CN103667184A - In vitro culturing method of goat skeletal muscle satellite cells - Google Patents
In vitro culturing method of goat skeletal muscle satellite cells Download PDFInfo
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- CN103667184A CN103667184A CN201310662855.6A CN201310662855A CN103667184A CN 103667184 A CN103667184 A CN 103667184A CN 201310662855 A CN201310662855 A CN 201310662855A CN 103667184 A CN103667184 A CN 103667184A
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Abstract
The invention belongs to the field of biotechnology, and especially relates to an in vitro culturing method of goat skeletal muscle satellite cells. The in vitro culturing method comprises following steps: (1) sampling; (2) digestion; (3) filtration; (4) primary culturing; and (5) induction culturing. According to the in vitro culturing method, goat skeletal muscle satellite cells can be quickly obtained via in vitro culturing, and are uniform in cellular morphology, and high in breeding vitality; and after differentiation induction, myotubes can be obtained via fusion of the goat skeletal muscle satellite cells. The in vitro culturing method of goat skeletal muscle satellite cells is simple in operation, is capable of reducing culturing cost greatly, possesses certain economic significance, and can be used for obtaining a large amount of cells with excellent subculturing stability. In vitro culturing of goat skeletal muscle satellite cells is capable of providing exploration on molecular mechanisms of muscle differentiation approaches and production of transgenic animals with improved muscle properties with abundant cell resources, and possesses theoretical values and application values in a certain degree.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of extracorporeal culturing method of goat skeletal muscle satellite cell.
Background technology
Sarcoplast in skeletal muscle tissue is to exist with the form of muscle satellite cell (satellite cell, SC); Experiment shows that the muscle satellite cell in skeletal muscle has self and becomes the abilities such as flesh, skeletonization, one-tenth fat, is also referred to as " muscle stem cell ", and this cell is to the directed a kind of precursor cell of sarcoplast system; Anathrepsis process is mainly completed by muscle satellite cell; Muscle satellite cell, between myofibrillar sarolemma and basilar membrane, is generally the flat monocyte of little fusiformis; When as impaired in skeletal muscle when muscle tissue is upset, satellite cell will be activated, and a large amount of divisions, propagation mutual fusion form regenerated muscle fibers; The satellite cell activating and damaged cytogamy are with repairing damage cell, or several sarcoplast fusion formation myotube, and then generate new myocyte.The activation of satellite cell, propagation and differentiation are also the important mechanisms that Skeletal Muscle Cell is loose, number increases and myofiber transforms.Muscle satellite cell Isolation and culture becomes the important technological platform of the molecular mechanism of research muscle differentiation pathway and character improvement, for improveing domestic animal meat quality and livestock industry production field, establishes Research foundation.
At present, the separation and Culture of goat muscle satellite cell yet there are no report, and the functional study of myofiber satellite cell has the important meaning to Goat Breeding, and the extracorporeal culturing method of setting up a set of complete goat myofiber satellite cell is very necessary.
Summary of the invention
The object of the invention is at present still without the defect of the extracorporeal culturing method of goat myofiber satellite cell, a kind of goat skeletal muscle satellite cell extracorporeal culturing method is provided, it can realize the amplification in vitro of goat skeletal muscle satellite cell, for further molecular biology research provides a kind of method basis.
For achieving the above object, the technical solution used in the present invention is: a kind of goat skeletal muscle satellite cell extracorporeal culturing method, comprises the steps:
(1) sampling: under aseptic condition, get goat longissimus dorsi muscle and put into ice chest, take back immediately laboratory;
(2) digestion: meat sample is shredded with scissors, add in the D-Hanks liquid that contains pancreatin and NTx enzyme, digest 60～120min under 37 ℃ of conditions, obtain cell suspending liquid;
(3) filter: filtration cell suspension, then centrifugal, gained cell precipitation is resuspended and centrifugal with D-Hanks liquid, then uses Growth of Cells nutrient solution re-suspended cell;
(4) first culture: the cell of suspension is inoculated in in the coated Tissue Culture Flask of poly-lysine, adopt differential attachment method culturing cell pp1 to pp6, by the cell cultures in pp6, to converging rate, be 95%, then adopting mass concentration is that 0.25% pancreatin goes down to posterity, and completes the Isolation and culture of goat muscle satellite cell;
(5) induction differentiation.
