CN115181722A - In-vitro separation and culture method of goose skeletal muscle satellite cells - Google Patents
In-vitro separation and culture method of goose skeletal muscle satellite cells Download PDFInfo
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Abstract
The invention discloses an in vitro separation and culture method of a goose skeletal muscle satellite cell, wherein the goose skeletal muscle satellite cell is prepared by in vitro separation, culture and purification; wherein, the separation of the goose skeletal muscle satellite cells comprises digestion, separation, culture of the satellite cells and differential adherent purification of the satellite cells. The goose skeletal muscle satellite cells are obtained by dispersing and digesting goose skeletal muscles by combined action of Dispase II and collagenase II for the first time, and filtering and separating erythrocyte cracking treatment. The differentiation potential of satellite cells was maintained during in vitro culture using DMEM/F12 growth complete medium containing 20% fbs and supplemented with bFGF, and the dishes involved in the culture were specially coated with matrigel. The goose skeletal muscle satellite cells with high differentiation ability obtained by the method can form mature myotubes through in vitro induced differentiation, and the differentiation index is up to more than 78%.
Description
Technical Field
The invention belongs to the technical field of biology and new medicines, and particularly relates to an in-vitro separation and culture method of a goose skeletal muscle satellite cell.
Background
With the improvement of living standard, the requirements of people on the quality of livestock meat are increasingly improved. Goose meat is an ideal nutritional food with high protein, low fat and low cholesterol, and is classified as one of the major health products developed in the 21 st century by food and agriculture organization of the united nations as early as 2002. In China, goose is highly favored by consumers. Therefore, the goose meat production performance variety improvement is developed, the functional gene of goose muscle growth is explored, the molecular mechanism of goose skeletal muscle growth and development regulation is explored, the research of goose muscle genetic breeding is developed, and the economic effect is great.
The acquisition of goose skeletal muscle satellite cells with high differentiation potential is the premise and the basis for developing the research on goose muscle tissue growth and development regulation mechanism. Skeletal muscle satellite cells are derived from a portion of the myoknives as muscle progenitor cells, located between the sarcolemma and the basement membrane. These cells remain undifferentiated in the muscle tissue of the postnatal individual and have a strong differentiation potential. As early as 1961, myogenic satellite cells were discovered by scientists by electron microscopy in the peripheral region of frog skeletal muscle fibers (Mauro, 1961). In 1974, bischoff et al first isolated satellite cells from adult rat skeletal muscle (Bischoff, 1974). Subsequently, satellite cells of different species, such as human (Blau and Webster, 1981), sheep (Dodson et al, 1986), chicken (Bennett et al, 1986), cattle (Dodson et al, 1987), etc., were gradually isolated by continuous optimization of the isolation procedure. However, few studies have been made on the isolation and culture of goose skeletal muscle satellite cells and the differentiation induction of goose skeletal muscle satellite cells, and the satellite cells isolated by the existing methods have a limited differentiation ability (Wang et al, 2021). Therefore, the research provides a novel method for separating goose skeletal muscle satellite cells in vitro, and experiments prove that the goose skeletal muscle satellite cells obtained by separation have high differentiation potential, can be used as an in vitro induced differentiation model of muscle tissues, and provides a research material for the research on growth and development regulation molecular mechanisms of the goose muscle tissues.
Disclosure of Invention
The invention aims to provide a goose skeletal muscle satellite cell with high differentiation potential and a separation and culture method thereof.
The purpose of the invention is realized by the following modes:
a method for in vitro isolation and culture of goose skeletal muscle satellite cells, comprising the following steps:
(1) Digestion: cutting the muscle tissue of the washed goose into a minced meat shape, and adding a digestive working solution for digestion for 50-60 min;
(2) Separation: adding high-glucose DMEM medium containing 3% fetal calf serum to stop digestion; after the digestive juice is fully dispersed, filtering the digestive juice by using a 100 mu m cell sieve and a 40 mu m cell sieve in sequence, centrifuging and removing the upper solution; resuspending the centrifuged cells by using erythrocyte lysate, gently blowing and beating the cells for multiple times, standing the cells for 2 to 5min, adding DPBS buffer solution, centrifuging the cells, discarding the upper solution, and collecting the precipitate;
(3) Culturing satellite cells: suspending and precipitating by using a skeletal muscle satellite cell growth complete culture medium, gently blowing and beating uniformly, transferring to a culture dish coated by matrix glue, and culturing primary satellite cells in an incubator by using a skeletal muscle satellite cell growth complete culture medium; adding part of new complete growth culture medium for the goose skeletal muscle satellite cells every two days without changing the liquid; in the primary culture process, part of satellite cells slowly adhere to the wall, and part of satellite cells are lost if liquid is changed;
(4) Differential adherent purification of satellite cells: the confluence degree of the primary satellite cells in a culture dish coated by matrigel reaches more than 90 percent within 5-7 days, and passage is carried out; digesting with pancreatin, collecting cells into a centrifuge tube, centrifuging, collecting precipitate, suspending the precipitate by using a goose skeletal muscle satellite cell growth complete culture medium, transferring the precipitate to a culture dish which is not coated with matrix glue, placing the culture dish into an incubator for standing for 45-60 min, absorbing an upper culture medium in the culture dish into a new culture dish which is coated with the matrix glue, mixing uniformly in a cross shape, and placing the culture dish into the incubator for continuous culture.
