CN104152406B - Preparation for enhancing differentiation capacity of chicken skeletal muscle myoblast and application thereof - Google Patents
Preparation for enhancing differentiation capacity of chicken skeletal muscle myoblast and application thereof Download PDFInfo
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- CN104152406B CN104152406B CN201410400520.1A CN201410400520A CN104152406B CN 104152406 B CN104152406 B CN 104152406B CN 201410400520 A CN201410400520 A CN 201410400520A CN 104152406 B CN104152406 B CN 104152406B
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Abstract
The invention discloses a preparation for enhancing differentiation capacity of chicken skeletal muscle myoblast and application thereof, and belongs to the technical field of biological engineering. The preparation provided by the invention is composed of a muscle derived fibroblast extractive, pyruvate and calcium salt. The muscle derived fibroblast extractive is obtained from the steps of: conducting ultrasonic cracking on chicken muscle derived fibroblast, isolating to obtain a protein solution less than 10KDa, and conducting vacuum drying. Calcium salt and pyruvate can be calcium pyruvate. The preparation provided by the invention has the characteristics of simple and easily available composition and simple and effective usage, and can significantly improve the fusion rate of chicken skeletal muscle myoblast and promote the increase of skeletal muscle fiber diameter and expression of functional contractile protein. The preparation provided by the invention can be used for culturing myoblast; and the fusion rate of the myoblast is increased by 32.01%, the average number of nuclei in myotubes is increased by 91.63%, and average diameter of myotubes is increased by 45.59%.
Description
Technical field
The present invention relates to a kind of preparation for improving chicken skeletal myoblast differentiation capability and its application, belong to bioengineering
Technical field.
Background technology
Musculature is widely present in animal body, is one of tissue of earliest formation in embryo development procedure, is thin research
The good model of the basic biological phenomena such as the decision of born of the same parents' destiny, cell differentiation, form generation.But so far, to muscle development
And the elaboration of regenerative system is only tip of the iceberg.Although the foundation into muscle model simulates the regeneration of muscle well in vivo
Journey, but also the research to related mechanism increased complexity, hence set up the Skeletal Muscle Cell culture system in vitro of efficient stabilization
To be that skeletal development, regenerative system is expanded on further to lay the foundation.
It is a continuous physiology course that muscle is formed.Embryonic period, embryonic phase, muscle precursor cell --- myoblast proliferation, point
Change, be fused to primary myotube and progressively maturation；After birth, although the number of myotube no longer changes, when musculature is impaired,
The satellite cell of tranquillization is activated, so divide, fusion forms new myotube, muscle be regenerated (Brian etal.,
2008).By INVENTIONModern cell separation, culture technique, researcher can utilize embryo sarcoblast or adult satellite cell body
Outer culture obtains primary myotube, but gained myotube type randomness is strong, and oxidative metabolism level is different, generally only in form or function
Some aspects meet internal into flesh process.This will be for future medically by cellular transplantation therapy myopathy, such as cardiac muscle stalk
Extremely, ischemic heart failure, large area muscle damage, duchenne's progressive muscular dystrophy etc. cause technical obstacle.Additionally, poultry
In animal husbandry production, the body weight gains of livestock and poultry, Lean mass and body fat content are extremely important for the livestock and poultry of different breeding objectives
Inspection target.For example, poultry requires there is body weight gains faster and Lean mass higher, to obtain economic benefit higher；
And it is then opposite to plant fowl, it is desirable to which body weight is small, weightening is slow, is beneficial to the semen quality of public fowl and the egg laying performance of female bird；Some places
Kind requires the certain muscle of control and body fat ratio to ensure its good to eat local flavor again.Therefore, correlated process is formed to muscle
Research with mechanism also will provide theoretical reference for the application on Animal Breeding.
Musculature is not that, by the cellularity of single type, the interaction between muscle cell and other cell types exists
Whole (Donoghue and Sanes, 1994 into playing a significant role during flesh；Krauss et al.,2005).Either
All there is flesh source fibroblast in the musculature in puberty or maturity period, they are located in the matrix outside muscle cell,
It is not only into flesh and provides the skeleton structure that can adhere to, is also largely responsible for extracellular matrix secretion and growth factor
(Idea etal.,2009).The existing effect not accounted for mostly into flesh cultivating system in vivo into flesh microenvironment, thus build
It is vertical a kind of close in vivo into the research and medical treatment and herding of the culture system in vitro for muscle development and regenerative system of flesh pattern
The application of production field is respectively provided with positive effect.
