CN106282100A - A kind of method of the purification tissue-derived Preadipocyte In Vitro of cattle fetal skeletal muscle - Google Patents
A kind of method of the purification tissue-derived Preadipocyte In Vitro of cattle fetal skeletal muscle Download PDFInfo
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- 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
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Abstract
A kind of method that the invention discloses purification tissue-derived Preadipocyte In Vitro of cattle fetal skeletal muscle, the material that described method uses is, cattle 3 monthly age fetus longissimus dorsi muscle tissue, agents useful for same is, collagenase, hyclone, low sugar Da Erbai kirschner MEM, cell sorting buffer, antiplatelet source property growth factor receptors Alpha antibodies, dexamethasone, 3 isobutyl group 1 methylxanthine, bovine insulin and oil red O.
Description
Technical field
The invention belongs to technological field of biochemistry, be specifically related to a kind of tissue-derived front body fat of purification cattle fetal skeletal muscle
The method of fat cell.
Background technology
Stem cell is can to maintain replicate and and can be divided into a class cell of dissimilar cell ability, stem-cell research
It is very important for understanding generation and the differentiation regulation and control of cell and tissue.It addition, stem-cell research is for medical domain
There are huge potentiality equally.As a lot of other organs, skeletal muscle comprises a lot of cell type, can produce the satellite of muscle-derived
Cell and the adipose cell of fatty tissue source property, these are all critically important to Animal husbandry production.It is known that satellite cell pair
After birth, the regeneration of the skeletal muscle of the growth of skeletal muscle and adult is the most critically important.The satellite to separation in nearly 50 years of past is thin
The research of born of the same parents is concentrated mainly on activation and the suppression of its propagation, regulates and controls their activity in vitro, raw with other cell such as blood vessel
Become intercellular interaction, the potentiality between subgroup, and as the potentiality of carrier in gene therapy.In beef cattle production, animal
The growth promoter of skeletal muscle and the deposition of intramuscular fat be the main factor affecting beef production and price.But, at present
Till research that early stage muscle development and intramuscular fat are grown relatively fewer, the development of stem cells of especially different cells comes
Source, therefore identifies the stem cell surface molecular marker characteristic for distinguishing different, and sets up Differentiation Induction in vitro model
System is significant for studying domestic animal muscle and intramuscular fat differentiation.
Embryonic stem cell still has the most indefinite to the molecular mechanism becoming flesh, one-tenth fat, one-tenth fiber orientation.
But, the confirmed embryonic stem cell of quality is separated from a limited number of mammalian species.And, 1981
Embryonic stem cell is being separated from inhuman experiment house mouse.Mice pluripotent stem cell is built thoroughly
Vertical, by normal growth in embryo transfer to female mice body until being born after being injected in blastaea.Additionally, mouse embryonic stem
After the genome of cell can be modified by transgenic and homologous recombination technique.Genetically engineered cell can be after system genitale be delivered to
Generation.The human embryonic stem cell line of pluripotency is successfully established the most, is likewise supplied with being divided into the ability of variety classes cell.To the greatest extent
Pipe to having put into substantial amounts of effort on agricultural species embryonic stem cell, but with Mus and the mankind effort compared be not already becoming very much
Merit.Difficulty of this work may have following some, one is that agricultural species is compared the growth before Embryonic limb bud cell and deposited with mice
At the specificity of species, second is to be not fully understood for the somatomedin required for agricultural animal fetal development, three be
In mice, whether effective stem cell labeling is applicable to agricultural animal and does not still know.But, the stem cell field of Quick Extended
Knowledge, the research of agricultural species aspect embryonic stem cell also will make progress.It is true that the embryonic stem cell development of agricultural species
Research has a unique chance, because they can be used to carry out the test of system genitale, by being injected into involution of embryo
Move on in the parent of replace-conceive, but this test is forbidden in hESC.In addition, mouse embryonic stem
Cell should be possible (having this ability to go to be operated in terms of hereditism's angle), as satellite cell development and functional study
Platform.In the future, the embryonic stem cell germline if from agricultural animal is developed, and mouse stem cells research may be just
Can transfer in the kinetics applied research of agricultural species skeletal development.