Further: in described step (2), D-Hanks liquid is by the NaCl of 8g, the Na of 0.6g
2hPO
4.2H
2the KCl of O, 4g, the KH of 0.6g
2pO
4, 3.5g NaHCO
3, the mycillin of 5g, the amphotericin B of 1.0g and 1000mL tri-distilled water form.
Further: in described step (2), in D-Hanks liquid, pancreatin content is 0.25g, and NTx enzyme content is 0.2g.
Further: in described step (3), filtering screen-aperture is 400 orders.
Further: described step (4) Growth of Cells nutrient solution is comprised of DMEM high glucose medium, the FBS of 20ml, the mycillin of the foetal calf serum of 10ml, 1g and the amphotericin B of 1g of 70ml, and pH value is 7.2.
Further: the ratio going down to posterity in described step (4) is 1: 1.
Further: described step (5) induction differentiation comprises the steps:
The high sugared nutrient solution of DMEM of A, employing 100 μ l is hatched respectively 5min to the PEI transfection reagent of the pEGFP-IRES-Myf6 plasmid of 4 μ g and 8 μ l, then mixes and hatches 15min, obtains PEI-plasmid composite;
B, get the goat muscle satellite cell of Isolation and culture and be cultured to that to converge rate be 75%, discard nutrient solution, with the high sugared nutrient solution of DMEM, clean goat muscle satellite cell twice, then add the high sugared nutrient solution of DMEM of 1.8ml, again PEI-plasmid composite be dropwise added drop-wise to goat muscle satellite cell surface and act on 4h, then be replaced by Growth of Cells nutrient solution A and cultivate 24h, then be replaced by Growth of Cells nutrient solution B, in temperature, be 37 ℃ and 5%CO is housed
2incubator in induction differentiation 48～72h, complete the induction differentiation of goat muscle satellite cell.
Further: in described step B, Growth of Cells nutrient solution A is comprised of DMEM high glucose medium, the FBS of 20ml, the mycillin of the foetal calf serum of 10ml, 1g and the amphotericin B of 1g of 70ml, and pH value is 7.2.
Further: in described step B, Growth of Cells nutrient solution B is comprised of the high sugared nutrient solution of DMEM of 98ml, the foetal calf serum of 2ml, and pH value is 7.2.
Useful technique effect of the present invention is:
(1) the inventive method can obtain goat skeletal muscle satellite cell by vitro culture fast, and institute's cultured cells form homogeneous is bred energeticly, has the myotube of being fused into and obtain ability after induction.The extracorporeal culturing method of goat muscle satellite cell of the present invention is not only easy and simple to handle, and has greatly saved cultivation cost, has certain economic implications, and can obtain the cell that a large amount of mitotic stabilities are good.
(2) vitro culture of goat muscle satellite cell can be produced abundant cell derived is provided for the transgenic animal of exploring the molecular mechanism of muscle differentiation pathway and improving Muscle Traits, has certain theory and using value.
Embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, rather than whole embodiment.Embodiment based in the present invention, those of ordinary skills, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment mono-
Goat skeletal muscle satellite cell extracorporeal culturing method, comprises the steps:
(1) sampling: under aseptic condition, get goat longissimus dorsi muscle and put into ice chest, take back immediately laboratory;
(2) digestion: meat sample is shredded to (1mm with scissors
2), add the D-Hanks(that contains 0.25g pancreatin and 0.2g NTx enzyme by the NaCl of 8g, the Na of 0.6g
2hPO
4.2H
2the KCl of O, 4g, the KH of 0.6g
2pO
4, 3.5g NaHCO
3, the mycillin of 5g, the amphotericin B of 1.0g and 1000mL tri-distilled water form) in liquid, under 37 ℃ of conditions, digest 60～120min, obtain cell suspending liquid;
(3) filter: utilizing sieve aperture is 400 object filter sieve filtration cell suspensions, then centrifugal, gained cell precipitation is resuspended and centrifugal with D-Hanks liquid, use again Growth of Cells nutrient solution (the DMEM high glucose medium of 70ml, the FBS of 20ml, the mycillin of the foetal calf serum of 10ml, 1g and the amphotericin B of 1g form, and pH value is 7.2) re-suspended cell;
