CN103881966A - Preparation method of mouse myoblasts and application thereof - Google Patents
Preparation method of mouse myoblasts and application thereof Download PDFInfo
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- CN103881966A CN103881966A CN201410157261.4A CN201410157261A CN103881966A CN 103881966 A CN103881966 A CN 103881966A CN 201410157261 A CN201410157261 A CN 201410157261A CN 103881966 A CN103881966 A CN 103881966A
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Abstract
The invention discloses a preparation method of mouse myoblasts, and the method comprises the steps of adopting mechanical tissue separation, culturing by use of a primary culture medium and differentiating the culture medium when cells form the abundance of 40-80% till a myotube is induced to form the mouse myoblasts. By adopting the method, about 1*10<8> cells can be separated from each gram of mouse muscle, wherein the proportion of the myoblasts to satellite cells is up to above 90%. The myoblasts play an important role in the aspect of researching the formation of the myotube, muscle development cells and a molecular mechanism.
Description
Invention field:
The present invention relates to myoblastic preparation method, more particularly, the present invention relates to the preparation method of mouse muscle-forming cell, and the application of the method.
The description of prior art:
Sarcoplast is a kind of embryo's progenitor cell, and it can break up becomes muscle cell.In the time that sarcoplast merges, will form skeletal muscle fibre, therefore myofiber has multiple nucleus, and each nucleogenesis is in single sarcoplast.It is skeletal muscle special (for example biceps muscle) that sarcoplast is merged, and cardiac muscle and unstriated muscle are not like this.Those are not divided into myofibrillar sarcoplast and dedifferente as satellite cell.These satellite cells are close on myofiber, are present between sarcolemma and endomysium.Muscle bundle is divided into single myofiber (Kuang S, Cell Stem Cell, 2008,2 (1): 22-31 by these conjunctive tissues; Sacco A, Nature, 2008,456 (7221): 502-506).Bone sarcoplast provides the instrument (Nguyen TH, BMC Cell Biol, 2010,11:57) of a good in vitro study muscle cell multiplication and differentiation for a long time for investigator.The research of sarcoplast differentiation is very important to understanding muscle cell growth and repairing (regeneration).Sarcoplast in cultivation can show the feature of muscle generating process, comprises propagation, and migration is merged, and myotube forms and shrinks.Converge even if grow in the muscle cell containing in 10% foetal calf serum substratum, also continued propagation; And in they grow in containing the substratum of 2-5% horse serum time, but there is to merge and form myotube.Mouse cell lines C2C12 and C2F3 are the clone that is widely used for studying sarcoplast propagation and differentiation.Current scientific research requires investigator from laboratory animal, to separate and cultivate primary sarcoplast.Thomas A.Rando is by the processing of several " adhere to-depart from " of taking turns, separate and enrichment sarcoplast, abandoned myofibroblast (Rando TA, J Cell Biol, 1994,125 (6): 1275-1287).The Moira A.Lawson step that equally also usage variance adheres to, the sarcoplast (Lawson MA, Cell Tissues Organs, 2000,167 (2-3): 130-137) of separation and enrichment chicken.Yu Zhang report myofibroblast can be protected sarcoplast, to avoid the inherent apoptosis that is attended by differentiation (Zhang Y, Dev Growth Differ, 2010,52 (8): 725-733).
Find under study for action, the method that adopts Rando to describe separates and to break up the sarcoplast of newborn mice very difficult, and success ratio is low.Along with the increase of experimental procedure, the sarcoplast obtaining is fewer and feweri, has lost a lot of sarcoplasts and satellite cell in process, and satellite cell is that sarcoplast differentiation is necessary, and is myoblastic source; The survive sarcoplast growth of getting off and slowly of minority, is very easy to occur apoptosis.
Invention technology contents:
First object of the present invention is to provide a kind of preparation method of mouse muscle-forming cell, adopt the method, can obtain quickly and easily a large amount of mouse muscle-forming cells, surviving rate is high, grow vigorous, can perform well in being divided into the research of myotube, the vary stable of its cytologic characteristic and developed by molecule.