In mammal, major part structure of skeletal muscles completes at period of fetus growth and development stage.Main muscle fiber is opened
Beginning formation is period of embryo, and secondary myofibrillogenesis is in people's mid trimester of pregnancy and later stage, the late period of Mus and neonatal period.Myocyte
Generate and controlled by a series of transcription factor, include MyoD, Myf-5 including Pax3, Pax7, Gli and four muscle-derived regulatory factors,
Myogenin and MRF-4.Secondary muscle fiber is formed at adipose cell, and it is people that fiber generates the formation of lap, pig,
Cattle, sheep, horse, the mid trimester of pregnancy of chicken and rodent latter half of gestation.Muscle-derived, fatty is by embryo with fibroid stem cell
The differentiation of tire stem cell produces.Skeletal muscle stem Cells is converted into fatty by muscle-derived may increase intramuscular fat deposition.One
Fiber/fat grandmother's cell there may be in skeletal muscle, piles up intramuscular fat and has an impact in terms of disease just as fiber.
Muscle cell and adipose cell produce from mesenchymal cell, and mesenchymal cell is present in the getting up early stage of development, especially
It is in the skeletal muscle of period of fetus and newborn stage.When most Derived from Mesenchymal Stem Cells becomes myocyte time, its
The difference of middle sub-fraction becomes the basis that adipose cell intramuscular fat is piled up.In cell fate, key factor is Wnt family
Race's albumen, these albumen are paracrine growth regulators, there may be different functions: Wnt signal may in cell development
Cause cell proliferation, apoptosis, determine that cell fate, variation, or precursor are safeguarded.Classical Wnt path is beta-catenin
Dependent: the FZ of combination activates in disorder family protein, family protein have activated glycogen synthase kinase 3, resistance
The beta-catenin having stopped phosphorylation is degraded subsequently, causes the accumulation of beta-catenin.Not having Wnt to stimulate, axle albumen/glycogen closes
Become kinase enzyme 3/ to be combined aspirin complex and can be improved the antigen degradation of beta-catenin by the phosphorylation of GSK-3 β.
Stable beta-catenin is special with the T cell factor in transcription factor/lymph enhancer effect deexcitation after entering nucleus
Fixed target gene.In the mescenchymal stem cell coming from bone marrow, it is raw that the activation of the signal path of Wnt strengthens myocyte
Become, it is suppressed that lipogenesis.Suppression beta-catenin path can reduce the total number of myocyte.In Preadipocyte In Vitro
Wnt signal is highly expressed, and suppresses the generation of fat by blocking C/EBP and PPAR γ.Stable beta-catenin with become
The Adipose Differentiation suppression of myocyte is relevant with the lipogenesis ability of the muscle satellite cell increased with the age.
Skeletal muscle stem Cells is present in all skeletal muscle, owing to position or function are different, may control different hypertrophy
Type/differentiation capability.Postnatal skeletal muscle is affected extremely sensitive by environment and physiological signal, it is possible to grow up according to required amendment
And functional characteristic, such as, motion, injured or damage starts regenerating and repairing of skeletal muscle, although skeletal muscle is by greatly
Measure and form through mitotic multinuclear muscle fiber.The plasticity of skeletal muscle very big most be from present in skeletal muscle
Population of stem cells, it is common that satellite cell.Major part in vitro study be the back of rodent and the muscle of hind leg divides
Satellite cell from muscle-derived.Nearest one shows with the non-Separation Research ruminating meat animals about ruminating, satellite cell
Have been described with specific skeletal muscle to separate, but have weak point to need to go to determine, if control from different muscle
Satellite cell goes to separate.But, substantial amounts of report display adipose cell behavior difference depends on the position being separated cell place,
This shows that position may affect the activity of different tissues cell.Such as, different adipose tissue sites has the growth of uniqueness,
Growing and regulation feature, the dependent enzyme of animal is lived.It is similar to recently and studies the fat stem cell of the purification cultivation being use simultaneously
In the research that cell and two levels of molecule are carried out.