(4) first culture: the cell of suspension is inoculated in in the coated Tissue Culture Flask of poly-lysine, adopt differential attachment method culturing cell pp1 to pp6, by the cell cultures in pp6, to converging rate, be 95%, then adopting mass concentration is that 0.25% pancreatin goes down to posterity (1: 1), completes the Isolation and culture of goat muscle satellite cell;
(5) induction differentiation
The high sugared nutrient solution of DMEM of A, employing 100 μ l is hatched respectively 5min to the PEI transfection reagent of the pEGFP-IRES-Myf6 plasmid of 4 μ g and 8 μ l, then mixes and hatches 15min, obtains PEI-plasmid composite;
B, get the goat muscle satellite cell of Isolation and culture and be cultured to that to converge rate be 75%, discard nutrient solution, with the high sugared nutrient solution of DMEM, clean goat muscle satellite cell twice, then add the high sugared nutrient solution of DMEM of 1.8ml, again PEI-plasmid composite be dropwise added drop-wise to goat muscle satellite cell surface and act on 4h, then be replaced by the DMEM high glucose medium of Growth of Cells nutrient solution A(70ml, the FBS of 20ml, the foetal calf serum of 10ml, the mycillin of 1g and the amphotericin B of 1g form, pH value is 7.2) cultivation 24h, be replaced by again the high sugared nutrient solution of Growth of Cells nutrient solution B(DMEM, the foetal calf serum of 2ml forms, pH value is 7.2), in temperature, be 37 ℃ and 5%CO is housed
2incubator in induction differentiation 48～72h, complete the induction differentiation of goat muscle satellite cell.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (9)
1. goat skeletal muscle satellite cell extracorporeal culturing method, is characterized in that, comprises the steps:
(1) sampling: under aseptic condition, get goat longissimus dorsi muscle and put into ice chest, take back immediately laboratory;
(2) digestion: meat sample is shredded with scissors, add the D-Hanks liquid that contains pancreatin and NTx enzyme, digest 60～120min under 37 ℃ of conditions, obtain cell suspending liquid;
(3) filter: filtration cell suspension, then centrifugal, gained cell precipitation is resuspended and centrifugal with D-Hanks liquid, then uses Growth of Cells nutrient solution re-suspended cell;
(4) first culture: the cell of suspension is inoculated in in the coated Tissue Culture Flask of poly-lysine, adopt differential attachment method culturing cell pp1 to pp6, by the cell cultures in pp6, to converging rate, be 95%, then adopting mass concentration is that 0.25% pancreatin goes down to posterity, and completes the Isolation and culture of goat muscle satellite cell;
(5) induction differentiation.
2. goat skeletal muscle satellite cell extracorporeal culturing method according to claim 1, is characterized in that: in described step (2), D-Hanks liquid is by the NaCl of 8g, the Na of 0.6g
2hPO
4.2H
2the KCl of O, 4g, the KH of 0.6g
2pO
4, 3.5g NaHCO
3, the mycillin of 5g, the amphotericin B of 1.0g and 1000mL tri-distilled water form.
3. goat skeletal muscle satellite cell extracorporeal culturing method according to claim 1, is characterized in that: in described step (2), in D-Hanks liquid, pancreatin content is 0.25g, and NTx enzyme content is 0.2g.
4. goat skeletal muscle satellite cell extracorporeal culturing method according to claim 1, is characterized in that: in described step (3), filtering screen-aperture is 400 orders.
5. goat skeletal muscle satellite cell extracorporeal culturing method according to claim 1, it is characterized in that: described step (3) Growth of Cells nutrient solution is comprised of DMEM high glucose medium, the FBS of 20ml, the mycillin of the foetal calf serum of 10ml, 1g and the amphotericin B of 1g of 70ml, and pH value is 7.2.
6. goat skeletal muscle satellite cell extracorporeal culturing method according to claim 1, is characterized in that: the ratio going down to posterity in described step (4) is 1: 1.