Second object of the present invention be to provide obtained sarcoplast research myotube form and muscle development cell, molecule mechanism aspect application.
The preparation method who the invention discloses a kind of mouse muscle-forming cell, the method comprises the following steps:
A) by the just in vitro and mouse leg muscle that shreds by volume 1:2 add dispersion agent, put upside down shake 5-10min at 37 ℃, the centrifugal 5min of 1000rpm, removes supernatant, obtains fragment of tissue;
B) in fragment of tissue, 1:2 adds dispersion agent by volume, becomes starchiness at 37 ℃ of shake 5-10min that turn upside down, and 1:10 adds serum to stop enzymic digestion by volume;
C) filter the aseptic nylon wire of 80 μ m, remove large fragment of tissue, the centrifugal 5min of filtrate 1000rpm, removes supernatant, obtains precipitation;
D) precipitation is put in the coated culture dish of collagen protein, adds primary sarcoplast growth medium, at 37 ℃, in the incubator of 5%CO2, cultivates;
E) in the time that sarcoplast grows into 40-80% abundance, remove growth medium, add division culture medium, change division culture medium every day and continue to cultivate sarcoplast, until myotube is induced to form;
Wherein, dispersion agent composition is 0.2% collagenase and 0.05% Dispase, and surplus is PBS, pH=7.5-7.8; Primary sarcoplast growth medium is by 80%F10,19%FBS, 1% penicillin and Streptomycin sulphate, 2.5-5.0ng/mL Prostatropin bFGF composition; Division culture medium is by 94-97%DMEM, 2-5% horse serum, 1% penicillin and Streptomycin sulphate composition.
The invention also discloses a kind of preferred preparation method, the method comprises the following steps:
A) by the just in vitro and mouse leg muscle that shreds by volume 1:2 add dispersion agent, put upside down shake 10min at 37 ℃, the centrifugal 5min of 1000rpm, removes supernatant, obtains fragment of tissue;
B) in fragment of tissue, 1:2 adds dispersion agent by volume, becomes starchiness at 37 ℃ of shake 10min that turn upside down, and 1:10 adds serum to stop enzymic digestion by volume;
C) filter the aseptic nylon wire of 80 μ m, remove the large centrifugal 5min of fragment of tissue filtrate 1000rpm, remove supernatant;
D) precipitation is put in the coated culture dish of collagen protein, and adds primary sarcoplast growth medium, at 37 ℃, in the incubator of 5%CO2, cultivates;
E) in the time that sarcoplast grows into 80% abundance, remove growth medium, add division culture medium, change division culture medium every day and continue to cultivate sarcoplast, until myotube is induced to form;
Wherein, dispersion agent composition is 0.2% collagenase and 0.05% Dispase, and surplus is PBS, pH=7.8; Primary sarcoplast growth medium is by 80%F10,19%FBS, 1% penicillin and Streptomycin sulphate, and 5.0ng/mL Prostatropin bFGF composition; Division culture medium is 100% horse serum.
The method disclosed in the present is applicable to separate and be divided into from normal wild type mouse, knock out mice and transgenic mice myocyte.
The preparation method who the invention also discloses mouse muscle-forming cell is divided into the application aspect myotube MAML1 transgenic mice shank sarcoplast.
The newborn mice using in the present invention is for being born 2 days with interior mouse.From 1 gram of mouse muscle, approximately can be separated to 1 × 10
8individual sarcoplast or satellite cell.The bFGF using in the present invention, its concentration can not be lower than 2.5ng/mL.Use collagenase and Dispase digestive muscular fragment in 15mL centrifuge tube time, should tightly seal the mouth of pipe, control liquid spills.
After Cell abundance exceedes 80%, sarcoplast differentiation capability weakens, and after abundance exceedes 95%, cell can not form myotube substantially.Cell abundance is lower than 40% time, and cell quantity is on the low side, is also unfavorable for differentiation.
In division culture medium, can not use foetal calf serum, calf serum and bovine serum, they all can not induce myoblastic differentiation well.