The content of the invention
To solve the above problems, the invention provides a kind of preparation for improving chicken skeletal myoblast differentiation capability, institute
Take technical scheme as follows：
It is an object of the present invention to provide a kind of preparation for improving chicken skeletal myoblast differentiation capability, said preparation is
It is made up of flesh source fibroblast extract, acetonate and calcium salt.
The flesh source fibroblast extract be chicken flesh source fibroblast through ultrasonic treatment after, it is isolated to be less than
The vacuum dried acquisition of protein liquid of 10KDa.
The preparation is used to cultivate chicken skeletal myoblast, improves the differentiation capability of chicken skeletal myoblast.
The acetonate can be CALCIUM PYRUVIC, Sodium Pyruvate, Potassium pyruvate；The calcium salt can for CALCIUM PYRUVIC,
Calcium chloride, calcium sulfate；Acetonate and calcium salt can be simultaneously CALCIUM PYRUVIC.
Preferably, acetonate and calcium salt are simultaneously CALCIUM PYRUVIC.
The flesh source fibroblast extract is individually packed with CALCIUM PYRUVIC.
It is a further object to provide a kind of method using the preparation culture chicken skeletal myoblast, should
Method is to be added in myogenic differentiation culture medium to use after flesh source fibroblast is mixed with CALCIUM PYRUVIC.
The flesh source fibroblast extract mixes with CALCIUM PYRUVIC, flesh source fibroblast extract and CALCIUM PYRUVIC
Mass ratio be 10-120:1.
Described being added in myogenic differentiation culture medium uses, final concentration of 600-2400mg/ of the flesh source into fiber extract
L, the final concentration of 5-80mg/L of CALCIUM PYRUVIC.
Preferably, final concentration of 2400mg/L, the final concentration of 20mg/L of CALCIUM PYRUVIC of the flesh source into fiber extract.
The preferred cultivation temperature of the chicken skeletal myoblast differentiation condition of culture is 39 DEG C, CO2Concentration is 5%, the cycle
It is 8-10 days, changed liquid once every 2 days therebetween.
The myogenic differentiation culture medium preferably comprise 2% horse serum, 2mML- glutamine, 100U/ml penicillin,
The DMEM in high glucose culture medium of 100ug/ml streptomysins.
Beneficial effect of the present invention：
In muscle forming process, substantial amounts of flesh source fibroblast is distributed with the extracellular matrix of sarcoblast, they
Regulate and control whole into flesh process by secrete cytokines, including suppress sarcoblast apoptosis, promote sarcoblast migration and myotube
Maturation etc..The present invention uses biological extractive technique, using flesh source fibroblast extract as induction chicken skeletal myoblast
The culture component of differentiation, on the one hand simulate in internal growth course into flesh microenvironment；On the other hand prior art is avoided
Middle use co-culture of cells mode causes the possibility that sarcoblast pollutes, and disclosure satisfy that research with clinical rank cell culture
Need.Calcium ion is the medium molecule of muscle cell excitement-contraction coupling, for promoting muscle cell maturation and feature to receive
Contracting is played an important role.Pyruvic acid is the intermediate product of body glycometabolism, can replace glucose for cellular oxidation energy supply is carried
For carbon source, there is positive role for improving the phosphorylation level of muscle cell, strengthening mechanical performance etc..
The preparation of the raising chicken skeletal myoblast differentiation capability that the present invention is provided, composition is simple and easy to get, easy to use
Effectively, the fusion rate of chicken skeletal myoblast is remarkably improved, the increase and feature for promoting skeletal muscle fibre diameter are shunk
The expression of albumen, so as in form and functionally improve the differentiation capability of chicken skeletal myoblast simultaneously, different from existing
Be only capable of promote the Phenetic loose cultural method of muscle cell.To be solution for the exploration and optimization in vitro into flesh condition of culture
The formation of analysis muscle and regenerative system etc. lay the foundation, with good research and application value.
Brief description of the drawings
Fig. 1 is the myotube form formed after sarcoblast breaks up culture 8 days in each group culture medium；
(100 ×, A contains myogenic differentiation culture medium；B, contains myogenic differentiation culture medium, 20mg/L CALCIUM PYRUVICs；C, contains
There are myogenic differentiation culture medium, 2400mg/L fleshes source fibroblast extract；D, contains myogenic differentiation culture medium, 5mg/L acetone
Sour calcium, 2400mg/L fleshes source fibroblast extract；E, contains myogenic differentiation culture medium, 20mg/L CALCIUM PYRUVICs, 2400mg/
L fleshes source fibroblast extract；F, containing myogenic differentiation culture medium, 80mg/L CALCIUM PYRUVICs, 2400mg/L fleshes source into fiber
Cell extract).
