CN105647857A - Separation and purification method for skeletal muscle satellite cells in goats - Google Patents
Separation and purification method for skeletal muscle satellite cells in goats Download PDFInfo
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Abstract
The invention discloses a separation and purification method for skeletal muscle satellite cells in goats. The separation and purification method comprises separation of the skeletal muscle satellite cells in the goats and purification of the skeletal muscle satellite cells in the goats. According to the method, trypsin digestion and a tissue mass method are combined for separation of the skeletal muscle satellite cells, a differential adhesion method is used continuously for purification of the skeletal muscle satellite cells in the goats during cell passage and is used for purification of the skeletal muscle satellite cells in the goats during more than 5 successional generations in late passage process, and the skeletal muscle satellite cells with higher purity are obtained.
Description
Technical field
The present invention relates to the isolation and purification method of a kind of goat skeletal muscle satellite cell.
Background technology
There is abundant Goats Breeds resource in China, but due to the impact by breeding potential and growth performance, the cultivation of China goat also has very big room for promotion. Along with improving constantly of national life level, owing to Carnis Naemorhedi is nutritious, delicious meat, it is subject to the favor of increasing consumer.
First skeletal muscle satellite cell is found in 1961 by Mauro, it is a kind of Muscle-derived Stem Cells with propagation and differentiation capability between muscle cell membrane and basement membrane, it is further characterized by muscle satellite cell later and take part in growth and the reparation of skeletal muscle, there is the ability of oneself's increment and Multidirectional Differentiation, maintain stablizing of internal muscle stem cell pond, play an important role in the growth and regeneration of skeletal muscle. Since being found to have dryness, it is always up study hotspot, how to separate from animal skeletal muscle tissue and obtain some and the high skeletal muscle satellite cell of purity is the target that all scholars of research skeletal muscle satellite cell pursue. In recent years the separation method of skeletal muscle satellite cell system is in progress and is not as big, the present invention is by the optimization to goat skeletal muscle satellite cell system Isolation and culture technology, to obtaining high-quality skeletal muscle satellite cell, for the tool materials that the research offer of skeletal muscle is important. Separation and Culture for skeletal muscle satellite cell can help us to understand the growth and development process of skeletal muscle in depth. Us can also be helped simultaneously to be more fully understood that the pathogenesis of some diseases, in the production efficiency of improvement livestock products, provide reliable theories integration simultaneously.
The isolation technics development of skeletal muscle satellite cell is long-standing, was separated by Bischoff early than 1974 and to obtain. Skeletal muscle satellite cell has in succession been isolated afterwards in each species. The method separating skeletal muscle satellite cell at present mainly has two step enzyme methods and tissue block method, but cuts both ways. In recent years research shows, skeletal muscle satellite cell has unlimited competence for added value and belongs to stem cell, has Multidirectional Differentiation ability. The foundation of skeletal muscle satellite cell system contributes to understanding the growth course of muscle, provides theoretical foundation for Biology Breeding.
Summary of the invention
The technical problem to be solved in the present invention is to provide the isolation and purification method of a kind of goat skeletal muscle satellite cell.