The horse serum concentration that uses of differentiation can not be lower than 2%, otherwise sarcoplast growth is bad, and apoptosis even can occur.Its concentration can not be higher than 5%, otherwise the ability that induction myotube forms reduces.
Horse serum can be induced sarcoplast to break up and be formed myotube.In experiment, find that round and little satellite cell is essential to the differentiation of newborn mice sarcoplast and formation myotube, as Fig. 3, shown in 4 and 5.If there is no satellite cell, sarcoplast breeds continuation and is converted into inoblast, and they can not break up and form myotube.Under correct condition, almost 90% sarcoplast of cultivating can break up and form myotube as shown in Figure 5.As shown in Figure 4, growth medium is being changed into the division culture medium that contains 2-5% horse serum after one day, wild-type mice sarcoplast only forms several little myotubes in culture dish, but the sarcoplast of MAML1 transgenic mice but can form more myotube.Two days later, the myotube that has MAML1 transgenosis sarcoplast to form becomes longer and more sturdy.After three days, wherein some myotube extends longly especially.After four days, the myotube being formed by wild-type sarcoplast starts atrophy degraded, and those myotubes that formed by MAML1 transgenosis sarcoplast also start atrophy, but comparatively gentle, and they can continue to maintain to exist and exceed five days, then just degrades.
At Rando(1994) experimental program in, they by mouse tissue fragment through going down to posterity several times, to remove the inoblast of quick wall attaching, and slowly adherent sarcoplast of enrichment.They knock an angle very doughtily from the side of culture dish, and with the sarcoplast that comes off, and inoblast still sticks on plate.But adopt after this method, results are to cell less and less.Although these similar myoblastic short and little cells can be survived 2 weeks, finally all apoptosis have occurred, can not resemble Rando and form clone described.We are changing foetal calf serum from 10% to 20%, CO
2from 5% to 10%, or the concentration of bFGF from 1ng/ml to 10ng/ml, change the mode of going down to posterity-from striking an angle to enzymic digestion, after having changed plate coating protein-from collagen protein to Laminin ELISA, still final apoptosis of the similar cell of this one-tenth flesh.
Can obtain purer mouse muscle-forming cell by the technology of the present invention, comprise a large amount of can budding into myoblastic satellite cell, be rendered as spheroidal; And a large amount of sarcoplasts, be rendered as short fusiformis.Myosin Myosin and MyoD are that sarcoplast is different from the albumen that inoblast characteristic is expressed, and we identify sarcoplast with this, as shown in Fig. 1 and Fig. 6.In Fig. 1, the cell of green-emitting fluorescence is mouse muscle-forming cell and the satellite cell of high expression level MyoD albumen, myoblastic ancester cell.In experiment, use the two anti-of the primary antibodie of anti-mouse MyoD and FITC mark.In Fig. 6, by the sarcoplast induction differentiation from wild-type WT and MAML1 transgenosis type TG mouse three days, be then gathered into myocyte and myotube, prepare total protein, and carrying out myosin detection with Western blot and antibody, Internal loading is selected internal reference.
In the present invention, dispersion agent compound method is: weigh 0.2 gram of collagenase and 0.05 gram of Dispase, be fully dissolved in 100 milliliters of phosphoric acid buffer PBS, use aseptic membrane filtration, filtrate is kept at 4 ℃.
In the present invention, the compound method of primary sarcoplast growth medium is: in the F10 substratum of 80 milliliters, add the penicillin of 1 milliliter and the mixed solution of Streptomycin sulphate, add again the Prostatropin bFGF that weighs and dilute, final concentration reaches 5.0ng/mL, add the foetal calf serum of 19-20 milliliter, mix, aseptic membrane filtration, filtrate is kept at 4 ℃.
In the present invention, the compound method of division culture medium is: penicillin and the Streptomycin sulphate mixed solution of the DMEM substratum of 94 milliliters and 1 milliliter are mixed, add the horse serum of 5 milliliters, mix, and aseptic membrane filtration, filtrate is kept at 4 ℃.
