CN106282100B - A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle - Google Patents
A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The invention discloses a kind of methods for purifying the tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle, the material that the method uses is, 3 monthly age of ox fetus longissimus dorsi muscle tissue, agents useful for same is clostridiopetidase A, fetal calf serum, low sugar Dulbecco modified Eagle medium, cell sorting buffer, antiplatelet source property growth factor receptors Alpha antibodies, dexamethasone, 3-isobutyl-1-methylxanthine, bovine insulin and oil red O.
Description
Technical field
The invention belongs to technological field of biochemistry, and in particular to a kind of tissue-derived preceding body fat of purifying ox fetal skeletal muscle
The method of fat cell.
Background technique
Stem cell is can to maintain duplication and and can be divided into a kind of cell of different type cell ability, stem-cell research
The generation and differentiation regulation that understand cell and tissue are very important.In addition, stem-cell research is for medical domain
Equally there are huge potentiality.As a lot of other organs, skeletal muscle includes many cell types, can generate the satellite of muscle-derived
The fat cell of cell and adipose tissue source property, these are all critically important to Animal husbandry production.It is well known that satellite cell pair
The regeneration of the skeletal muscle of the growth and adult of skeletal muscle is all critically important after birth.Past nearly 50 years thin to isolated satellite
The research of born of the same parents is concentrated mainly on the activation and inhibition of its proliferation, regulates and controls their activity in vitro, raw with other cells such as blood vessel
At intercellular interaction, potentiality between subgroup, and as the potentiality of carrier in gene therapy.In beef cattle production, animal
The growth and development of skeletal muscle and the deposition of intramuscular fat be influence beef production and price main factor.However, at present
Until the research developed for early stage muscle development and intramuscular fat it is relatively fewer, the development of stem cells of especially different cells comes
Source, therefore the stem cell surface molecular marker characteristic for distinguishing different is identified, and establish Differentiation Induction in vitro model
System is of great significance for studying domestic animal muscle and intramuscular fat differentiation.
Embryonic stem cell still has significantly indefinite to the molecular mechanism at orientation flesh, at rouge, at fiber.
However, the embryonic stem cell that quality is proved is separated from a limited number of mammalian species.Moreover, 1981
Embryonic stem cell is separated from inhuman experiment house mouse.Mouse pluripotent stem cell is thoroughly built
It is vertical, by normal growth in embryo transfer to female mice body until birth after being injected into blastaea.In addition, mouse embryonic stem
After the genome of cell can be by transgenosis and homologous recombination technique modification.Genetically engineered cell can be after system genitale be transmitted to
Generation.The human embryonic stem cell line of pluripotency has also been successfully established, and is likewise supplied with the ability for being divided into variety classes cell.To the greatest extent
Pipe to having put into a large amount of effort on agricultural species embryonic stem cell, but be not yet compared with the effort of mouse and the mankind very at
Function.The difficulty of this work may have the following, deposit first is that agricultural species compare the development before embryo implantation with mouse
In the specificity of species, second be growth factor required for agricultural animal embryonic development is not fully understood, third is that
Whether effective stem cell labeling is suitable for agricultural animal and does not know still in mouse.However, the stem cell field of Quick Extended
The research of knowledge, agricultural species aspect embryonic stem cell will also make progress.In fact, the embryonic stem cell of agricultural species develops
There is a unique chance, because they can be used to carry out the test of system genitale, by being injected into involution of embryo in research
It moves on in the parent of replace-conceive, however this test is forbidden in hESC.In addition to this, mouse embryonic stem
Cell should be possible and (have this ability to go to be operated in terms of science of heredity angle), as satellite cell development and functional study
Platform.In the future, if the embryonic stem cell germline from agricultural animal is developed, mouse stem cells research may be just
It can be transferred in the dynamics application study of agricultural species skeletal development.
