaortic fat and its hetero-organization, and sustainer is hung on syringe needle rapidly, is knotted with the end of a thread and fixed, syringe needle
Position no more than aortic root；No. 1 liquid is sucked with the syringe that capacity is 1ml to go out the blood in heart；Remove
Syringe needle, is hung on Langendorff perfusion systems, removes bubble in irrigator, with No. 1 perfusion 2min, then changes No. 2 liquid digestion
15min, softens to heart tissue and collapses, and perfusion flow velocity is accelerated, and heart color is shallow by red change；Stop digestion, by cardiac scissors
Under in 60mm wares, cardiac scissors is broken to several bulks by plus No. 2 liquid of 2-5ml, is dispelled with pasteur pipet；The filtering of 50 mesh filters contains
The digestive juice of cell, removes undigested heart tissue, and to No. 3 liquid of 10ml are added in filtrate, to terminate digestion；Will be mixed
Close in liquid immigration sterile centrifugation tube, 1min is centrifuged under centrifugal force 50g, abandon supernatant, add the multiple calcium of No. 3 liquid of 5ml, abandon No. 3
Liquid, adds No. 4 liquid of 10ml to continue multiple calcium 10min, abandons No. 4 liquid, adds the hyclone (FBS) that mass fraction is 10%
DMEM in high glucose culture medium re-suspended cell, and count.
2nd, mark ACM nucleus and co-culture ACM and NRVM, use living cells work station Follow-up observation cardiac muscle cell
Division
DRAQ5TMFluorescence probe mark ACM nucleus is far infrared fluorescence (5mM/L processes 10min), and by NRVM and ACM
With 20:1 ratio is seeded in copolymerization Jiao's capsule, and wherein NRVM is used as trophocyte.
The image forming program and imaging parameters of living cells work station are set, fluorescence intensity during picture is adjusted to, the time for exposure, are made
The influence of imaging clearly and reduction to the state of cell.Usual each visual field carries out multi channel imaging, and to multiple visuals field
Imaging.Usual imaging time interval can be according to experiment purpose from for 10 minutes to 1 hour.
Copolymerization Jiao's capsule is put into the cell culture cell of living cells work station and fixed, closing cell culture cell,
Humidity is 95%, and temperature is 37 DEG C, CO2Concentration be 5% under conditions of cultivate 7d, while carrying out cell imaging.
Acquired results are shown as shown in Figure 2, Figure 3 and Figure 4, wherein, as shown in Figure 2, cardiac muscle cell can be observed in incubation
Death, survival and division, its ratio are respectively 53.6%, 39.4% and 7%, compared to depositing for individually culture adult cardiomyocytes
Motility rate is significantly enhanced；From Fig. 3, Fig. 4, monokaryon does not have notable difference with the proliferation rate of double-core cardiac muscle cell, and
The cardiac muscle cell of division and cardiac muscle cell's size no significant difference of nondividing, illustrate cardiomyocyte proliferation and cell nuclei and
Cell size is without obvious correlation.
3rd, immunostaining
New life ACM in co-culture system is taken, after fixing cell 15min with the paraformaldehyde fixer that mass fraction is 4%,
Rinsed 3 times with PBS, each 5min；Then, immunostaining confining liquid is added dropwise, 30min is closed, unnecessary confining liquid is washed away, adds
TnI antibody (1:100, addition flooded cell), 4 DEG C of incubation 12h are then rinsed 3 times, each 5min with PBS, are added
Cy3 marks goat anti-rabbit antibodies (1:100, addition flooded cell), 37 DEG C are incubated 1 hour, PBS flushings 3 times, every time
5min, is eventually adding DAPI dye liquors, and 20min is incubated at 25 DEG C, and PBS is rinsed 3 times, each 5min.
To cardiac muscle cell Ki67, PH3 positive rates are counted, as a result as shown in figure 5, as shown in Figure 5, in incubation,
Cardiac muscle cell's Ki67 and PH3 positive takes the lead in raising, and then reduces, the positive rate highest at the 4th day and the 5th day.
Expression, sarcomere structure to Cardiac-specific albumen (troponin) label cTnI are formed and detected, are tied
Fruit is shown in Table 2.