Unless definition especially, the term using in the present invention has common meaning with writing a Chinese character in simplified form.For example, " mm " refers to millimeter, and " min " refers to minute, " mL " refers to milliliter, " ℃ " refer to degree Celsius, " ng " refers to nanogram, " F10 substratum " refers to Ham ' s F-10Nutrient Medium, and " DMEM " refers to Dulbecco's Modified Eagle Medium, and " rpm " refers to rev/min.
The present invention compared with prior art, has the following advantages:
1. can be separated to more, purer mouse muscle-forming cell and satellite cell.From every gram of mouse muscle, can be separated to approximately 1 × 10
8individual cell, wherein the ratio of sarcoplast and satellite cell reaches more than 90%.
2. the Growth of Cells being separated to is vigorous, can under the induction of horse serum, be differentiated to form a large amount of typical myotubes, and quantity is that other method forms the more than 10 times of myotube.
3. operation is simpler, and the time is shorter, cell better quality.Myoblastic separation only needs 1.5-2 hour with obtaining, and only need to use routine instrument device, and 90% above cultured cells can be differentiated to form myotube.
4. the expression that characteristic protein can be detected in sarcoplast separation and atomization changes, and meets bibliographical information.
Accompanying drawing explanation
Fig. 1 is the expression figure of characteristic protein MyoD in sarcoplast.
Fig. 2 is the expression figure of MAML1 gene in transgenic mice.
Fig. 3 is the myoblastic figure that is divided into myotube after going down to posterity several times.
Fig. 4 is sarcoplast differentiation and myotube formation figure after secondary goes down to posterity.
Fig. 5 is the figure that does not go down to posterity and directly sarcoplast is broken up.
Fig. 6 is myosin expression figure in the sarcoplast of differentiation.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be appreciated that these embodiment are only for the present invention is described, and can not limit the scope of the invention.
Dispersion agent composition is 0.2% collagenase and 0.05% Dispase, and surplus is PBS, pH=7.8; Primary sarcoplast growth medium is by 80%F10,19%FBS, 1% penicillin and Streptomycin sulphate, 5.0ng/mL Prostatropin bFGF composition; Division culture medium is by 94%DMEM, and 5% horse serum, also can adopt 100% horse serum to make division culture medium.F10 and DMEM are commercial substratum, and investigator can buy from market.
Dispersion agent compound method is: weigh 0.2 gram of collagenase and 0.05 gram of Dispase, be fully dissolved in 100 milliliters of phosphoric acid buffer PBS, use aseptic membrane filtration, filtrate is kept at 4 ℃.
The compound method of primary sarcoplast growth medium is: in the F10 substratum of 80 milliliters, add the penicillin of 1 milliliter and the mixed solution of Streptomycin sulphate, add again the Prostatropin bFGF that weighs and dilute, final concentration reaches 2.5-5.0ng/mL, add the foetal calf serum of 19-20 milliliter, mix, aseptic membrane filtration, filtrate is kept at 4 ℃.
The compound method of division culture medium is: penicillin and the Streptomycin sulphate mixed solution of the DMEM substratum of 94 milliliters and 1 milliliter mixed, adds the horse serum of 5 milliliters, mix, and aseptic membrane filtration, filtrate is kept at 4 ℃; Or directly adopt 100% horse serum to make division culture medium.
The bFGF using in the present invention, its concentration can not be lower than 2.5ng/mL.