In order to solve above-mentioned technical problem, the technical solution used in the present invention is, the isolation and purification method of goat skeletal muscle satellite cell comprises the following steps:
(1) gather goat muscular tissue, first sterilize with alcohol washes, then with putting in DMEM/F12 culture medium after normal saline flushing, within 12h, carry out the separation of skeletal muscle satellite cell;
(2) by the normal saline flushing muscular tissue containing penicillin and streptomycin, it is cut into the big piece of tissue of 0.5 �� 0.5cm;
(3) the big piece of tissue obtained is put in culture dish, add 0.25% trypsin-EDTA and digest, it is ensured that trypsin can not have excessive piece of tissue completely, under 4 DEG C of conditions, stand 12h, add proliferated culture medium and terminate digestion; Being transferred in the centrifuge tube of 50ml by the big piece of tissue in culture dish together with culture fluid afterwards, 1000r/min is centrifuged 8min, absorbs supernatant, more resuspended with enrichment culture liquid, is transferred in culture dish with aseptic straw by big piece of tissue and culture fluid;
(4) the big piece of tissue that step (3) obtains is cut into 0.5��1mm again3Small tissue blocks, add appropriate enrichment culture liquid, small tissue blocks is spread out equably in culture dish, spacing between each small tissue blocks is at 3mm, suck culture medium, after small tissue blocks is fully adsorbed in culture bottle, again culture bottle is turned whether observation small tissue blocks is fully adsorbed on culture bottle, it is slowly added into the culture fluid being preheated to 37 DEG C again, it is replaced in cell culture incubator and carries out original cuiture, every day is observation of cell form and growing state under inverted microscope, within every two days, changes culture fluid; When cell length to 80%��90% degree of converging, sucking old culture fluid in culture bottle, with the PBS cell being preheated to 37 DEG C, to remove remaining serum and piece of tissue and dead cell, repeating 2 times, thus obtaining unpurified goat skeletal muscle satellite cell;
(5) adopt differential attachment method to carry out the purification of Primary caprine skeletal muscle satellite cell: the non-purification attached cell DPBS first step (4) obtained washs 3 times, trypsinization 3min, add 2 times of volume enrichment culture liquid and terminate digestion; 1500r/min is centrifuged 7min, collects cell, adds enrichment culture liquid resuspended; By 5 �� 104��8 �� 104Individual being seeded to is coated with on the culture dish of gelatin in advance and cultivates 1h, the not adherent suspension cell renewed vaccination in upper strata is taken to new culture dish when there being a large amount of cell attachment, now adherent cell is mainly fibroblast, owing to the adherent speed of skeletal muscle satellite cell is slower, this step repeats 2��3 times, within the 4th day, changes liquid, removes hemocyte and not adherent dead cell, and observation of cell growing state, the hereafter continuous purification carrying out skeletal muscle satellite cell for 5 days.
As preferably, in step (2), the concentration of described penicillin is 500IU/ml, and the concentration of described streptomycin is 500 �� g/ml.
As preferably, in step (3), the composition of described proliferated culture medium is 80%DMEM/F12+10% hyclone+10% horse serum.
As preferably, in step (3), the 0.25% trypsin-EDTA adding 3ml digests.
The invention has the beneficial effects as follows: adopt trypsinization and tissue block method to combine and carry out the separation of skeletal muscle satellite cell, proceed differential attachment method when passage and carry out the purification of goat skeletal muscle satellite cell, and by more than 5 generations continuous in later stage succeeding generations continuing the purification adopting differential attachment method to carry out goat skeletal muscle satellite cell, obtain the skeletal muscle satellite cell that purity is higher.
Detailed description of the invention
The present embodiment is the isolation and purification method of a kind of goat skeletal muscle satellite cell, comprises the following steps:
1, the separation of goat skeletal muscle satellite cell
(1) first by sterilizations such as the laboratory table used in laboratory, equipment, preparing corresponding reagent, reagent includes containing dual anti-PBS, containing dual anti-normal saline, cell culture fluid etc.
(2) muscular tissue of in vitro Huanghuai Goats is gathered, first with the abundant cleaning and sterilizing of the ethanol of 75%, by the normal saline flushing muscular tissue containing penicillin and streptomycin, the wherein concentration of penicillin and streptomycin respectively 500IU/ml and 500 �� g/ml, then fat and connective tissue are removed, wash away bloodstain and the spot on surface as far as possible, be cut into the piece of tissue of 0.5 �� 0.5cm size, and require to carry out the separation of primary cell within two hours.
(3) after the tissue fetched being delivered to laboratory, first piece of tissue is placed in the ethanol of 75% and soaks 15 minutes, again with containing dual anti-normal saline flushing surface, finally spray surface with ethanol, then rinse histioid surface 3 times with containing dual anti-PBS, finally rinse one time with without dual anti-PBS.