7. goat skeletal muscle satellite cell extracorporeal culturing method according to claim 1, is characterized in that: described step (5) induction differentiation comprises the steps:
The high sugared nutrient solution of DMEM of A, employing 100 μ l is hatched respectively 5min to the PEI transfection reagent of the pEGFP-IRES-Myf6 plasmid of 4 μ g and 8 μ l, then mixes and hatches 15min, obtains PEI-plasmid composite;
B, get the goat muscle satellite cell of Isolation and culture and be cultured to that to converge rate be 75%, discard nutrient solution, with the high sugared nutrient solution of DMEM, clean goat muscle satellite cell twice, then add the high sugared nutrient solution of DMEM of 1.8ml, again PEI-plasmid composite be dropwise added drop-wise to goat muscle satellite cell surface and act on 4h, then be replaced by Growth of Cells nutrient solution A and cultivate 24h, then be replaced by Growth of Cells nutrient solution B, in temperature, be 37 ℃ and 5%CO is housed
2incubator in induction differentiation 48～72h, complete the induction differentiation of goat muscle satellite cell.
8. goat skeletal muscle satellite cell extracorporeal culturing method according to claim 7, it is characterized in that: in described step B, Growth of Cells nutrient solution A is comprised of DMEM high glucose medium, the FBS of 20ml, the mycillin of the foetal calf serum of 10ml, 1g and the amphotericin B of 1g of 70ml, and pH value is 7.2.
9. goat skeletal muscle satellite cell extracorporeal culturing method according to claim 7, is characterized in that: in described step B, Growth of Cells nutrient solution B is comprised of the high sugared nutrient solution of DMEM of 98ml, the foetal calf serum of 2ml, and pH value is 7.2.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104237533A (en) * | 2014-09-23 | 2014-12-24 | 中国科学院东北地理与农业生态研究所 | Method for preliminarily screening transgenic ewe capable of breeding offspring |
| CN105200005A (en) * | 2014-08-13 | 2015-12-30 | 中国科学院海洋研究所 | Paralichthys olivaceus muscle satellite cell line establishing method, specific primer for identifying paralichthys olivaceus muscle satellite cell marker gene and application of specific primer |
| CN107475182A (en) * | 2017-08-30 | 2017-12-15 | 长沙学院 | The applications in the medium of grass carp FSRP 3 and grass carp muscle satellite cell special culture media |
| CN108048396A (en) * | 2017-12-29 | 2018-05-18 | 山西农业大学 | A kind of separation method of Sheep Muscle stem cell |
| CN110628766A (en) * | 2019-09-23 | 2019-12-31 | 中国农业科学院北京畜牧兽医研究所 | LncRNA coding gene related to sheep skeletal muscle development and application thereof |
| CN110760475A (en) * | 2019-12-04 | 2020-02-07 | 扬州大学 | Isolated culture method of chicken muscle satellite cells |
| KR20220132095A (en) * | 2021-03-22 | 2022-09-30 | 강원대학교산학협력단 | A simplified method for isolation, characterization, and culture optimization of bovine muscle satellite cells and use of the same |
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2013
- 2013-12-10 CN CN201310662855.6A patent/CN103667184A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105200005A (en) * | 2014-08-13 | 2015-12-30 | 中国科学院海洋研究所 | Paralichthys olivaceus muscle satellite cell line establishing method, specific primer for identifying paralichthys olivaceus muscle satellite cell marker gene and application of specific primer |
| CN104237533A (en) * | 2014-09-23 | 2014-12-24 | 中国科学院东北地理与农业生态研究所 | Method for preliminarily screening transgenic ewe capable of breeding offspring |
| CN107475182A (en) * | 2017-08-30 | 2017-12-15 | 长沙学院 | The applications in the medium of grass carp FSRP 3 and grass carp muscle satellite cell special culture media |
| CN108048396A (en) * | 2017-12-29 | 2018-05-18 | 山西农业大学 | A kind of separation method of Sheep Muscle stem cell |
| CN110628766A (en) * | 2019-09-23 | 2019-12-31 | 中国农业科学院北京畜牧兽医研究所 | LncRNA coding gene related to sheep skeletal muscle development and application thereof |
| CN110760475A (en) * | 2019-12-04 | 2020-02-07 | 扬州大学 | Isolated culture method of chicken muscle satellite cells |
| KR20220132095A (en) * | 2021-03-22 | 2022-09-30 | 강원대학교산학협력단 | A simplified method for isolation, characterization, and culture optimization of bovine muscle satellite cells and use of the same |
| KR102594006B1 (en) | 2021-03-22 | 2023-10-25 | 강원대학교 산학협력단 | A simplified method for isolation, characterization, and culture optimization of bovine muscle satellite cells and use of the same |
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Application publication date: 20140326 |