The separation of embodiment 2. mouse muscle-forming cells:
On ice, birth is shredded with the fresh limb muscle of interior mouse for 2 days.Muscle fragments shifts in aseptic 1.5mL Eppendorf pipe, adds 0.5mL0.2% collagenase collagenase and 0.05% Dispase dispase mixed solution, continues to shred several minutes.At 37 ℃ of shake 5-10min that turn upside down, centrifugal 5 minutes of 1000rpm, removes supernatant.Fragment of tissue proceeds in the 15mL centrifuge tube that is added with 2mL collagenase/dispase mixed solution, sealing.At 37 ℃ of shake 10-20min that turn upside down.Fragment of tissue is digested to starchiness.Proceed in 15mL centrifuge tube, use microsyringe to draw fragment of tissue and blow and beat several times towards tube wall, with broken agglomerate.At 37 ℃ of shake 5-10min that again turn upside down.Supernatant liquor is transferred in 15mL pipe, adds 1mL serum to stop the digestion of enzyme.Fragment of tissue is filtered to the aseptic nylon wire of 80 μ m, to remove large fragment of tissue.Again be transferred in another 15mL pipe, the centrifugal 5min of 1000rpm, removes supernatant, uses the resuspended precipitation of the primary sarcoplast growth medium of 2-4mL F10, the cell being separated to is positioned in the coated culture dish of 35-60mm collagen protein, and in 37 ℃, 5%CO
2incubator in cultivate.
The newborn mice using in the present invention is for being born 2 days with interior mouse.From 1 gram of mouse muscle, approximately can be separated to 1 × 10
8individual sarcoplast or satellite cell.The bFGF using in the present invention, its concentration can not be lower than 2.5ng/mL.Use collagenase and Dispase digestive muscular fragment in 15mL centrifuge tube time, should tightly seal the mouth of pipe, control liquid spills.
Can obtain purer mouse muscle-forming cell by the technology of the present invention, comprise that a large amount of can budding into is rendered as spheroidal cell under myoblastic satellite cell-microscope, and under a large amount of sarcoplast-microscopes, be rendered as short fusiformis.Myosin Myosin and MyoD are that sarcoplast is different from the albumen that inoblast characteristic is expressed, and we identify sarcoplast with this, as shown in Fig. 1 and Fig. 6.In Fig. 1, the cell of green-emitting fluorescence is mouse muscle-forming cell and the satellite cell of high expression level MyoD albumen--myoblastic ancester cell.In experiment, use the two anti-of the primary antibodie of anti-mouse MyoD and FITC mark.In Fig. 6, by the sarcoplast induction differentiation from wild-type-WT and MAML1 transgenosis type-TG mouse three days, be then gathered into myocyte and myotube, prepare total protein, and carrying out myosin detection with Western blot and antibody, Internal loading is selected internal reference.
The maintaining and preserving of embodiment 3. mouse muscle-forming cells:
The mouse muscle-forming cell being separated to can directly add the cells frozen storing liquid freeze-stored cell of 90% foetal calf serum and 10% dimethyl sulfoxide (DMSO) DMSO composition in liquid nitrogen, also can directly carry out myotube differentiation.In the time that reaching 70%, sarcoplast abundance induces differentiation.
After Cell abundance exceedes 80%, sarcoplast differentiation capability weakens, and after abundance exceedes 95%, cell can not form myotube substantially.Cell abundance is lower than 40% time, and cell quantity is on the low side, is also unfavorable for differentiation.
The differentiation of embodiment 4 mouse muscle-forming cells:
4.1 in the time that sarcoplast grows into 40% abundance, growth medium is changed by 95-98%DMEM into 2-5% horse serum, the division culture medium of 1% penicillin and Streptomycin sulphate composition.Change division culture medium every day and continue to cultivate sarcoplast, until myotube is induced to form.Generally, induction differentiation just has little myotube to form after 24 hours, and about 90% above cultured cells can be divided into myotube.
4.2 in the time that sarcoplast grows into 80% abundance, growth medium is changed into the division culture medium of 100% horse serum composition.Change division culture medium every day and continue to cultivate sarcoplast, until myotube is induced to form.Generally, induction differentiation just has little myotube to form after 24 hours, and about 90% above cultured cells can be divided into myotube.