(4) adequately disinfected muscular tissue is placed on the pallet of sterilization, sterilizing, delivers in the super-clean bench shining ultraviolet together with pallet. Under stereomicroscope, tear muscle package afterwards, and extrude gently with Glass rod, to discharge the part heteroproteose cells such as fibroblast.
(5) it is subsequently adding 0.25% appropriate pancreas enzyme-EDTA to digest, stand 12h under 4 DEG C of conditions, then with suction pipe, piece of tissue is transferred in the centrifuge tube of 50ml, the myofilament obtained is washed 2��3 times in containing dual anti-PBS, each 1��2min, sops up last cleaning mixture. Piece of tissue is cut into 1mm3The piece of tissue of size, add 2ml enrichment culture liquid, with aseptic straw, piece of tissue is transferred in the culture bottle processed, and spread out equably in culture bottle, the spacing between every fritter, at 5mm, is inverted after sucking culture medium, after piece of tissue is fully adsorbed in culture bottle, upset culture bottle, allows culture fluid not have piece of tissue, then is slowly added into the culture fluid 5ml of 37 DEG C of preheatings. It is replaced in 5%CO2Carrying out original cuiture in the saturated incubator of concentration, every day is observation of cell form and growing state under inverted microscope, within every two days, changes culture fluid. Wherein the composition of proliferated culture medium is 80%DMEM/F12+10% hyclone+10% horse serum.
(6) when cell length to 80%��90% degree of converging, suck old culture fluid in culture bottle, cell is cleaned with the DPBS of 37 DEG C of preheating, to remove remaining serum and piece of tissue and dead cell, repeat 2 times, the pancreas enzyme-EDTA 1ml of 0.25% is added in culture bottle, then culture bottle is placed in 37 DEG C of constant incubators and preheats 3min, take out culture bottle and be put in the digestion situation of basis of microscopic observation cell, treat cell rounding, come off, the enrichment culture liquid adding two volumes immediately terminates digestion, draw culture medium with suction pipe and repeatedly blow and beat the cell of bottle wall, the formation cell suspension so as to come off from bottle wall, going down to posterity of cell is carried out in the ratio of 1:2, put back to cell culture incubator to continue to cultivate.
2, the purification of goat skeletal muscle satellite cell
The purification of Primary caprine skeletal muscle satellite cell adopts differential attachment method, first after cell is cultivated 3 days, washs 3 times with DPBS, each 10��20s, trypsinization 3��4min, adds 2 times of volume enrichment culture liquid and terminates digestion; 1500 leave heart 7min, collect cell, and addition enrichment culture liquid is resuspended, by 5 �� 104Individual being seeded to is coated with on the culture dish of gelatin in advance and cultivates 1 hour, and we are by groping the shortest fibroblastic adherent time, it is determined that the Best Times of 1h. The not adherent suspension cell renewed vaccination in upper strata is taken to new culture dish when there being a large amount of cell attachment, now adherent cell is mainly fibroblast, owing to the ferrum wall speed of skeletal muscle satellite cell cell is slower, this step can repeat 2-3 time, within 4th day, change liquid, remove hemocyte and not adherent dead cell observation of cell growing state, the hereafter continuous purification carrying out cell for 5 days.
3, the going down to posterity of goat skeletal muscle satellite cell
Growth of Cells to 80%��90% converge time go down to posterity, discard the culture medium in culture dish, add the pancreas enzyme-EDTA adding 500 �� l0.25% after the PBS preheated washs 1��2 time to digest, basis of microscopic observation, when the culture medium adding 1ml after most of cell detachment immediately terminates digestion, being inoculated in new culture medium by 1:3,3d goes down to posterity once, hereafter in continuous 5 generations, all adopt differential attachment method to collect the not adherent cell of 20min, to remove fibroblast.
