CN109486752A - A kind of method of Qinchuan cattle intramuscular fat cell separation - Google Patents
A kind of method of Qinchuan cattle intramuscular fat cell separation Download PDFInfo
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- CN109486752A CN109486752A CN201811547543.XA CN201811547543A CN109486752A CN 109486752 A CN109486752 A CN 109486752A CN 201811547543 A CN201811547543 A CN 201811547543A CN 109486752 A CN109486752 A CN 109486752A
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- C—CHEMISTRY; METALLURGY
- 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
- C12N5/0653—Adipocytes; Adipose tissue
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C—CHEMISTRY; METALLURGY
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The present invention relates to a kind of Qinchuan cattle intramuscular fat cell isolate and purify and identification method, Qinchuan cattle longissimus dorsi muscle is digested using II Collagenase Type, it is neutralized through DMEM/F12 culture medium of the postdigestive tissue block of II Collagenase Type containing 10% fetal calf serum, it filtered, be centrifuged to obtain cell, cell is washed again, is resuspended in the DMEM/F12 culture medium containing 10%FBS after centrifugation, after counting, by cell inoculation in culture dish, in CO2It is cultivated 1.5 hours in incubator, is washed with sterile phosphate buffer saline solution and remove non-adherent cell, then by cell culture in 37 DEG C, CO2In the incubator that concentration is 5%, a subculture was changed every 2 days, after cell confluency degree reaches 70%-80%, carries out cellular immunofluorescence label positive cell identification.The beef cattle intramuscular fat cell PECTORAL LIMB SKELETON positive rate with higher that this method obtains lays a good foundation not increase subcutaneous and visceral fat deposition research while raising intramuscular fat deposition.
Description
Technical field
The invention belongs to cell biologies, cell engineering field, and in particular to a kind of Qinchuan cattle intramuscular fat is thin
The separation method of born of the same parents.
Background technique
There are 4 primary fat tissue locations: internal organ in domestic animal body, subcutaneously, between intramuscular and muscle.Intramuscular adipose tissue
Accumulation be very important, and adipose tissue is greatly increased in the burden that the excess accumulation of other positions is to husbandry sector
Production cost, common phenotype are that certain animals are more much bigger than species, are primarily not the accumulation of intramuscular adipose tissue,
And mainly subcutaneous or interior fat accumulation.However, up to the present, the mechanism for leading to intramuscular fat preferred accumulation is still fixed
Justice is indefinite.
Fat cell, especially intramuscular fat cell possess during fetus muscle development with myogenous cells common
Progenitor cells.Explore mesenchymal stem/progenitor cells will be helpful to understand to the mechanism of muscle-derived or fatty fibroblast pedigree early differentiation
Intramuscular fat cell preferentially forms, thus the mechanism for promoting marbling to deposit.It is consistent with this concept, more and more
Evidence shows that intramuscular fat cell is different with the subcutaneous performance compared with visceral adipocytes, therefore, explores intramuscular fat cell
Uniqueness development origin and property realize enhancing beef marbling, and the whole fat target without improving animal, is application
One of the project of people's research.
Summary of the invention
The object of the present invention is to provide a kind of separation methods of Qinchuan cattle intramuscular fat cell, to probe into intramuscular rouge
The deposition regulatory mechanism of fat lays the foundation.
In order to realize that above-mentioned task, the present invention take following technical solution:
A kind of separation method of Qinchuan cattle intramuscular fat cell, which is characterized in that follow these steps to implement:
1) the 4 age in days Qinchuan cattle longissimus dorsi muscle tissues that will be separated under aseptic condition, with the penicillin and chain of PBS plus 2%
Mycin cleaning, is then cut into 1mm3~2mm3Tissue block；
2) tissue block is added in centrifuge tube, is digested with II Collagenase Type, used through the postdigestive tissue block of II Collagenase Type
DMEM/F12 culture medium containing 10%FBS neutralizes, and is then filtered, will be obtained with the stainless steel mesh of 70 mesh and 200 mesh
Filtrate with 1500rpm centrifugation 10 minutes, discard supernatant liquid, obtain cell；
3) obtained cell is resuspended with serum-free DMEM/F12 culture medium and is centrifuged 10 minutes with 1500rpm, after centrifugation
It washes twice；
4) cell after washing is resuspended in the DMEM/F12 culture medium containing 10%FBS, after counting, by cell with 2.5
×105The density of a cell is seeded in culture dish, and culture dish is placed in CO21.5 hours in the incubator that concentration is 5%, then
It is washed with sterile PBS to remove non-adherent cell, and replace culture dish with the fresh DMEM/F12 culture medium containing 10%FBS
In former culture medium；
5) by cell culture in 37 DEG C, CO2In the incubator that concentration is 5%, was changed every 2 days and primary fresh contain 10%
The DMEM/F12 culture medium of FBS is logical using PECTORAL LIMB SKELETON specific marker Pref-1 after cell confluency degree reaches 70%
The method for crossing cellular immunofluorescence carries out positive cell identification.