Horse serum can be induced sarcoplast to break up and be formed myotube.In experiment, find that round and little satellite cell is essential to the differentiation of newborn mice sarcoplast and formation myotube, as Fig. 3, shown in 4 and 5.If there is no satellite cell, sarcoplast breeds continuation and is converted into inoblast, and they can not break up and form myotube.Under correct condition, almost 90% sarcoplast of cultivating can break up and form myotube as shown in Figure 5.As shown in Figure 4, growth medium is being changed into the division culture medium that contains 2-5% horse serum after one day, wild-type mice sarcoplast only forms several little myotubes in culture dish, but the sarcoplast of MAML1 transgenic mice but can form more myotube.Two days later, the myotube that has MAML1 transgenosis sarcoplast to form becomes longer and more sturdy.After three days, wherein some myotube extends longly especially.After four days, the myotube being formed by wild-type sarcoplast starts atrophy degraded, and those myotubes that formed by MAML1 transgenosis sarcoplast also start atrophy, but comparatively gentle, and they can continue to maintain to exist and exceed five days, then just degrades.
Myoblastic differentiation is very important to the formation of muscle.Although existing bibliographical information separates, and cultivates and break up the sarcoplast of mouse and chicken, method is wherein difficult to follow, and can not obtain the gedanken experiment result that we want.If only have several sarcoplasts, even if they can not form myotube in the division culture medium that contains 2-5% horse serum yet.Sarcoplast propagation is very fast, and very high abundance exists, but this has hindered sarcoplast formation myotube.Particularly, through several take turns go down to posterity and for a long time cultivate after, sarcoplast can be lost the ability that forms in vitro myotube.Inoblast is helpful to sarcoplast differentiation, and satellite cell can change the good sarcoplast of differentiation capability into.
Embodiment 5MAML1 transgenic mice leg muscle sarcoplast forms myotube through horse serum induction
5.1 laboratory animal, antibody and other reagent
The genetically modified C57BL/6 mouse of wild-type and MAML1 system is raised by this research department.Anti-Actin muscle (β-actin) antibody, anti-myosin (Myosin) antibody and horse serum are purchased from Sigma company.F10 substratum, bFGF, Proteinase K and RNase H are purchased from Invitrogen company.DMEM is purchased from Cellgro company, and foetal calf serum, penicillin and Streptomycin sulphate are purchased from HyClone company, and collagenase (collagenase), Dispase (dispase) and Laminin ELISA (laminin) are purchased from Gibco company, and collagen (collagen) is purchased from Upstate company.
5.2 gene identification
4 week age of clip 0.5cm or newborn wild-type or MAML1 transgenic mice tail, put into and contain 0.3mL DNA digestion damping fluid (50mM Tris-HCl pH8.0,100mM EDTA pH8.0,100mM NaCl and 1%SDS) and 2 μ L Proteinase K (20mg/mL, final concentration is 0.5mg/mL) Eppendorf pipe in, seal with sealed membrane, 50-55 ℃ of overnight incubation.Then in cell lysate, add 1.5 μ L RNase H(0.5mg/mL, do not contain DNA enzyme), the centrifuge tube 5 times of turning upside down, high speed centrifugation several seconds, is then placed on 37 ℃ and hatches 15-60min.Adopt conventional phenol/chloroform/primary isoamyl alcohol (25:24:1) method to extract genomic dna, after measurement DNA concentration, carry out the expression of identification of M AML1 gene with PCR.The PCR upstream primer sequence of mouse Maml1 gene is 5 ' GCCACTCCCGCCACCAAA AAC3 ', and downstream primer sequence is 5 ' TTTCCGACCTCATTCTTTACA3 '.PCR upstream primer (exon 2) sequence of people MAML1 gene is 5 ' CGAGCAGAACTCCCTGTTTC3 ', and downstream primer (exon 4) sequence is 5 ' CCC TGT GAA CTG TCC AAC CT3 '.Genotype identification PCR circulation is: 1 circulation (96 ℃ of 1min, 62 ℃ of 1min, 72 ℃ of 45sec) adds 35 circulations (94 ℃ of 50sec, 62 ℃ of 1min, 72 ℃ of 45sec); 72 ℃ extend 5min, and end product is placed room temperature.Amplified band size is MAML1,415bp; Maml1,534bp.