Fig. 2 is that sarcoblast breaks up Skeletal Muscle Contraction protein expression level after culture 8 days in each group culture medium
Western Blot testing results；
(A contains myogenic differentiation culture medium；B, contains myogenic differentiation culture medium, 20mg/L CALCIUM PYRUVICs；C, containing into flesh
Differential medium, 2400mg/L fleshes source fibroblast extract；D, containing myogenic differentiation culture medium, 5mg/L CALCIUM PYRUVICs,
2400mg/L fleshes source fibroblast extract；E, contains myogenic differentiation culture medium, 20mg/L CALCIUM PYRUVICs, 2400mg/L fleshes source
Fibroblast extract；F, carries containing myogenic differentiation culture medium, 80mg/L CALCIUM PYRUVICs, 2400mg/L fleshes source fibroblast
Take thing).
Fig. 3 be sarcoblast after differentiation is cultivated 8 days in the A groups and E group culture mediums cytoskeletal filament albumen in myotube
The Immunofluorescence test result of distribution；
(200 ×, A contains myogenic differentiation culture medium；E, containing myogenic differentiation culture medium, 20mg/L CALCIUM PYRUVICs,
2400mg/L fleshes source fibroblast extract, wherein, upper figure is the microfilament protein of phalloidine mark；Figure below is
The nucleus of Hochest33342 marks；The part that dotted line is irised out is myotube).
Specific embodiment
With reference to specific embodiment, the present invention will be further described, but the present invention should not be limited by the examples.
Method in following embodiments is conventional method without special instruction.
Instrument and reagent are the conventional instrument that can be obtained by commercial sources without special instruction in following embodiments
Device and reagent.
The acquisition of the chicken skeletal myoblast of embodiment 1 and Multiplying culture
Fresh kind egg is hatched 11 days in 38 DEG C in the incubator of 63% humidity.After eggshell is through 70% ethanol disinfection,
Broken shell is taken out chicken embryo and is placed in culture dish.Chicken embryo skin of chest is pushed aside with tweezers, separates pectoralis major, and removed under anatomical lens
Ligament, connective tissue and blood vessel.Hank's liquid cleaning breast muscle 3 times, the clostridiopetidase A I of addition 0.1% is in 38 after fully shredding
DEG C be incubated 30 minutes, every 10 minutes of period blow and beat once.Centrifugation, after adding Hank's liquid to dispel cell, with the stainless steel of 200 mesh
Strainer filtering cell is obtaining single cell suspension.
20%, 27.5% and 40% Percoll solution is prepared, it is sequentially added 15ml centrifugations from high to low by concentration
In pipe (action is light and slow), then above-mentioned cell suspension is slowly layered on 20%Percoll liquid levels.1500g, 4 DEG C of levels from
The heart 8 minutes, the cell of the liquid level intersection of careful collection 27.5%/40%, with containing 10%FBS, 2mML- glutamine, 100U/
Ml penicillin, 100ug/ml streptomysins, DMEM in high glucose into flesh proliferated culture medium re-suspended cell and be inoculated in culture dish, 39 DEG C training
Support 2 hours, collect not yet adherent cell and be sarcoblast.
Sarcoblast is inoculated in the culture dish of 0.1% gelatin treatment, inoculum density is 5000 cell/cm2,39 DEG C
Liquid is changed in culture after 1 day, continued culture and reached within 2-3 days 70% cell confluency.
The acquisition of the flesh source fibroblast extract of embodiment 2
Fresh kind egg is hatched 11 days in 38 DEG C in the incubator of 63% humidity.After eggshell is through 70% ethanol disinfection,
Broken shell is taken out chicken embryo and is placed in culture dish.Chicken embryo skin of chest is pushed aside with tweezers, separates pectoralis major, and removed under anatomical lens
Ligament, connective tissue and blood vessel.Hank's liquid cleaning breast muscle 3 times, the clostridiopetidase A I of addition 0.1% is in 38 after fully shredding
DEG C be incubated 30 minutes, every 10 minutes of period blow and beat once.Centrifugation, after adding Hank's liquid to dispel cell, with the stainless steel of 200 mesh
Strainer filtering cell is obtaining single cell suspension.With the height sugar containing 10%FBS, 100U/ml penicillin, 100ug/ml streptomysins
DMEM re-suspended cells are simultaneously inoculated in culture dish, 39 DEG C cultivate 2 hours, adherent cell collecting and pass on purifying culture 3-5 generation obtain
Flesh source fibroblast.