When needed, the function that satellite cell merges by starting adult terminal differentiation program to realize.Interesting
Being that we constantly find that new somatomedin affects the propagation of satellite cell, this shows likely have extra mechanism to wait to be sent out
Existing.When muscle fiber hypertrophy or repairing, core can be contributed out by satellite cell and existing muscle fiber merges.Work as flesh
The when that fiber being remained indifferent or apathetic for damage, satellite cell carries out merging the new myotube of formation each other and finally takes for muscle fiber.When
So, skeletal muscle is that a dynamic organization is made up of a large amount of key elements, including blood vessel, and neural and connective tissue.In Skeletal Muscle Growth and
During regeneration, these key elements need growth and repair, and need between functional unit muscle fiber thoroughly mutually to grow one
Coordinate.This is supported by previous studies, needs to relate to myocyte generate to remove to recover suitable muscle function, blood vessel
Generate the cooperation between neural formation.Cooperate the most possible between myocyte and other cells.Recently, research worker is
This idea is supported through presenting evidence.For describing the interaction between them, research worker have developed the most common
The model being made up of microvessel fragment and satellite cell cultivated.In this system, the separation being suspended on collagen gel
MVF cultivates on the basis of Mus SC monolayer culture.In the presence of SC, cultivate alone with MVF compared with MVF illustrate higher
Angiogenesis index.Show that satellite and endotheliocyte are tight in muscle more recently by Christov and other people data
Close arranged side by side, it is proposed that directly contact is probably and important means intercellular exchange.In a word, these preliminary results show one
The satellite cell effect not being found before individual is to start to generate program before blood vessel.
But a lot of reports have recorded the regulation and control that postnatal muscle-derived satellite cell is external He intrinsic, plasticity bone
Bone flesh carries out illustration by production capacity and the response to various cytokine.Compose according to inflammation and cytokine, bone
Bone flesh will respond in catabolism and anabolism.Such as, skeletal muscle is infecting the supporter process relevant to existence
Stage will decompose.On the contrary, inflammation eventually results in muscle hypertrophy plus exercise.Up to now, have studied in satellite cell
The effect of the multiple types cytokine in activity provides the result of mixing, may be with cell type, and dosage is relevant with the time.
At cellular level with the presence of two physiological components.One is lipid metabolism, refers to into or out adipose cell (fat
Fat generates and steatolysis) energy stream, it is movable that it need not stem cell.Second is physiology's component, referred to as lipogenesis,
Being the cells switch that can identify, it is shunk by fusiform stem cell, is initially formed a precursor fatty lacking lipid thin
Born of the same parents, then define the adipose cell that multiple fat drip, and eventually form the adipose cell of maturation.In view of much deliver every year
About lipid metabolism and lipogenetic article, substantially can not obtain one and effectively reduce internal oils and fats by exogenous process
Or the method reducing Adipocyte Differentiation.Really, the article that the major part delivered in adipose cell formation field is delivered is thought,
Once Preadipocyte In Vitro starts oil accumulation, and cell becomes one participate in lipid metabolism by continuing to be carried forward terminal differentiation
Adipose cell.In most Animal fat, the adipocyte number possessing oil synthesis and storage capacity does not the most all have
Clear and definite represents.Further, after birth, adipocytic cell growth is hypertrophy and loose double effects, and the effect of every kind of change is because of fat position
Put and different.It is interesting that should be that more adipose cell needs to deposit at specific part according to Traditional Thinking, and fibroblast
Dimension cell is at connective tissue portions turn lipoblast.
The peroxidase proliferation receptor (PPAR γ) of in vitro study display activation and CCAAT-enhancer-associated proteins
(C/EBPs) it is all to control the decisive factor that adipose cell generates.Their induced expression is raw from embryonic stem cell fat
Become.The most issued a large amount of evidences relevant to agricultural animal support this viewpoint, but with the mankind and rodent the most not
Same regulatory mechanism.Lipogenesis originates in period of fetus, ruminant mid trimester of pregnancy, pig and rodent later stage.Lipogenesis
Difference time initial is mainly due to different plant species new born animal difference in terms of Maturity.Lipogenesis is by several passes
Key transcription factor controls, including PPAR γ and C/EBPs.C/EBP β and-δ first passes through lipogenesis stimulus object and induces, and connects
Get off is by the increase in terms of expression at PPAR γ and C/EBP α.C/EBP α and PPAR γ forces each other and opens fat shape
Specific program is become to go to promote lipogenesis.The PPAR γ-value adipogenic main regulation factor.PPAR γ is at retinoids x receptor alpha
(RXR α) and the peroxisome Proliferators combined react and define heterodimer at target base in the cooperation of original paper
On activating.Therefore, retinoic acid can affect lipogenesis by the interaction of RXR α and RXR α and PPAR γ.PPAR γ is
One activation part of transcription factor.In disabled state, PPAR γ and co-repressor contact the transcription activity suppressing it.Knot
Closing part causes co-repressor to replace co-activator to go to arrange the combination of histone acetyltransferase activity such as cAMP response element
Albumen ((CBP/p300)).The acetylation of histone causes chromatin division herein and gene to be expressed.Fatty acid is
The receptor of PPAR γ, compared with normal fatty acid, oxidized fatty acid seems there is higher ability deexcitation PPAR γ.