In mammals, most of structure of skeletal muscles is completed in foetal period growth and development stage.Main muscle fibre is opened
Beginning to be formed is in embryonic period, embryonic phase, and secondary myofibrillogenesis is in people's second trimester of pregnancy and later period, the advanced stage and neonatal period of mouse.Myocyte
Generation is controlled by a series of transcription factors, including Pax3, Pax7, and Gli and four muscle-derived regulatory factor includes MyoD, Myf-5,
Myogenin and MRF-4.Secondary muscle fibre is formed in fat cell, fiber generate lap formation be in people, pig,
Ox, sheep, horse, the second 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 generates.Skeletal muscle stem Cells, which are converted into fatty by muscle-derived, may will increase intramuscular fat deposition.One
Fiber/fat grandmother cell has an impact to intramuscular fat accumulation there may be in skeletal muscle just as fiber in terms of disease.
Muscle cell and fat cell are generated from mesenchymal cell, and mesenchymal cell is present in the getting up early stage of development, especially
It is in the skeletal muscle of foetal period and newborn stage.When most Derived from Mesenchymal Stem Cells becomes myocyte,
Middle sub-fraction difference becomes fat cell --- the basis of intramuscular fat accumulation.Key factor is Wnt family in cell fate
Race's albumen, these albumen are paracrine growth regulators, and there may be different functions in cell development: Wnt signal may
Cause cell Proliferation, apoptosis determines cell fate, variation or precursor maintenance.Classical Wnt access is beta-catenin
Dependence: in conjunction with frizzled protein activate in disorder family protein, family protein has activated glycogen synthase kinase 3, resistance
The beta-catenin for having stopped phosphorylation is then degraded, and the accumulation of beta-catenin is caused.There is no Wnt stimulation, axis albumen/glycogen closes
The antigen degradation of beta-catenin can be improved by the phosphorylation of GSK-3 β at the compound aspirin compound of kinase enzyme 3/.
Stable beta-catenin, which enters after nucleus, deactivates spy with the T cell factor in transcription factor/lymph enhancer effect
Fixed target gene.In the mescenchymal stem cell from marrow, it is raw that the activation of the signal path of Wnt strengthens myocyte
At, it is suppressed that fat generates.Inhibit beta-catenin access that can reduce the total number of myocyte.In Preadipocyte In Vitro
Wnt signal is highly expressed, and inhibits the generation of fat by blocking C/EBP and PPAR γ.Stable beta-catenin at
The Adipose Differentiation of myocyte inhibits related with the fatty generative capacity of the muscle satellite cell increased with the age.
Skeletal muscle stem Cells are present in all skeletal muscle, since position or function are different, can control different hyperplasia
Type/differentiation capability.Postnatal skeletal muscle is influenced extremely sensitive by environment and physiological signal, can be grown up according to required modification
And functional characteristic, for example, movement, injured or damage starts regenerating and repairing for skeletal muscle, although skeletal muscle is by big
Amount is formed by mitotic multicore muscle fibre.Very most is come from present in skeletal muscle to the plasticity of skeletal muscle greatly
Population of stem cells, usually satellite cell.Most of in vitro study is that the back of rodent and the muscle of hind leg divide
Satellite cell from muscle-derived.Nearest one shows satellite cell with the non-Separation Research for ruminating meat animals about ruminating
Specific skeletal muscle separation has been described, but there are shortcoming needs to go to determine, if control is from different muscle
Satellite cell goes to separate.However, a large amount of report display fat cell behavioral difference is depended on by the position where separation cell,
This shows that position may will affect the activity of different tissues cell.For example, different adipose tissue sites have unique growth,
Development and adjusting feature, the enzyme activity of animal dependence.Similar research recently be using purifying culture fat stem cell simultaneously
In cell and the molecule two horizontal researchs carried out.