According to the present invention, cellular immunofluorescence identification positive cell operating procedure is as follows:
Cell inoculation is added to the DMEM/F12 culture medium culture containing 10%FBS in 6 well culture plates using preceding, when thin
Born of the same parents converge when reaching 70%, discard the DMEM/F12 culture medium containing 10%FBS and wash cell with PBS, at room temperature with dense
The cells are fixed 30 minutes for the paraformaldehyde that degree is 4%, and is washed again with PBS；
The Triton X-100 that the BSA and concentration for being at room temperature 0.1% with concentration are 0.5% divides cell-permeant 60
Clock discards liquid, is then washed again with PBS, is closed 60 minutes using the BSA that concentration is 3% at room temperature；
Lock solution is discarded, and the diluted mouse Pref-1 antibody (primary antibody) of sufficient amount is added, and is incubated overnight at 4 DEG C,
Then mouse Pref-1 antibody is discarded, diluted anti-mouse HRP fluorescence secondary antibody is added, 37 DEG C of incubation 80min use PBST after discarding secondary antibody
It washes 3 times, each 3min.DAPI is added dropwise afterwards and is protected from light incubation 5min, washes 4 removals extra DAPI, each 5min with PBST.
Finally liquid is blotted, and in fluorescence microscopy microscopic observation result.
The separation method of Qinchuan cattle intramuscular fat cell of the invention has the advantage that compared with prior art
(1) the Qinchuan cattle intramuscular fat cell obtained has high PECTORAL LIMB SKELETON positive rate；
(2) pollution-free；
(3) the intramuscular fat cell that 4 age in days Qinchuan cattle longissimus dorsi muscle tissues obtain is digested by using II Collagenase Type,
The positive cell ratio for obtaining intramuscular fat cell from musculature is significantly improved, and greatly reduces fat cell pollution
Risk.Meanwhile using pref-1 antibody mediated immunity fluorescent marker PECTORAL LIMB SKELETON, provided for the separation identification of Preadipocyte In Vitro
A kind of new method does not yet increase subcutaneous and visceral fat deposition research and establishes to improve while intramuscular fat deposition
Basis.
Detailed description of the invention
Fig. 1 is the 0d after Qinchuan cattle intramuscular fat cell inoculation, and 2d, 4d (label is D2, D4 in figure) are micro- afterwards
The photo (200 ×) obtained under the microscope；
Fig. 2 is the immunofluorescence photograph (40 using PECTORAL LIMB SKELETON specifically expressing factor Pref-1 identification PECTORAL LIMB SKELETON
×)；
Fig. 3 is the expression quantity of Western-blot detection PECTORAL LIMB SKELETON specifically expressing factor Pref-1 albumen.
The present invention is described in further detail with reference to the accompanying drawings and examples.
Specific embodiment
Applicant pass through in the course of the research test it was found that, using II Collagenase Type digestion Qinchuan cattle calf back most
Long flesh can obtain more positive PECTORAL LIMB SKELETONs, digest with other methods such as collagenase type I, or with adult ox back longest
Flesh be sample obtain intramuscular fat cell there is some difference.
Applicant through using different age group ox back longest muscular tissue, different clostridiopetidase As, different digestions time with
And the different differential velocity adherent time is compared discovery, the longissimus dorsi muscle tissue of ox of coming into being (birth 0-1 weeks) passes through II Collagen Type VI
It digests within enzyme 1.5 hours, more positive PECTORAL LIMB SKELETONs can be obtained by replacing fresh culture after 1.5 hours after inoculation.Meanwhile
Using the methods of immunofluorescence, the reliability for identifying intramuscular Preadipocyte In Vitro is further improved.