In a word, skeletal muscle tissue growth is the central factor of agricultural animal production capacity.Improve skeletal muscle yield and intramuscular
Fat content is again the important research content during beef raising produces.Although having carried out dry thin about agricultural domestic animal in a large number
The research of born of the same parents, but world scientific research personnel is also carrying out extensive work for setting up preferable external model at present.In order to point
From with purification skeletal muscle origin become flesh/one-tenth fat stem cell, and then be used for setting up the model of induction differentiation in vitro always
It is also one of extremely urgent work that agricultural animal scientific research personnel carries out for a long time.
Summary of the invention
For deficiency of the prior art, it is an object of the invention to provide a kind of purification skeletal muscle origin fat precursors thin
The method of born of the same parents.
To achieve these goals, the present invention adopts the technical scheme that by cell surface marker molecule platelet derived
Skeletal muscle origin pluripotent stem cell is sorted by growth factor receptors α.
A kind of method of the purification tissue-derived Preadipocyte In Vitro of cattle fetal skeletal muscle, the material that described method uses is,
Cattle 3 monthly age fetus longissimus dorsi muscle tissue, agents useful for same is, the improvement Iger training of collagenase, hyclone, low sugar Da Erbai kirschner
Support base, cell sorting buffer, antiplatelet source property growth factor receptors Alpha antibodies, dexamethasone, 3-isobutyl group-1-methyl yellow
Purine, bovine insulin and oil red O.
Said method comprising the steps of:
Step one, the separation and Culture of cell；
Step 2, cell are cultivated；
Step 3, the adipogenic induction of cell；
Step 4, oil red O；
Step 5, oil red O stain result of the test.
Described step one specifically includes following steps:
Step (1) by cattle fetus with 75% ethanol and rinse several times containing dual anti-phosphate buffer；
Step (2) is then placed in gnotobasis carefully breaking amniotic membrane, takes out fetus, cuts off back skin with eye scissors
Skin, isolates muscle of back with eye scissors, tweezers；
The muscular tissue that step (3) takes out 2 pieces of Semen phaseoli radiati grain sizes with tweezers is put in culture dish, adds in advance in culture dish
Enter containing dual anti-phosphate buffer, wash down 3 times with phosphate buffer and remove hemocyte；
Step (4) is then transferred in EP pipe, adds 1mL 0.1% type Ⅳ collagenase, shreds to meat paste shape with eye scissors；
The tissue shredded is put into digestion 45min, period every 15min piping and druming group in 37 DEG C of gas thermostatic shaking tables by step (5)
Knit once；
Cytoplasm being filtered with the cell filtration net of 40 μm after step (6) 45min, 400rpm is centrifuged 5min, supernatant discarded
Liquid, adds cell sorting buffer suspension cell；
Step (7) hatches 15min after adding 2 μ L antiplatelet source property growth factor receptors Alpha antibodies, 300rcf is centrifuged
10min；
After step (8) adds 1mL cell sorting buffer, 300rcf is centrifuged 10min, abandons supernatant, is repeated once；
Step (9) adds 80 μ L cell sorting buffer after sucking supernatant, add 20 μ L anti-rabbit source immunoglobulins micro-
Pearl, mixes in latter 4 DEG C and hatches 15min；
After step (10) adds 1mL cell sorting buffer, 300rcf is centrifuged 10min, abandons supernatant, is repeated once；
Step (11) adds 500 μ L cell sorting buffer suspension cells；
MS is sorted post as on magnetic frame by step (12), is joined by suspension on ready MS sorting post, collects
The negative cells flowed down, rinses sorting post 3 times, every time with 500 μ L cell sorting buffer, collects the liquid and the first step flowed down
Merge；
MS is sorted post and takes off from magnetic frame and be placed in new collecting pipe by step (13), adds 1mL cell sorting buffering
After liquid, quickly it is pushed in sorting post with piston, collects the liquid flowed out and be positive cell.