As shown in Figure 2, Maml1 represents mouse mastermind-like1 gene, and MAML1 represents that mankind mastermind-like1 gene ， – represents negative control ,+representing positive control, M is a 100bp DNA marker.Mankind MAML1 gene has inserted transgenic mice 1,2, and in 4,5 and 6 genome, corresponding PCR product size is 415bp.The PCR product size of endogenic mouse Maml1 gene is 534bp.Mouse 3 is wild-types, does not insert mankind MAML1 gene.The mRNA sequence of mankind MAML1 gene has 95% and the Maml1 DNA homolog of mouse.
5.3 separate and cultivate newborn mice sarcoplast
Newborn mice is carried out to CO
2and Ethanol Treatment, to decaptitate, clip tail is for genotype identification.Use aseptic scissors to take off four limbs, put into freezing HBBS or PBS damping fluid.Muscle is separated with bone with skin with aseptic tweezers, change HBBS or PBS damping fluid.On ice, muscle is shredded.Muscle fragments shifts in aseptic 1.5mL Eppendorf pipe, adds 0.5mL0.2%collagenase and 0.05%dispase mixed solution, continues to shred several minutes.Carry out the digestion of enzyme at 37 ℃ of shake 5min that turn upside down, centrifugal 5 minutes of 1000rpm, removes supernatant.Fragment of tissue proceeds in the 15mL centrifuge tube that is added with 2mL collagenase/dispase mixed solution, sealing.At 37 ℃ of shake 10-20min that turn upside down.Fragment of tissue uses microsyringe absorption fragment of tissue to blow and beat several times towards tube wall after being digested to starchiness, with broken agglomerate.At 37 ℃ of shake 5-10min that again turn upside down.Supernatant liquor is transferred in 15mL pipe, adds 1mL serum to stop the digestion of enzyme.Fragment of tissue is filtered to 80 aseptic μ m nylon wires, to remove large fragment of tissue.Again filtrate (containing cell) is transferred in another 15mL pipe to the centrifugal 5min of 1000rpm.Remove supernatant, use the primary sarcoplast growth medium of 2-4mL F10 resuspended precipitation, sarcoplast is inoculated in to diameter 35-60mm, in the coated culture dish of collagen protein, in 37 ℃, 5%CO
2incubator in cultivate.
5.4 sarcoplast differentiation
In the time that sarcoplast grows into 80% abundance, growth medium is changed by 95-98%DMEM into 2-5% horse serum, the division culture medium of 1% penicillin and Streptomycin sulphate composition.Change division culture medium every day and continue to cultivate sarcoplast, until myotube is induced to form.
Separating mouse skeletal myoblast going down to posterity exceedes after 5 times, is induced differentiation three days in 2% horse serum, and result as shown in Figure 3, can only form a small amount of myotube so, and in figure, scale line represents 40 microns.
If wild-type (WT) and MAML1 transgenosis type (TG) sarcoplast were gone down to posterity for twice after (being less than 4 times), after changing growth medium into division culture medium, induce sarcoplast differentiation five days, result as shown in Figure 4, can form more myotube, and in figure, scale line represents 40 microns.
If separate and cultivate skeletal myoblast from newborn mice, directly cell induced in 2% horse serum to differentiation three days and do not go down to posterity, almost each sarcoplast and satellite cell can break up and form myotube, and the quantity of the myotube forming is the more than 10 times of two kinds of methods above, and result as shown in Figure 5.WT represents wild-type, and TG represents MAML1 transgenosis type, and in figure, scale line represents 40 microns.
5.5Western?blot
After differentiation, use lysate (20mM Tris-Cl, 150mM NaCl, 10%Glycerol, 1%NP-40,0.4%NaF, 100mM Na3VO4,10 μ g/mL pepstatin, 10 μ g/mL leupeptin, 10 μ g/mL aprotinin, pH7.4) on ice, sarcoplast is carried out to cracking 30min.The centrifugal 10min of 10,000rpm.50 μ g total proteins, for SDS-PAGE electrophoresis, to polyvinylidene difluoride (PVDF) film, use antimyosin antibody reaction by protein delivery, and luminous with the colour developing of Pierce ECL reaction substrate, rinse X-ray photograph.