Below in an example, related animal tissue, reagent source are as follows:
4 age in days calves improve center experimental farm (Shaanxi, China Yang Ling) from national beef cattle；
II Collagenase Type, DAPI are purchased from GIBCO company.
FBS, DMEM/F12, PBS culture medium are purchased from Hyclone company.
Tissue Culture Dish and centrifuge tube are purchased from Corning company.
The paraformaldehyde and PBST that concentration is 4% are purchased from Suo Laibao company.
4', 6- diamidino -2-phenylindone (4', 6-diamidino-2-phenylindole, DAPI), BSA and it is non-from
Sub- surfactant Triton X-100 (hereinafter referred to as Triton X-100) is purchased from sigma company.
Mouse Pref-1 antibody (primary antibody) and anti-mouse HRP fluorescence secondary antibody are purchased from abcam company.
The separation method for the Qinchuan cattle intramuscular fat cell that the present embodiment provides, specifically follows these steps to implement:
1) the 4 age in days Qinchuan cattle longissimus dorsi muscle tissues that will be separated under aseptic condition, with the penicillin and chain of PBS plus 2%
Mycin cleaning, is then cut into 1mm3~2mm3Tissue block；
2) tissue block is added in centrifuge tube, is digested with II Collagenase Type, used through the postdigestive tissue block of II Collagenase Type
DMEM/F12 culture medium containing 10%FBS neutralizes, and is then filtered, will be obtained with the stainless steel mesh of 70 mesh and 200 mesh
Filtrate with 1500rpm centrifugation 10 minutes, discard supernatant liquid, obtain cell；
3) it by obtained cell serum-free DMEM/F12 culture medium and with 1500rpm centrifugation 10 minutes, is washed after centrifugation
Twice；
4) wash after cell be resuspended in the DMEM/F12 culture medium culture containing 10%FBS, after counting, by cell with
2.5×105The density of a cell is seeded in culture dish, and culture dish is placed in the CO that concentration is 5%21.5 hours in incubator,
Observation discovery under the microscope has a small amount of cell adherent, is then washed with sterile PBS to remove non-adherent cell, and with fresh
Former culture medium in DMEM/F12 culture medium replacement culture dish containing 10%FBS；Observation finds cell exponentially after being inoculated with 2d
Multiple increases, and observation finds that cell basically reaches 100% and converges after being inoculated with 4d, pollution-free phenomenon (Fig. 1).
5) it by cell culture in 37 DEG C, the incubator that CO2 concentration is 5%, was changed every 2 days and primary fresh contains 10%
The DMEM/F12 culture medium of FBS is logical using PECTORAL LIMB SKELETON specific marker Pref-1 after cell confluency degree reaches 70%
The method for crossing cellular immunofluorescence carries out positive cell identification.
Above-mentioned cellular immunofluorescence identification positive cell operating procedure is as follows:
Cell inoculation is added to the DMEM/F12 culture medium culture containing 10%FBS in 6 well culture plates using preceding, when thin
Born of the same parents converge when reaching 70%, discard the DMEM/F12 culture medium containing 10%FBS and wash cell with PBS, at room temperature with dense
The cells are fixed 30 minutes for the paraformaldehyde that degree is 4%, and is washed again with PBS；
The Triton X-100 that the bovine serum albumin(BSA) and concentration for being at room temperature 0.1% with concentration are 0.5% makes cell
Penetrating 60 minutes, liquid is discarded, is then washed again with PBS, is closed 60 minutes using the BSA that concentration is 3% at room temperature；
Lock solution is discarded, and the diluted mouse Pref-1 antibody (primary antibody) of sufficient amount is added, and is incubated overnight at 4 DEG C,
Then mouse Pref-1 antibody is discarded, diluted anti-mouse HRP fluorescence secondary antibody is added, 37 DEG C of incubation 80min use PBST after discarding secondary antibody
It washes 3 times, each 3min.DAPI is added dropwise afterwards and is protected from light incubation 5min, washes 4 removals extra DAPI, each 5min with PBST.