Described step 2 specifically includes following steps:
When primary cell growth is assembled to 70～80% when, suck old culture medium, and clean 2 with phosphate buffer
Secondary, after being added by 0.25% trypsin, put into about 30s in incubator, under inverted phase contrast microscope, observation of cell is rounded
Time, softly beat culture dish with hands, until after circular cells float, at once add culture medium terminate digestion reaction continue into
OK, soft piping and druming cell, carry out Secondary Culture with 1:3 ratio, it is 37 DEG C, CO that cell is put into temperature2Content is the perseverance of 5%
Temperature incubator is cultivated.
Described step 3 specifically includes following steps: when cell growth converges to 100%, discards culture medium, uses phosphoric acid
Salt buffer clean 2 times, add lipoblast induction liquid, growth medium add 0.5 μM of 3-isobutyl-1-methylxanthine, 10
μ g/mL insulin, 1 μM of dexamethasone, proceed by induction, and every 3d changes a not good liquor.
Described step 4 specifically includes following steps:
Step (a) discards old culture medium, is added in orifice plate by 4% paraformaldehyde, and room temperature fixes cell 20min；
Step (b) phosphate buffer cleans 3 times；
Step (c) adds the 10min that dyes under oil red O room temperature；
Step (d) discards oil red O stain liquid, cleans 3 times with phosphate buffer；
Step (e) is placed in staining conditions that under inverted microscope, in observation of cell, fat drips and takes pictures.
Described step 5 specifically includes following steps:
When cell induction 10d, to Platelet-derived growth factor α-receptor positive cell and platelet derived growth because of
Sub-receptor alpha negative cells carries out oil red O stain, and result shows, the former is more, bigger than the latter fat drips.
Beneficial effect
Fat precursors by molecular surface labelling Platelet-derived growth factor α-receptor screening positive group after purification is thin
Born of the same parents, define after induction differentiation and have the adipose cell that a large amount of fat drips, and the fat amount of dripping that negative group cell is formed is little.Cause
This, the method is for promoting that Preadipocyte purification has the effect of excellence.
Accompanying drawing explanation
The oil red O stain result of cell lipoblast induction differentiation after Fig. 1-2 sorting；
Wherein, Fig. 1 is that Platelet-derived growth factor α-receptor negative cells becomes oil red O stain result after fat differentiation；
Fig. 2 is that Platelet-derived growth factor α-receptor positive cell becomes oil red O stain result after fat differentiation；
Detailed description of the invention
Embodiment 1
A kind of method of the purification tissue-derived Preadipocyte In Vitro of cattle fetal skeletal muscle, the material that described method uses is,
Cattle 3 monthly age fetus longissimus dorsi muscle tissue, agents useful for same is, the improvement Iger training of collagenase, hyclone, low sugar Da Erbai kirschner
Support base, cell sorting buffer, antiplatelet source property growth factor receptors Alpha antibodies, dexamethasone, 3-isobutyl group-1-methyl yellow
Purine, bovine insulin and oil red O.
Said method comprising the steps of:
Step one, the separation and Culture of cell；
Step 2, cell are cultivated；
Step 3, the adipogenic induction of cell；
Step 4, oil red O；
Step 5, oil red O stain result of the test.