As shown in Table 2, Troponin I in the daughter cell for being produced after cardiac muscle cell's mitosis+/ sarcomere+, Troponin I+/ sarcomere-And Troponin I-/ sarcomere-Composition ratio respectively 32.25%, 19.35% and 48.38%, illustrate myocardium thin
The daughter cell produced after born of the same parents' division can again be divided into the cardiac muscle cell of functional structure.
In the present invention, except the cardiac muscle cell using β-actin-GFP transgenosis adult mices, α-MHC- are it is also possible to use
The cardiac muscle cell of MerCreMer-GFP pedigree spike mouse, the heart of α-MHC-MerCreMer-tdTomato pedigree spike mouse
One kind in the cardiac muscle cell of myocyte or α-MHC-MerCreMer-lacZ pedigree spike mouse.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
Cross above preferred embodiment to be described in detail the present invention, it is to be understood by those skilled in the art that can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (10)
1. a kind of extracorporeal culturing method for promoting Adult Mammals myocyte survival, it is characterised in that methods described is：Will
The cardiac muscle cell of neonatal mammal co-cultures with the cardiac muscle cell of Adult Mammals.
2. a kind of extracorporeal culturing method for promoting Adult Mammals myocyte survival as claimed in claim 1, its feature
It is that the cardiac muscle cell of the Adult Mammals is by the cell of fluorochrome label.
3. a kind of extracorporeal culturing method for promoting Adult Mammals myocyte survival as claimed in claim 2, its feature
It is that the cardiac muscle cell of the neonatal mammal is of the same race or heterologous mammalian with the cardiac muscle cell of Adult Mammals
Cardiac muscle cell.
4. a kind of extracorporeal culturing method for promoting Adult Mammals myocyte survival as claimed in claim 2, its feature
It is that the cardiac muscle cell of the neonatal mammal is the cardiac muscle cell of rat；The cardiac muscle cell of the Adult Mammals is
The cardiac muscle cell of mouse.
5. a kind of extracorporeal culturing method for promoting Adult Mammals myocyte survival as claimed in claim 4, its feature
It is that the cardiac muscle cell of the Adult Mammals is cardiac muscle cell, the α-MHC- of β-actin-GFP transgenic mices
The cardiac muscle cell of MerCreMer-GFP pedigree spike mouse, the heart of α-MHC-MerCreMer-tdTomato pedigree spike mouse
One kind in the cardiac muscle cell of myocyte or α-MHC-MerCreMer-lacZ pedigree spike mouse.
6. a kind of extracorporeal culturing method for promoting Adult Mammals myocyte survival as claimed in claim 4, its feature
It is that the cardiac muscle cell of the neonatal mammal is 20 with the inoculative proportion of the cardiac muscle cell of Adult Mammals:1.
7. it is a kind of monitor Adult Mammals cardiomyocyte proliferation method, it is characterised in that comprise the following steps：
(1) using the extracorporeal culturing method culture adult cardiac muscle of mammal cell described in claim any one of 1-6, the training
During supporting, the breeding of the Adult Mammals cardiac muscle cell is monitored using living cells work station；
(2) immunostaining treatment is carried out to the newborn adult cardiac muscle of mammal cell obtained in step (1), identifies the new life
The property of Adult Mammals cardiac muscle cell.
8. a kind of method for monitoring Adult Mammals cardiomyocyte proliferation as claimed in claim 7, it is characterised in that step
(1) in, it is described culture be humidity be 95%, temperature be 37 DEG C, CO2Concentration be 5% under conditions of cultivate 7d.
9. a kind of method for monitoring Adult Mammals cardiomyocyte proliferation as claimed in claim 7, it is characterised in that step
(1) in, when the utilization living cells work station is monitored, the imaging interval time is 10min-1h.
10. a kind of method for monitoring Adult Mammals cardiomyocyte proliferation as claimed in claim 7, it is characterised in that step
Suddenly in (2), the property of the newborn adult cardiac muscle of mammal cell includes the expression of Cardiac-specific protein marker cTnI
Formed with sarcomere structure.
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