Flesh source fibroblast is carried out into ultrasonic disruption, parameter is 450Hz, each ultrasound is stopped 10 seconds, 40 times totally for 6 seconds.
Using the protein concentration post (Millipore UFC901024 Amicon Ultra -15, by specification operation) of 15mL/10KD
The above-mentioned cell pyrolysis liquid of filtering and concentrating, collecting protein liquid of the molecular weight less than 10KD carries out vacuum drying treatment.
The differentiation culture of the chicken skeletal myoblast of embodiment 3
When the chicken skeletal myoblast cultivated by embodiment 1 reaches 70% to be converged, flesh proliferated culture medium is removed into,
PBS 3 times, the A-F group culture mediums shown in table 1 are slowly added to along culture dish inwall.Wherein, A groups are control group, are only contained
Have myogenic differentiation culture medium (myogenic differentiation culture medium be containing 2% horse serum, 2mM Glus, 100U/ml penicillin,
The DMEM in high glucose of 100ug/ml streptomysins)；B-F groups are experimental group, for the myogenic differentiation for being added with least one formulation components is trained
Support base.
The composition and proportioning of 1 A of table-F group culture mediums
| Group | Myogenic differentiation culture medium | Flesh source fibroblast extract (mg/L) | CALCIUM PYRUVIC (mg/L) |
| A | √ | — | — |
| B | √ | — | 20 |
| C | √ | 2400 | — |
| D | √ | 2400 | 5 |
| E | √ | 2400 | 20 |
| F | √ | 2400 | 80 |
It is 39 DEG C, CO that above-mentioned each group is placed in temperature2Concentration be 5% incubator in cultivate 8 days, therebetween every 2 days more
Change nutrient solution.
The chicken skeletal myoblast cell level of differentiation of embodiment 4 is detected
First, the morphologic detection of myogenic differentiation level
Myogenic differentiation includes two relatively independent processes：What myoblast fusion formed primary myotube and myotube enters one
Step is ripe.The maturation of myotube has counted the sarcoblast of above-mentioned each treatment group here along with a series of morphologic changes
The average diameter of average cell check figure and myotube in fusion rate, myotube, the maturity of each group myotube is evaluated with this.
Myoblast fusion rate refers to the percentage that cell check figure in myotube accounts for total cell check figure, is for evaluating into flesh
The index of differentiation efficiency.After through the differentiation of embodiment 3 culture 8 days, each group has myotube to be formed, and its representative configuration is as shown in figure 1, can
See that D, E, F group myotube are thick compared with A, B, C group, especially E groups.For the ease of nuclei count, culture is fixed through absolute methanol
Afterwards, dyeed 10 minutes with 10% Giemsa dye liquors, running water is fully cleaned after just putting basis of microscopic observation.Count into flesh thin
During born of the same parents' fusion rate, each treatment group randomly selects 6 visuals field and obtains data；Average cell check figure and myotube in statistics myotube
During average diameter, each treatment group randomly selects 6 visuals field, and each visual field randomly selects 10 myotubes and obtains data.Experiment number
Single factor test variance point is carried out according to using SPSS statistical softwares (SPSS for Windows, Release 10.0.1, SPSS Inc.)
Analysis, the aobvious author of variance analysis carries out Multiple range test using LSD methods.Result is as shown in table 2, it is seen that the myotube of test group (B-F groups)
Compared with control group (A groups) maturation, especially E groups, the fusion rate of its sarcoblast improves 32.01% compared with A groups, average thin in myotube
Karyon number improves 91.63% compared with A groups, and myotube average diameter increases by 45.59% compared with A groups.It can be seen that the preparation that the present invention is provided can be with
Effectively improve the level of differentiation of chicken skeletal myoblast.
The morphology of the myogenic differentiation level of table 2 is determined
Note：In mark in same row, the expression significant difference (P without same letter<0.05)
2nd, the feature detection of myogenic differentiation level
Rythmic contractions characteristic is the important embodiment of skeletal muscle function, is also the major way of skeletal muscle function.Bone
The contraction of flesh depends on the expression of its contractile protein and the correct assembling of cytoskeleton, Western Blot is used here and is immunized
The method of fluorescence have detected the positioning of the expression and cytoskeletal filament of skeletal muscle main shrinkage albumen in myotube respectively.