Described step one specifically includes following steps:
Step (1) by cattle fetus with 75% ethanol and rinse several times containing dual anti-phosphate buffer；
Step (2) is then placed in gnotobasis carefully breaking amniotic membrane, takes out fetus, cuts off back skin with eye scissors
Skin, isolates muscle of back with eye scissors, tweezers；
The muscular tissue that step (3) takes out 2 pieces of Semen phaseoli radiati grain sizes with tweezers is put in culture dish, adds in advance in culture dish
Enter containing dual anti-phosphate buffer, wash down 3 times with phosphate buffer and remove hemocyte；
Step (4) is then transferred in EP pipe, adds 1mL 0.1% type Ⅳ collagenase, shreds to meat paste shape with eye scissors；
The tissue shredded is put into digestion 45min, period every 15min piping and druming group in 37 DEG C of gas thermostatic shaking tables by step (5)
Knit once；
Cytoplasm being filtered with the cell filtration net of 40 μm after step (6) 45min, 400rpm is centrifuged 5min, supernatant discarded
Liquid, adds cell sorting buffer suspension cell；
Step (7) hatches 15min after adding 2 μ L antiplatelet source property growth factor receptors Alpha antibodies, 300rcf is centrifuged
10min；
After step (8) adds 1mL cell sorting buffer, 300rcf is centrifuged 10min, abandons supernatant, is repeated once；
Step (9) adds 80 μ L cell sorting buffer after sucking supernatant, add 20 μ L anti-rabbit source immunoglobulins micro-
Pearl, mixes in latter 4 DEG C and hatches 15min；
After step (10) adds 1mL cell sorting buffer, 300rcf is centrifuged 10min, abandons supernatant, is repeated once；
Step (11) adds 500 μ L cell sorting buffer suspension cells；
MS is sorted post as on magnetic frame by step (12), is joined by suspension on ready MS sorting post, collects
The negative cells flowed down, rinses sorting post 3 times, every time with 500 μ L cell sorting buffer, collects the liquid and the first step flowed down
Merge；
MS is sorted post and takes off from magnetic frame and be placed in new collecting pipe by step (13), adds 1mL cell sorting buffering
After liquid, quickly it is pushed in sorting post with piston, collects the liquid flowed out and be positive cell.
Described step 2 specifically includes following steps:
When primary cell growth is assembled to 70～80% when, suck old culture medium, and clean 2 with phosphate buffer
Secondary, after being added by 0.25% trypsin, put into about 30s in incubator, under inverted phase contrast microscope, observation of cell is rounded
Time, softly beat culture dish with hands, until after circular cells float, at once add culture medium terminate digestion reaction continue into
OK, soft piping and druming cell, carry out Secondary Culture with 1:3 ratio, it is 37 DEG C, CO that cell is put into temperature2Content is the perseverance of 5%
Temperature incubator is cultivated.
Described step 3 specifically includes following steps: when cell growth converges to 100%, discards culture medium, uses phosphoric acid
Salt buffer clean 2 times, add lipoblast induction liquid, growth medium add 0.5 μM of 3-isobutyl-1-methylxanthine, 10
μ g/mL insulin, 1 μM of dexamethasone, proceed by induction, and every 3d changes a not good liquor.
Described step 4 specifically includes following steps:
Step (a) discards old culture medium, is added in orifice plate by 4% paraformaldehyde, and room temperature fixes cell 20min；
Step (b) phosphate buffer cleans 3 times；
Step (c) adds the 10min that dyes under oil red O room temperature；
Step (d) discards oil red O stain liquid, cleans 3 times with phosphate buffer；
Step (e) is placed in staining conditions that under inverted microscope, in observation of cell, fat drips and takes pictures.
Described step 5 specifically includes following steps:
When cell induction 10d, to Platelet-derived growth factor α-receptor positive cell and platelet derived growth because of
Sub-receptor alpha negative cells carries out oil red O stain, and result shows, the former is more, bigger than the latter fat drips.
It is last that it is noted that obviously above-described embodiment is only for clearly demonstrating the application example, and also
The non-restriction to embodiment.For those of ordinary skill in the field, can also do on the basis of the above description
Go out change or the variation of other multi-form.Here without also cannot all of embodiment be given exhaustive.And thus drawn
What Shen went out obviously changes or among the variation protection domain still in the application type.
Claims (7)
1. the method for the purification tissue-derived Preadipocyte In Vitro of cattle fetal skeletal muscle, it is characterised in that: described method uses
Material be that cattle 3 monthly age fetus longissimus dorsi muscle tissue, agents useful for same is, collagenase, hyclone, low sugar Da Erbai kirschner change
Good Eagle's medium, cell sorting buffer, antiplatelet source property growth factor receptors Alpha antibodies, dexamethasone, 3-isobutyl
Base-1-methylxanthine, bovine insulin and oil red O.
Method the most according to claim 1, it is characterised in that said method comprising the steps of:
Step one, the separation and Culture of cell；
Step 2, cell are cultivated；
Step 3, the adipogenic induction of cell；
Step 4, oil red O；
Step 5, oil red O stain result of the test.