Myosin is the mark that muscle occurs.If will, from the sarcoplast induction differentiation of wild-type (WT) and MAML1 transgenosis type (TG) mouse three days, then be gathered into myocyte and myotube, to prepare total protein, and carry out myosin detection with Western blot and antibody, result is as shown in Figure 6.In the myotube forming the sarcoplast being separated with MAML1 transgenic mice by wild-type, MAML1 transgenosis sarcoplast can be expressed more myosin.Myosin expression amount at first day is maximum, and afterwards, expressing quantity starts to decline.The result consistent (Shen H, Genes Dev, 2006,20 (6): 675-688) that these results also obtain with the C2C12 clone of expressing MAML1.
Above-mentioned application example result demonstration, the mouse muscle-forming cell that the present invention obtains can form good myotube, reaches the requirement of muscle cell growth research and screening of medicaments.
Reference (References)
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Claims (2)
1. a preparation method for mouse muscle-forming cell, the method comprises the following steps:
A) by the just in vitro and mouse leg muscle that shreds by volume 1:2 add dispersion agent, put upside down shake 5-10min at 37 ℃, the centrifugal 5min of 1000rpm, removes supernatant, obtains fragment of tissue;
B) in fragment of tissue, 1:2 adds dispersion agent by volume, becomes starchiness at 37 ℃ of shake 5-10min that turn upside down, and 1:10 adds serum to stop enzymic digestion by volume;
C) filter the aseptic nylon wire of 80 μ m, remove large fragment of tissue, the centrifugal 5min of filtrate 1000rpm, removes supernatant, obtains precipitation;
D) precipitation is put in the coated culture dish of collagen protein, adds primary sarcoplast growth medium, at 37 ℃, in the incubator of 5%CO2, cultivates;
E) in the time that sarcoplast grows into 40-80% abundance, remove growth medium, add division culture medium, change division culture medium every day and continue to cultivate sarcoplast, until myotube is induced to form;
Wherein, dispersion agent composition is 0.2% collagenase and 0.05% Dispase, and surplus is PBS, pH=7.5-7.8; Primary sarcoplast growth medium is by 80%F10,19%FBS, 1% penicillin and Streptomycin sulphate, 2.5-5.0ng/mL Prostatropin bFGF composition; Division culture medium is by 94-97%DMEM, 2-5% horse serum, 1% penicillin and Streptomycin sulphate composition.
2. according to the preparation method in claim 1, the method comprises the following steps:
A) by the just in vitro and mouse leg muscle that shreds by volume 1:2 add dispersion agent, put upside down shake 5-10min at 37 ℃, the centrifugal 5min of 1000rpm, removes supernatant, obtains fragment of tissue;
B) in fragment of tissue, 1:2 adds dispersion agent by volume, becomes starchiness at 37 ℃ of shake 5-10min that turn upside down, and 1:10 adds serum to stop enzymic digestion by volume;
C) filter the aseptic nylon wire of 80 μ m, remove large fragment of tissue, the centrifugal 5min of filtrate 1000rpm, removes supernatant, obtains precipitation;
D) precipitation is put in the coated culture dish of collagen protein, adds primary sarcoplast growth medium, at 37 ℃, in the incubator of 5%CO2, cultivates;
E) in the time that sarcoplast grows into 80% abundance, remove growth medium, add division culture medium, change division culture medium every day and continue to cultivate sarcoplast, until myotube is induced to form;
Wherein, dispersion agent composition is 0.2% collagenase and 0.05% Dispase, and surplus is PBS, pH=7.8; Primary sarcoplast growth medium is by 80%F10,19%FBS, 1% penicillin and Streptomycin sulphate, and 5.0ng/mL Prostatropin bFGF composition; Division culture medium is 100% horse serum.
3) sarcoplast of claim 1 or 2 preparation research myotube form and muscle development cell, molecule mechanism aspect application.
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