When needed, satellite cell realizes the function of fusion by starting adult terminal differentiation program.Interesting
It is that we, which constantly discover new growth factor, influences the proliferation of satellite cell, this shows to be possible to additional mechanism and waits being sent out
It is existing.In muscle fibre hypertrophy or repairing, core can be contributed and be merged with existing muscle fibre by satellite cell.Work as flesh
When fiber is for damaging unmoved, satellite cell carries out fusion and forms new myotube finally taking for muscle fibre each other.When
So, skeletal muscle is that a dynamic organization is made of a large amount of elements, including blood vessel, nerve and connective tissue.In Skeletal Muscle Growth and
During regeneration, these elements need to grow and repair, in order to grow need between a thorough functional unit muscle fibre it is mutual
Coordinate.This is supported by previous studies, needs to be related to myocyte's generation, blood vessel to remove to restore muscle function appropriate
Generate the cooperation between nerve formation.Cooperate between myocyte and other cells seemingly possible.Recently, researcher has been
It is presented evidence to support this idea.To describe the interaction between them, researcher has developed common in vitro
The model of culture being made of microvessel fragment and satellite cell.In this system, it is suspended in the separation on collagen gel
MVF is cultivated on the basis of mouse SC monolayer cultivation.In the presence of SC, MVF is illustrated higher compared with MVF is cultivated alone
Angiogenesis index.Satellite is shown more recently by Christov and other people data and endothelial cell is tight in muscle
It is close arranged side by side, it is proposed that directly contact, which may be important, means intercellular exchange.In short, these are preliminary the result shows that one
The satellite cell effect not being found before a is that starting generates program before blood vessel.
However many reports have recorded the external and intrinsic regulation of postnatal muscle-derived satellite cell, plasticity bone
Bone flesh carries out illustration by production capacity and to the response of various cell factor.It is composed according to inflammation and cell factor, bone
Bone flesh will respond in catabolism and anabolism.For example, skeletal muscle is infecting supporter process relevant to existence
It will be decomposed in stage.On the contrary, inflammation eventually results in muscle hypertrophy plus exercise.So far, it has studied in satellite cell
The effect of a variety of type cytokines in activity provide mixing as a result, may be with cell type, dosage is related with the time.
In cellular level, there are two physiological components to exist.One is lipid metabolism, refers into or out fat cell (rouge
Fat generate and lipolysis) energy stream, it does not need stem cell activity.Second is physiology component, referred to as Adipogenesis,
It is the cells switch that can be identified, is shunk by fusiform stem cell, the precursor fatty for being initially formed a shortage lipid is thin
Then born of the same parents form the fat cell of a multiple fat drips, eventually form mature fat cell.In view of what is much delivered every year
About lipid metabolism and lipogenetic article, cannot obtain one substantially effectively reduces internal grease by exogenous processing
Or the method for reducing Adipocyte Differentiation.Really, the article that the major part that field is delivered is delivered is formed in fat cell to think,
Once Preadipocyte In Vitro starts oil accumulation, cell will continue to be carried forward terminal differentiation as participation lipid metabolism
Fat cell.In most animal tallow, the adipocyte number for having oil synthesis and storage capacity does not all have at birth
Explicitly show.Also, adipocytic cell growth is hyperplasia and loose double effects after being born, and the effect of every kind of variation is because of fatty position
It sets and different.It is interesting that should be more fat cells needs in privileged site deposition according to Traditional Thinking, and at fibre
Cell is tieed up in connective tissue portions turn lipoblast.
