CN103667177A - Kit used for muscle fiber in vitro culturing - Google Patents
Kit used for muscle fiber in vitro culturing Download PDFInfo
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- CN103667177A CN103667177A CN201310663442.XA CN201310663442A CN103667177A CN 103667177 A CN103667177 A CN 103667177A CN 201310663442 A CN201310663442 A CN 201310663442A CN 103667177 A CN103667177 A CN 103667177A
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Abstract
The invention belongs to the field of biotechnology, and specifically relates to a kit used for muscle fiber in vitro culturing. The kit comprises a digestive enzyme, D-Hanks liquid, myocyte nutrient solution A, and myocyte nutrient solution B. The kit is capable of obtaining skeletal muscle muscle fiber satellite cells rapidly via in vitro culturing; morphology of the obtained cells is uniform, reproductive capacity is high, and after differentiation induction, myotubes can be obtained via fusion of the skeletal muscle fiber satellite cells; a method used for in vitro culturing of the skeletal muscle fiber satellite cells using the kit is simple in operation, is capable of reducing culturing cost greatly, possesses certain economic significance, and can be used for obtaining of a large amount of cells with excellent subculturing stability.
Description
Technical field
The present invention relates to biological technical field, is a kind of myofiber vitro culture test kit specifically.
Background technology
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.
Myocyte is the precursor cell of muscular tissue, easily cultivates, plasticity-is strong, has many important biomolecule characteristics of skeletal muscle, for the research of muscle satellite cell, becomes a popular domain in bioengineered tissue.The number of different ages, different muscle types Satellite cells is different, therefore draws materials very important for the former culture of muscle satellite cell; In general, the satellite cell content of young individuality is abundanter.Develop a kind of myofiber vitro culture test kit significant to myofibrillar research.
Summary of the invention
The object of this invention is to provide a kind of myofiber vitro culture test kit.
For achieving the above object, the technical solution used in the present invention is: a kind of myofiber vitro culture test kit, is characterized in that: comprise digestive ferment, D-Hanks liquid, myocyte's nutrient solution A and myocyte's nutrient solution B.
Further: described digestive ferment comprises pancreatin and NTx enzyme.
Further: described 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: described 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.
Further: described Growth of Cells nutrient solution B is comprised of the high sugared nutrient solution of DMEM of 98ml, the foetal calf serum of 2m.
Further: described Growth of Cells nutrient solution pH value is 7.2.
Useful technique effect of the present invention is: substratum of the present invention can obtain skeletal muscle fiber 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; Utilize test kit of the present invention to carry out the method for vitro culture muscle satellite cell not only easy and simple to handle, and greatly saved cultivation cost, there is certain economic implications, and can obtain the cell that a large amount of mitotic stabilities are good.
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-
Myofiber vitro culture test kit, comprises digestive ferment, D-Hanks liquid, myocyte's nutrient solution A and myocyte's nutrient solution B.
Wherein: digestive ferment comprises pancreatin and NTx enzyme.
Wherein: 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.
Wherein: 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.
Wherein: 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.
Utilize the method steps of culture medium culturing goat myofiber satellite cell of the present invention as follows:
(1) sampling: under aseptic condition, get goat longissimus dorsi muscle and put into ice chest, take back immediately laboratory;
(2) digestion: muscle sample is shredded with scissors, add the D-Hanks(D-Hanks liquid 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, with 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 Growth of Cells nutrient solution A(70ml, form, pH value is 7.2 again) 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 (6)
1. myofiber vitro culture test kit, is characterized in that: comprise digestive ferment, D-Hanks liquid, myocyte's nutrient solution A and myocyte's nutrient solution B.
2. myofiber vitro culture test kit according to claim 1, is characterized in that: described digestive ferment comprises pancreatin and NTx enzyme.
3. myofiber vitro culture test kit according to claim 1, is characterized in that: described 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.
4. myofiber vitro culture test kit according to claim 1, is characterized in that: described 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.
5. myofiber vitro culture test kit according to claim 1, is characterized in that: described Growth of Cells nutrient solution B is comprised of the high sugared nutrient solution of DMEM of 98ml, the foetal calf serum of 2ml.
6. according to myofiber vitro culture test kit described in claim 4 or 5, it is characterized in that: described Growth of Cells medium pH value is 7.2.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310663442.XA CN103667177A (en) | 2013-12-10 | 2013-12-10 | Kit used for muscle fiber in vitro culturing |
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| CN201310663442.XA CN103667177A (en) | 2013-12-10 | 2013-12-10 | Kit used for muscle fiber in vitro culturing |
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| CN103667177A true CN103667177A (en) | 2014-03-26 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255815A (en) * | 2015-10-29 | 2016-01-20 | 广州赛莱拉干细胞科技股份有限公司 | Fibroblast cleaning solution and preparation method thereof |
-
2013
- 2013-12-10 CN CN201310663442.XA patent/CN103667177A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255815A (en) * | 2015-10-29 | 2016-01-20 | 广州赛莱拉干细胞科技股份有限公司 | Fibroblast cleaning solution and preparation method thereof |
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Application publication date: 20140326 |