4, goat skeletal muscle satellite cell is frozen
During the degree of converging of the goat skeletal muscle satellite cell length to 90% after to be purified, carry out the frozen of goat skeletal muscle satellite cell. Firstly the need of preparation cells frozen storing liquid, 40%FBS+10%DMSO+50% culture fluid, the DPBS1ml being firstly added preheating cleans cell, add the pancreas enzyme-EDTA digestion 3��5min of 0.25%, terminating digestion when becoming round to most cells, 1200r/min is centrifuged 3min, careful Aspirate supernatant, then resuspended with frozen stock solution, resuspended cell is undertaken frozen by the amount of each cryopreservation tube 500 �� l. Cryopreservation tube is first put into-80 DEG C of refrigerator 4h, then transfers in liquid nitrogen container and preserve.
5, the qualification of goat skeletal muscle satellite cell
The qualification of goat skeletal muscle satellite cell uses three kinds of methods to carry out, including RT-PCR, immunofluorescence staining and carry out into flesh induction. First designing the amplimer of distinctive Pax7, Desmin, MyoD gene of skeletal muscle satellite cell, the total serum IgE of the skeletal muscle satellite cell of extraction purification when carrying out RT-PCR, then reverse transcription becomes cDNA, expands finally by regular-PCR; Utilize immunofluorescence staining: utilize the primary antibodie of Pax7 and Desmin to carry out; Becoming flesh induction system is that 2% horse serum+DMEM/F12 carries out into flesh induction, the method utilizing immunofluorescence dyeing after inducing 7 days, and MyoG and MyHC primary antibodie carries out dyeing and identifies myocyte. Result shows that the present invention can obtain the goat skeletal muscle satellite cell system that purity is higher.
Invention described above embodiment, is not intended that limiting the scope of the present invention. Any amendment, equivalent replacement and improvement etc. made within the spirit and principles in the present invention, should be included within the claims of the present invention.
Claims (4)
1. the isolation and purification method of goat skeletal muscle satellite cell, comprises the following steps:
(1) gather goat muscular tissue, first sterilize with alcohol washes, then with putting in DMEM/F12 culture medium after normal saline flushing, within 12h, carry out the separation of skeletal muscle satellite cell;
(2) by the normal saline flushing muscular tissue containing penicillin and streptomycin, it is cut into the big piece of tissue of 0.5 �� 0.5cm;
(3) the big piece of tissue obtained is put in culture dish, add 0.25% trypsin-EDTA and digest, it is ensured that trypsin can not have excessive piece of tissue completely, under 4 DEG C of conditions, stand 12h, add proliferated culture medium and terminate digestion; Being transferred in the centrifuge tube of 50ml by the big piece of tissue in culture dish together with culture fluid afterwards, 1000r/min is centrifuged 8min, absorbs supernatant, more resuspended with enrichment culture liquid, is transferred in culture dish with aseptic straw by big piece of tissue and culture fluid;
(4) the big piece of tissue that step (3) obtains is cut into 0.5��1mm again3Small tissue blocks, add appropriate enrichment culture liquid, small tissue blocks is spread out equably in culture dish, spacing between each small tissue blocks is at 3mm, suck culture medium, after small tissue blocks is fully adsorbed in culture bottle, again culture bottle is turned whether observation small tissue blocks is fully adsorbed on culture bottle, it is slowly added into the culture fluid being preheated to 37 DEG C again, it is replaced in cell culture incubator and carries out original cuiture, every day is observation of cell form and growing state under inverted microscope, within every two days, changes culture fluid;When cell length to 80%��90% degree of converging, sucking old culture fluid in culture bottle, with the PBS cell being preheated to 37 DEG C, to remove remaining serum and piece of tissue and dead cell, repeating 2 times, thus obtaining unpurified goat skeletal muscle satellite cell;
(5) adopt differential attachment method to carry out the purification of Primary caprine skeletal muscle satellite cell: the non-purification attached cell DPBS first step (4) obtained washs 3 times, trypsinization 3min, add 2 times of volume enrichment culture liquid and terminate digestion; 1500r/min is centrifuged 7min, collects cell, adds enrichment culture liquid resuspended; By 5 �� 104��8 �� 104Individual being seeded to is coated with on the culture dish of gelatin in advance and cultivates 1h, the not adherent suspension cell renewed vaccination in upper strata is taken to new culture dish when there being a large amount of cell attachment, now adherent cell is mainly fibroblast, owing to the adherent speed of skeletal muscle satellite cell is slower, this step repeats 2��3 times, within the 4th day, changes liquid, removes hemocyte and not adherent dead cell, and observation of cell growing state, the hereafter continuous purification carrying out skeletal muscle satellite cell for 5 days.