The peroxidase proliferation receptor (PPAR γ) and CCAAT- enhancer-binding protein of in vitro study display activation
It (C/EBPs) is all the decision sex factor for controlling fat cell and generating.Their induced expression is raw from embryonic stem cell fat
At.Issued a large amount of evidences relevant to agricultural animal support this viewpoint, but slightly not with the mankind and rodent
Same adjustment mechanism.Fat, which generates, originates in foetal period, ruminant second trimester of pregnancy, pig and rodent later period.Adipogenesis
Difference when starting is mainly the difference due to different plant species new born animal in terms of maturity.Adipogenesis is by several passes
The control of key transcription factor, including PPAR γ and C/EBPs.C/EBP β and-δ passes through Adipogenesis stimulant first and is induced, and connects
Getting off is increase by PPAR γ and C/EBP α in terms of expression.C/EBP α and PPAR γ are forced go to open fatty shape each other
It goes to promote Adipogenesis at specific program.The adipogenic main regulation factor of PPAR γ value.PPAR γ is in retinoids x receptor alpha
Heterodimer is formd in target base in the cooperation of (RXR α) and combined peroxisome Proliferators reaction original part
Because in activation.Therefore, vitamin A acid can influence Adipogenesis by the interaction of RXR α and RXR α and PPAR γ.PPAR γ is
One activation ligand of transcription factor.In disabled state, PPAR γ and corepressor contact the transcription activity for suppressing it.Knot
Closing ligand causes corepressor that co-activator is replaced to go to dominate histone acetyltransferase activity such as cAMP response element combination
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, the fatty acid being oxidized seems have higher ability to deactivate PPAR γ.
In short, skeletal muscle tissue development is the central factor of agricultural animal production capacity.Improve skeletal muscle yield and intramuscular
Fat content is the important research content in beef raising production again.Although having carried out largely dry thin about agriculture domestic animal
The research of born of the same parents, but world scientific research personnel is also carrying out extensive work to establish ideal external model at present.In order to divide
From, at flesh/at rouge stem cell, and then the model for being used to establish induction differentiation in vitro is always with purifying skeletal muscle origin
It is also extremely urgent one of work that agricultural animal scientific research personnel carries out for a long time.
Summary of the invention
Aiming at the shortcomings in the prior art, the purpose of the present invention is to provide a kind of purifying skeletal muscle origin fat precursors are thin
The method of born of the same parents.
To achieve the goals above, the technical solution adopted by the present invention is that it is platelet derived by cell surface marker molecule
Growth factor receptors α sorts skeletal muscle origin pluripotent stem cell.
A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle, the material that the method uses is,
3 monthly age of ox fetus longissimus dorsi muscle tissue, agents useful for same are that clostridiopetidase A, fetal calf serum, low sugar Dulbecco improve Iger training
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.
It the described method comprises the following steps:
Step 1: cell is separately cultured;
Step 2: cell culture;
Step 3: the adipogenic induction of cell;
Step 4: oil red O;
Step 5: oil red O stain test result.
The step 1 specifically includes the following steps:
Step (1) is rinsed several times by ox fetus with 75% alcohol and containing dual anti-phosphate buffer;
Step (2), which is then placed into gnotobasis, carefully breaks amnion, takes out fetus, cuts off back skin with eye scissors
Skin isolates muscle of back with eye scissors, tweezers;
Step (3) is put into culture dish with the musculature that tweezers take out 2 pieces of mung bean grain sizes, is added in advance in culture dish
Enter containing dual anti-phosphate buffer, washes down 3 removing haemocytes with phosphate buffer;
Step (4) is then transferred into EP pipe, and 0.1% type Ⅳ collagenase of 1mL is added, is shredded with eye scissors to meat gruel shape;
The tissue shredded is put into 37 DEG C of gas thermostatic shaking tables by step (5) digests 45min, during which every 15min piping and druming group
It knits primary;
Cytoplasm is filtered with 40 μm of cell filtering net after step (6) 45min, 400rpm is centrifuged 5min, discards supernatant
Cell sorting buffer suspension cell is added in liquid;
Step (7) is incubated for 15min after 2 μ L antiplatelet source property growth factor receptors Alpha antibodies are added, 300rcf centrifugation
10min;
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (8), abandons supernatant, is repeated once;
80 μ L cell sorting buffers are added after sucking supernatant in step (9), and it is micro- to add 20 μ L anti-rabbit source immunoglobulins
Pearl is incubated for 15min in 4 DEG C after mixing;
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (10), abandons supernatant, is repeated once;
500 μ L cell sorting buffer suspension cells are added in step (11);
MS sorting column as on magnetic frame, suspension is added on ready MS sorting column, is collected by step (12)
The negative cells flowed down rinse sorting column 3 times every time with 500 μ L cell sorting buffers and collect the liquid flowed down and the first step
Merge;
MS is sorted column and is placed in new collecting pipe from removing on magnetic frame by step (13), and 1mL cell sorting buffering is added
After liquid, quickly it is pushed into piston in sorting column, the liquid for collecting outflow is positive cell.