Finally liquid is blotted, and in fluorescence microscopy under the microscope as a result, identifying Preadipocyte In Vitro in separated cell
Ratio.
It is found after counting, Preadipocyte In Vitro immuning positive cell reaches 91.33% ± 0.03 (Fig. 2).It is connect to reaching
The cell that touching inhibits carries out induction differentiation, collects 0d, 2d, 4d respectively, and the cell of 6d (label is D2, D4, D6 in figure) is total
Albumen detects Preadipocyte In Vitro specific marker Pref-1 expressing quantity, discovery Pref-1 albumen (figure on a declining curve
3), show that separated cell is Preadipocyte In Vitro.
Claims (2)
1. a kind of separation method of Qinchuan cattle intramuscular fat cell, which is characterized in that follow these steps to implement:
1) the 4 age in days Qinchuan cattle longissimus dorsi muscle tissues that will be separated under aseptic condition, with the penicillin and streptomysin of PBS plus 2%
Cleaning, is then cut into 1mm3~2mm3Tissue block；
2) tissue block is added in centrifuge tube, is digested with II Collagenase Type, through the postdigestive tissue block of II Collagenase Type with containing
The DMEM/F12 culture medium of 10%FBS neutralizes, and is then filtered with the stainless steel mesh of 70 mesh and 200 mesh, the filter that will be obtained
Liquid was discarded supernatant liquid, is obtained cell with 1500rpm centrifugation 10 minutes；
3) obtained cell is resuspended with serum-free DMEM/F12 culture medium and is centrifuged 10 minutes with 1500rpm, washed after centrifugation
Twice；
4) cell after washing is resuspended in the DMEM/F12 culture medium containing 10%FBS, after counting, by cell with 2.5 × 105
The density of a cell is seeded in culture dish；Culture dish is placed in CO21.5 hours are cultivated in the incubator that concentration is 5%, then
It is washed with sterile PBS to remove non-adherent cell, and replace culture dish with the fresh DMEM/F12 culture medium containing 10%FBS
In former culture medium；
5) by cell culture in 37 DEG C, CO2In the incubator that concentration is 5%, changed every 2 days primary fresh containing 10%FBS's
DMEM/F12 culture medium, after cell confluency degree reaches 70%-80%, using PECTORAL LIMB SKELETON specific marker Pref-1 into
Row cellular immunofluorescence marks positive cell identification.
2. the method as described in claim 1, which is characterized in that the cellular immunofluorescence marks positive cell operating procedure
It is as follows:
Cell inoculation is added to the DMEM/F12 culture medium culture containing 10%FBS in 6 well culture plates using preceding, when cell converges
When conjunction reaches 70%, discards the DMEM/F12 culture medium containing 10%FBS and wash cell with PBS, be with concentration at room temperature
The cells are fixed 30 minutes for 4% paraformaldehyde, and is washed again with PBS；
The nonionic surfactant Triton that the bovine serum albumin(BSA) and concentration for being at room temperature 0.1% with concentration are 0.5%
X-100 makes cell-permeant 60 minutes, then discards liquid, is then washed again with PBS, the use of concentration is at room temperature 3%
BSA is closed 60 minutes；
Lock solution is discarded, and the diluted mouse Pref-1 antibody of sufficient amount is added, and is incubated overnight at 4 DEG C, mouse is then discarded
Pref-1 antibody, is added diluted anti-mouse HRP fluorescence secondary antibody, 37 DEG C of incubation 80min, discards and washes 3 times with PBST after secondary antibody, every time
3min, is added dropwise 4' afterwards, and 6- diamidino -2-phenylindone (4', 6-diamidino-2-phenylindole, DAPI) is protected from light incubation
5min washes 4 removals extra DAPI, each 5min with PBST；
Finally liquid is blotted, and in fluorescence microscopy microscopic observation image.
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Cited By (2)
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| WO2021056921A1 (en) * | 2019-09-25 | 2021-04-01 | 西北农林科技大学 | Cell co-culture apparatus and co-culture method of bovine muscle cells and adipose cells |
| CN113717932A (en) * | 2021-09-16 | 2021-11-30 | 四川农业大学 | Primary isolation culture and induced differentiation method for intramuscular precursor adipocytes of adult yaks |
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