2. method according to claim 1, it is characterised in that in step (2), the concentration of described penicillin is 500IU/ml, and the concentration of described streptomycin is 500 �� g/ml.
3. method according to claim 1, it is characterised in that in step (3), the composition of described proliferated culture medium is 80%DMEM/F12+10% hyclone+10% horse serum.
4. method according to claim 1, it is characterised in that in step (3), the 0.25% trypsin-EDTA adding 3ml digests.
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Cited By (10)
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| CN106770736A (en) * | 2016-12-01 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of chicken Ractopamine detection method and kit |
| 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 |
| CN112011502A (en) * | 2020-09-09 | 2020-12-01 | 扬州大学 | Method for efficiently separating and purifying porcine skeletal muscle satellite cells |
| CN112094806A (en) * | 2020-08-28 | 2020-12-18 | 内蒙古农业大学 | Method for improving adherent culture growth speed of equine skeletal muscle satellite cells and cell oxidative stress culture model |
| CN113337460A (en) * | 2021-06-10 | 2021-09-03 | 呼和浩特职业学院 | Efficient separation and purification method of sheep skeletal muscle satellite cells |
| CN113832098A (en) * | 2021-09-23 | 2021-12-24 | 中国海洋大学 | In-vitro culture method and identification method of lateolabrax japonicus skeletal muscle satellite cells |
| CN114606182A (en) * | 2022-05-11 | 2022-06-10 | 中国农业科学院北京畜牧兽医研究所 | Passage purification method of sheep embryo-derived myoblasts |
| CN115181722A (en) * | 2022-08-30 | 2022-10-14 | 江苏农牧科技职业学院 | In-vitro separation and culture method of goose skeletal muscle satellite cells |
| CN116355840A (en) * | 2022-12-27 | 2023-06-30 | 宁夏大学 | Livestock skeletal muscle satellite cell culture method and auxiliary device special for same |
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Cited By (12)
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| CN106770736A (en) * | 2016-12-01 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of chicken Ractopamine detection method and kit |
| 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 |
| CN112094806A (en) * | 2020-08-28 | 2020-12-18 | 内蒙古农业大学 | Method for improving adherent culture growth speed of equine skeletal muscle satellite cells and cell oxidative stress culture model |
| CN112011502A (en) * | 2020-09-09 | 2020-12-01 | 扬州大学 | Method for efficiently separating and purifying porcine skeletal muscle satellite cells |
| CN113337460A (en) * | 2021-06-10 | 2021-09-03 | 呼和浩特职业学院 | Efficient separation and purification method of sheep skeletal muscle satellite cells |
| CN113832098A (en) * | 2021-09-23 | 2021-12-24 | 中国海洋大学 | In-vitro culture method and identification method of lateolabrax japonicus skeletal muscle satellite cells |
| CN114606182A (en) * | 2022-05-11 | 2022-06-10 | 中国农业科学院北京畜牧兽医研究所 | Passage purification method of sheep embryo-derived myoblasts |
| CN115181722A (en) * | 2022-08-30 | 2022-10-14 | 江苏农牧科技职业学院 | In-vitro separation and culture method of goose skeletal muscle satellite cells |
| CN115181722B (en) * | 2022-08-30 | 2024-06-11 | 江苏农牧科技职业学院 | In-vitro separation and culture method of goose skeletal muscle satellite cells |
| CN116355840A (en) * | 2022-12-27 | 2023-06-30 | 宁夏大学 | Livestock skeletal muscle satellite cell culture method and auxiliary device special for same |
| CN116355840B (en) * | 2022-12-27 | 2024-05-14 | 宁夏大学 | Livestock skeletal muscle satellite cell culture method and auxiliary device special for same |
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Application publication date: 20160608 |