Method the most according to claim 2, it is characterised in that described step one specifically includes following steps:
Step (1) by cattle fetus with 75% ethanol and rinse several times containing dual anti-phosphate buffer；
Step (2) is then placed in gnotobasis carefully breaking amniotic membrane, takes out fetus, cuts off skin of back with eye scissors, uses
Muscle of back isolated by eye scissors, tweezers；
The muscular tissue that step (3) takes out 2 pieces of Semen phaseoli radiati grain sizes with tweezers is put in culture dish, is previously added and contains in culture dish
There is dual anti-phosphate buffer, wash down 3 times with phosphate buffer and remove hemocyte；
Step (4) is then transferred in EP pipe, adds 1mL 0.1% type Ⅳ collagenase, shreds to meat paste shape with eye scissors；
The tissue shredded is put into digestion 45min, period every 15min in 37 DEG C of gas thermostatic shaking tables and is blown and beaten tissue one by step (5)
Secondary；
Cytoplasm being filtered with the cell filtration net of 40 μm after step (6) 45min, 400rpm is centrifuged 5min, abandoning supernatant, adds
Enter cell sorting buffer suspension cell；
Step (7) hatches 15min after adding 2 μ L antiplatelet source property growth factor receptors Alpha antibodies, 300rcf is centrifuged 10min；
After step (8) adds 1mL cell sorting buffer, 300rcf is centrifuged 10min, abandons supernatant, is repeated once；
Step (9) adds 80 μ L cell sorting buffer after sucking supernatant, add 20 μ L anti-rabbit source immunoglobulin microballons, mixed
Even latter 4 DEG C hatch 15min；
After step (10) adds 1mL cell sorting buffer, 300rcf is centrifuged 10min, abandons supernatant, is repeated once；
Step (11) adds 500 μ L cell sorting buffer suspension cells；
MS is sorted post as on magnetic frame by step (12), is joined by suspension on ready MS sorting post, and collection flows down
Negative cells, rinse sorting post 3 times, every time with 500 μ L cell sorting buffer, collect the liquid and first step conjunction flowed down
And；
MS is sorted post and takes off from magnetic frame and be placed in new collecting pipe by step (13), after adding 1mL cell sorting buffer,
Quickly it is pushed in sorting post with piston, collects the liquid flowed out and be positive cell.
Method the most according to claim 2, it is characterised in that described step 2 specifically includes following steps:
When primary cell growth is assembled to 70～80% when, suck old culture medium, and clean 2 times with phosphate buffer,
After being added by 0.25% trypsin, put into about 30s in incubator, when under inverted phase contrast microscope, observation of cell is rounded,
Softly beat culture dish with hands, until after circular cells float, at once add culture medium and terminate digestion reaction and proceed,
Soft piping and druming cell, carries out Secondary Culture with 1:3 ratio, and it is 37 DEG C, CO that cell is put into temperature2Content is the constant temperature of 5%
Incubator is cultivated.
Method the most according to claim 2, it is characterised in that described step 3 specifically includes following steps: when cell is raw
When length converges to 100%, discard culture medium, clean 2 times with phosphate buffer, add lipoblast induction liquid, grown cultures
Base adds 0.5 μM of 3-isobutyl-1-methylxanthine, 10 μ g/mL insulins, 1 μM of dexamethasone, proceeds by induction, and every 3d changes
One not good liquor.
Method the most according to claim 2, it is characterised in that described step 4 specifically includes following steps:
Step (a) discards old culture medium, is added in orifice plate by 4% paraformaldehyde, and room temperature fixes cell 20min；
Step (b) phosphate buffer cleans 3 times；
Step (c) adds the 10min that dyes under oil red O room temperature；
Step (d) discards oil red O stain liquid, cleans 3 times with phosphate buffer；
Step (e) is placed in staining conditions that under inverted microscope, in observation of cell, fat drips and takes pictures.
Method the most according to claim 2, it is characterised in that described step 5 specifically includes following steps:
When cell induction 10d, Platelet-derived growth factor α-receptor positive cell and platelet-derived growth factor are subject to
Body α negative cells carries out oil red O stain, and result shows, the former is more, bigger than the latter fat drips.
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| 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 |
| CN113717932B (en) * | 2021-09-16 | 2023-03-14 | 四川农业大学 | Primary isolation culture and induced differentiation method for intramuscular precursor adipocytes of adult yaks |
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