The step 2 specifically includes the following steps:
When primary cell growth aggregation is to 70~80%, old culture medium is sucked, and clean 2 with phosphate buffer
It is secondary, after 0.25% trypsase is added, it is put into incubator about 30s, it is rounded that cell is observed under inverted phase contrast microscope
When, softly beat culture dish with hand, after circular cells float, at once be added culture medium come terminate digestion reaction continue into
Row, soft piping and druming cell carry out secondary culture with 1:3 ratio, and it is 37 DEG C, CO that cell, which is put into temperature,2The perseverance that content is 5%
It is cultivated in warm incubator.
The step 3 discards culture medium, uses phosphoric acid specifically includes the following steps: when cell growth converges to 100%
Salt buffer cleans 2 times, and lipoblast is added and induces liquid, growth medium adds 0.5 μM of 3-isobutyl-1-methylxanthine, 10
μ g/mL insulin, 1 μM of dexamethasone, start to be induced, every 3d changes a not good liquor.
The step 4 specifically includes the following steps:
Step (a) discards old culture medium, and 4% paraformaldehyde is added in orifice plate, the fixed cell 20min of room temperature;
Step (b) is cleaned 3 times with phosphate buffer;
Step (c) is added under oil red O room temperature and dyes 10min;
Step (d) discards oil red O stain liquid, is cleaned 3 times with phosphate buffer;
Step (e), which is placed under inverted microscope, to be observed the staining conditions of fat drips in cell and takes pictures.
The step 5 specifically includes the following steps:
When cell induces 10d, to Platelet-derived growth factor α-receptor positive cell and platelet derived growth because
Sub- receptor alpha negative cells carry out oil red O stain, the results showed that, the former is more than the latter fat drips, bigger.
Beneficial effect
Mark the fat precursors of the positive group of Platelet-derived growth factor α-receptor screening after purification thin by molecular surface
Born of the same parents form the fat cell with a large amount of fat drips after induction differentiation, and to be formed by fat drips amount seldom for negative group cell.Cause
This, the method is excellent for promoting Preadipocyte purifying to have the effect of.
Detailed description of the invention
The oil red O stain result of cell lipoblast induction differentiation after Fig. 1-2 sorting;
Wherein, Fig. 1 is oil red O stain result after Platelet-derived growth factor α-receptor negative cells break up at rouge;
Fig. 2 is oil red O stain result after Platelet-derived growth factor α-receptor positive cell breaks up at rouge;
Specific embodiment
Embodiment 1
A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle, the material that the method uses is,
3 monthly age of ox fetus longissimus dorsi muscle tissue, agents useful for same are that clostridiopetidase A, fetal calf serum, low sugar Dulbecco improve Iger training
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.
It the described method comprises the following steps:
Step 1: cell is separately cultured;
Step 2: cell culture;
Step 3: the adipogenic induction of cell;
Step 4: oil red O;
Step 5: oil red O stain test result.