1st, the expression of Skeletal Muscle Contraction albumen is determined
After through the differentiation of embodiment 3 culture 8 days, nutrient solution, PBS 3 times, by each group sample dissociation are removed.Lysate in
30000g, 4 DEG C be centrifuged 30 minutes, take supernatant and determine its protein concentration.Taking equal protein from each group carries out SDS-PAGE electrophoresis
Separate and transferring film, closed with the confining liquid containing 5% skim milk, 0.05% polysorbas20 afterwards.Primary antibody goes in 4 DEG C of overnight incubations
Except primary antibody, PBS washes film 30 minutes.The secondary antibody of HRP marks removed secondary antibody in incubation at room temperature 30 minutes, and PBS washes film 30 minutes, used
MTT colour reagents box (Pierce companies, by specification operation) colour developing.Wherein, primary antibody used is mouse anti alpha-sarcomeric
Anti- myosin heavy chain (MyHC) monoclonal antibody (Abcam) of actin monoclonal antibodies (Abcam), mouse and mouse are anti-
GAPDH monoclonal antibodies (Abcam)；GAPDH is used as internal reference.Result is as shown in Figure 2, it is seen that test group (B-F groups) skeletal muscle is received
The expression of pix protein α-sarcomeric actin and MyHC is significantly higher than control group (A groups), especially with the addition of whole
The test group (D-F groups) of formulation components, points out its functionalization higher level.
2nd, the Subcellular Localization of cytoskeletal filament
After control group (A groups) and test group (E groups) are cultivated 8 days through the differentiation of embodiment 3, removing nutrient solution, PBS 3 times,
4% paraformaldehyde room temperature fixes 30 minutes；PBS 3 times, adds 0.1%Triton-100 in 37 DEG C of permeable membranes 1 hour；PBS is clear
After washing 3 times, it is incubated 30 minutes with the confining liquid containing 1%BSA at room temperature, adds the ghost of the Rhodamine marks of confining liquid dilution
Lucifuge is incubated 1 hour at 37 DEG C of cyclic peptide (Sigma)；Hochest33342 dyeing liquors dye core 10 minutes；Fully added after cleaning
Anti- fluorescence quenching, observes and takes pictures under fluorescence inverted microscope (Olympus IX81).Result is as shown in figure 3, in A group myotubes
Microfilament skewness, in polarity distribution (as shown by arrows), and moves drawing, the nucleus in myotube due to lack stress fiber
Also it is in polarity distribution；Nucleus in E group myotubes is linearly distributed, and microfilament extends along the myotube longitudinal axis and forms structure at two ends
Clear and abundant stress fiber (as shown by arrows), is conducive to the Rythmic contractions characteristic of myotube.It can be seen that the preparation that the present invention is provided
The distribution of cytoskeletal filament can be effectively improved, promotes the perfect in shape and function of myotube.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this
The people of technology, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore protection of the invention
What scope should be defined by claims is defined.
Claims (8)
1. it is a kind of improve chicken skeletal myoblast differentiation capability preparation, it is characterised in that be to be carried by flesh source fibroblast
Take thing, acetonate and calcium salt composition；The flesh source fibroblast extract is that chicken flesh source fibroblast is split through ultrasonic wave
Xie Hou, the vacuum dried acquisition of the isolated protein liquid less than 10KDa.
2. preparation described in claim 1 improves the differentiation capability of chicken skeletal myoblast in culture chicken skeletal myoblast
In application.
3. preparation described in claim 1, it is characterised in that the acetonate and calcium salt can be simultaneously CALCIUM PYRUVIC.
4. preparation described in claim 3, it is characterised in that the flesh source fibroblast extract is individually wrapped with CALCIUM PYRUVIC
Dress.
5. the method for preparation culture chicken skeletal myoblast described in a kind of utilization claim 3, it is characterised in that by flesh source into
After the extract of fibrocyte mixes with CALCIUM PYRUVIC, it is added in myogenic differentiation culture medium and uses.
6. claim 5 methods described, it is characterised in that the flesh source fibroblast extract mixes with CALCIUM PYRUVIC, flesh
Source fibroblast extract is 10-120 with the mass ratio of CALCIUM PYRUVIC:1.
7. claim 5 methods described, it is characterised in that described being added in myogenic differentiation culture medium uses, flesh source is into fiber
The final concentration of 600-2400mg/L of cell extract, the final concentration of 5-80mg/L of CALCIUM PYRUVIC.
8. claim 7 methods described, it is characterised in that the final concentration of 2400mg/ of the flesh source fibroblast extract
L, the final concentration of 20mg/L of the CALCIUM PYRUVIC.
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| CN106754666B (en) * | 2017-01-23 | 2017-12-26 | 河南护理职业学院 | A kind of method of C2C12 myoblast differentiations culture |
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