The step 1 specifically includes the following steps:
Step (1) is rinsed several times by ox fetus with 75% alcohol and containing dual anti-phosphate buffer;
Step (2), which is then placed into gnotobasis, carefully breaks amnion, takes out fetus, cuts off back skin with eye scissors
Skin isolates muscle of back with eye scissors, tweezers;
Step (3) is put into culture dish with the musculature that tweezers take out 2 pieces of mung bean grain sizes, is added in advance in culture dish
Enter containing dual anti-phosphate buffer, washes down 3 removing haemocytes with phosphate buffer;
Step (4) is then transferred into EP pipe, and 0.1% type Ⅳ collagenase of 1mL is added, is shredded with eye scissors to meat gruel shape;
The tissue shredded is put into 37 DEG C of gas thermostatic shaking tables by step (5) digests 45min, during which every 15min piping and druming group
It knits primary;
Cytoplasm is filtered with 40 μm of cell filtering net after step (6) 45min, 400rpm is centrifuged 5min, discards supernatant
Cell sorting buffer suspension cell is added in liquid;
Step (7) is incubated for 15min after 2 μ L antiplatelet source property growth factor receptors Alpha antibodies are added, 300rcf centrifugation
10min;
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (8), abandons supernatant, is repeated once;
80 μ L cell sorting buffers are added after sucking supernatant in step (9), and it is micro- to add 20 μ L anti-rabbit source immunoglobulins
Pearl is incubated for 15min in 4 DEG C after mixing;
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (10), abandons supernatant, is repeated once;
500 μ L cell sorting buffer suspension cells are added in step (11);
MS sorting column as on magnetic frame, suspension is added on ready MS sorting column, is collected by step (12)
The negative cells flowed down rinse sorting column 3 times every time with 500 μ L cell sorting buffers and collect the liquid flowed down and the first step
Merge;
MS is sorted column and is placed in new collecting pipe from removing on magnetic frame by step (13), and 1mL cell sorting buffering is added
After liquid, quickly it is pushed into piston in sorting column, the liquid for collecting outflow is positive cell.
The step 2 specifically includes the following steps:
When primary cell growth aggregation is to 70~80%, old culture medium is sucked, and clean 2 with phosphate buffer
It is secondary, after 0.25% trypsase is added, it is put into incubator about 30s, it is rounded that cell is observed under inverted phase contrast microscope
When, softly beat culture dish with hand, after circular cells float, at once be added culture medium come terminate digestion reaction continue into
Row, soft piping and druming cell carry out secondary culture with 1:3 ratio, and it is 37 DEG C, CO that cell, which is put into temperature,2The perseverance that content is 5%
It is cultivated in warm incubator.
The step 3 discards culture medium, uses phosphoric acid specifically includes the following steps: when cell growth converges to 100%
Salt buffer cleans 2 times, and lipoblast is added and induces liquid, growth medium adds 0.5 μM of 3-isobutyl-1-methylxanthine, 10
μ g/mL insulin, 1 μM of dexamethasone, start to be induced, every 3d changes a not good liquor.
The step 4 specifically includes the following steps:
Step (a) discards old culture medium, and 4% paraformaldehyde is added in orifice plate, the fixed cell 20min of room temperature;
Step (b) is cleaned 3 times with phosphate buffer;
Step (c) is added under oil red O room temperature and dyes 10min;
Step (d) discards oil red O stain liquid, is cleaned 3 times with phosphate buffer;
Step (e), which is placed under inverted microscope, to be observed the staining conditions of fat drips in cell and takes pictures.
The step 5 specifically includes the following steps:
When cell induces 10d, to Platelet-derived growth factor α-receptor positive cell and platelet derived growth because
Sub- receptor alpha negative cells carry out oil red O stain, the results showed that, the former is more than the latter fat drips, bigger.
Finally, it should be noted that obviously, above-described embodiment is only intended to clearly illustrate the application example, and simultaneously
The non-restriction to embodiment.For those of ordinary skill in the art, it can also do on the basis of the above description
Other various forms of variations or variation out.There is no necessity and possibility to exhaust all the enbodiments.And thus drawn
Shen go out obvious changes or variations still in the protection scope of the application type among.
Claims (1)
1. a kind of method for purifying the tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle, it is characterised in that: the method uses
Material be 3 monthly age of ox fetus longissimus dorsi muscle tissue, agents useful for same is that clostridiopetidase A, fetal calf serum, low sugar Dulbecco change
Good Eagle's medium, cell sorting buffer, antiplatelet source property growth factor receptors Alpha antibodies, dexamethasone, 3- isobutyl
Base -1- methyl xanthine, bovine insulin and oil red O;
It the described method comprises the following steps: Step 1: cell is separately cultured;Specifically includes the following steps: step (1) is by ox tire
Youngster rinses several times with 75% alcohol and containing dual anti-phosphate buffer;
Step (2), which is then placed into gnotobasis, carefully breaks amnion, takes out fetus, cuts off skin of back with eye scissors, uses
Eye scissors, tweezers isolate muscle of back;
Step (3) is put into culture dish with the musculature that tweezers take out 2 pieces of mung bean grain sizes, is previously added and is contained in culture dish
There is dual anti-phosphate buffer, washes down 3 removing haemocytes with phosphate buffer;
Step (4) is then transferred into EP pipe, and 1mL0.1% type Ⅳ collagenase is added, is shredded with eye scissors to meat gruel shape;
The tissue shredded is put into 37 DEG C of gas thermostatic shaking tables by step (5) digests 45min, and during which every 15min blows and beats tissue one
It is secondary;
Cytoplasm is filtered with 40 μm of cell filtering net after step (6) 45min, 400rpm is centrifuged 5min, discards supernatant liquid, adds
Enter cell sorting buffer suspension cell;
Step (7) is incubated for 15min after 2 μ L antiplatelet source property growth factor receptors Alpha antibodies are added, and 300rcf is centrifuged 10min;
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (8), abandons supernatant, is repeated once;
80 μ L cell sorting buffers are added after sucking supernatant in step (9), add 20 μ L anti-rabbit source immunoglobulin microballons, mix
15min is incubated in 4 DEG C after even;
300rcf is centrifuged 10min after 1mL cell sorting buffer is added in step (10), abandons supernatant, is repeated once;
500 μ L cell sorting buffer suspension cells are added in step (11);
MS sorting column as on magnetic frame, suspension is added on ready MS sorting column, collection flows down by step (12)
Negative cells, rinse sorting column 3 times, use 500 μ L cell sorting buffers every time, collect the liquid that flows down and first step conjunction
And;
MS is sorted column and is placed in new collecting pipe from removing on magnetic frame by step (13), after 1mL cell sorting buffer is added,
It is quickly pushed into piston in sorting column, the liquid for collecting outflow is positive cell;
Step 2: cell culture;Specifically includes the following steps: being sucked when primary cell growth aggregation is to 70~80%
Old culture medium, and cleaned 2 times with phosphate buffer, after 0.25% trypsase is added, it is put into 30s in incubator, is being fallen
Set under phase contrast microscope observe cell it is rounded when, softly beat culture dish with hand, after circular cells float, at once plus
Enter culture medium to continue to terminate digestion reaction, soft piping and druming cell, secondary culture is carried out with 1:3 ratio, cell is put
Enter and is cultivated in the constant incubator that temperature is 37 DEG C, CO2 content is 5%;
Step 3: the adipogenic induction of cell;Specifically includes the following steps: discarding culture when cell growth converges to 100%
Base is cleaned 2 times with phosphate buffer, and lipoblast is added and induces liquid, the lipoblast induction liquid adds for growth medium
0.5 μM of 3-isobutyl-1-methylxanthine, 10 μ g/mL insulin, 1 μM of dexamethasone, start to be induced, every 3d is changed once
Liquid;
Step 4: oil red O;Specifically includes the following steps: step (a) discards old culture medium, 4% paraformaldehyde is added to orifice plate
In, the fixed cell 20min of room temperature;
Step (b) is cleaned 3 times with phosphate buffer;
Step (c) is added under oil red O room temperature and dyes 10min;
Step (d) discards oil red O stain liquid, is cleaned 3 times with phosphate buffer;
Step (e), which is placed under inverted microscope, to be observed the staining conditions of fat drips in cell and takes pictures;
Step 5: oil red O stain test result;Specifically includes the following steps: when cell induces 10d, to platelet derived
Growth factor receptors α positive cell and Platelet-derived growth factor α-receptor negative cells carry out oil red O stain, the results showed that,
The former is more than the latter fat drips, bigger.
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