In the digestion working solution in the step (1), every 10mL of the digestion working solution contains 6.9mL of high-sugar DMEM, 1.5mL of collagenase II stock solution, 1.5mL of LDispase II stock solution and 0.1mL of penicillin streptomycin mixed solution. The preparation of the Dispase II stock solution is that 1g of Dispase II (Dispase II) is dissolved by 50mL of DPBS, and the solution is filtered by a filter with the diameter of 0.2 mu m after being fully dissolved and is stored at the temperature of minus 20 ℃; the collagenase II stock solution was prepared by dissolving 5g of collagenase type II in 125mL of DPBS, filtering the solution through a 0.2 μm filter after sufficient dissolution, and storing the solution at-20 ℃. After 1.5mL of stock solution is added, the working concentration of collagenase II in the final digestion working solution is not more than 500U/mL; after 1.5mL of the Dispase II stock solution was added, the working concentration of Dispase II in the final digestion working solution did not exceed 2.4U/mL.
The primary satellite cells are fragile and easy to damage, and are easy to be stimulated by external sources to enter differentiation fate determination, so that the obtained satellite cells have limited proliferation capacity, do not have differentiation potential, and cannot be used as in-vitro induced differentiation models. Collagenase II is effective in connective tissue in muscle tissue, and is effective in separating satellite cells from dense muscle tissue; dispaseII, also known as dispase, is a mild cell digestive enzyme with little damage to cells and is capable of maintaining the integrity of cell membranes. The invention adopts the combined action of DispaseII and collagenase II, and has obvious effect on separating complete satellite cells from compact goose skeletal muscle containing more connective tissues.
Particularly, collagenase II with the working concentration not more than 500U/mL and Dispase II with the working concentration not more than 2.4U/mL are jointly digested for 50-60 min, so that the muscle tissues can be almost completely dispersed and digested, after uniform mixing, the digestive fluid flows in a sand shape, obvious granular muscle residues cannot be seen, and the living cell walls of the satellite cells are complete and the activity can be furthest reserved through microscopic observation.
The goose muscle tissue obtaining method in the step (1) is to take goose leg muscle tissue and immediately place the goose leg muscle tissue into a 50mL centrifuge tube filled with a high-sugar DMEM culture medium.
And (2) soaking and cleaning the muscle tissue of the cleaned goose in DPBS for 3 times.
Every 1cm described in step (1) 3 The muscle tissue is digested by 5-10 mL of digestive working solution under the condition of shaking and digesting at 37 ℃.
The medium for complete growth of goose skeletal muscle satellite cells described in the step (3) contains 20% FBS (fetal bovine serum) and DMEM/F12 to which bFGF has been added. The preparation method of the goose skeletal muscle satellite cell growth complete culture medium comprises the following steps: to 39.5mL of DMEM/F12 medium, 10mL of FBS and 0.5mL of penicillin streptomycin mixture were added to obtain bFGF at a final concentration of 5ng/mL, and the mixture was mixed well.
The complete goose skeletal muscle satellite cell growth culture medium adopted by the invention has the following advantages:
1. only the basic fibroblast growth factor (bFGF) of the growth factor is added exogenously, and the working concentration of the bFGF is only 5ng/mL, the proliferation of satellite cells can be promoted, and the low-concentration bFGF can reduce the culture cost.
2. At present, few methods for culturing goose skeletal muscle satellite cells are available, and the differentiation capacity of the cultured goose skeletal muscle satellite cells is limited, which indicates that the growth medium does not well maintain the differentiation potential of the satellite cells. In the process of culturing satellite cells of other species, such as mouse skeletal muscle satellite cells, CEE chick embryo extract (chick embryo extract) is added, which significantly increases the culture cost. In the present invention, the high differentiation potential of goose skeletal muscle satellite cells was maintained by using 20% FBS and 5ng/mL final concentration of bFGF. Experiments also prove that the satellite cells cultured by the culture system can form mature large myotubes after induced differentiation. Moreover, compared with other culture media, the DMEM/F12 culture medium selected by the invention is low in price and can still maintain the differentiation potential of satellite cells.
The coating step of the culture dish coated by the matrigel in the step (3) is as follows: adding the matrigel coating solution into a culture dish, covering the bottom of the culture dish, placing the culture dish into an incubator, standing for more than 3min, and recovering the matrigel coating solution. The preparation of the matrigel coating solution adopted in the goose skeletal muscle satellite cell culture is as follows: the matrigel and high-sugar DMEM were mixed at a volume ratio of 1.
After the culture dish is coated by matrigel, the short-term adherence capacity of the satellite can be greatly increased, so that the survival and proliferation level of satellite cells can be improved. If the culture dish is not coated with matrigel, only a small amount of goose skeletal muscle satellite cells can adhere to the wall in a small amount, and the number of the satellite cells adhering to the wall in 24 hours is reduced by about 70 percent. Moreover, even with satellite cells that have already adhered to the wall, the cells that have undergone division and proliferation have the same problem, and are difficult to adhere to the wall, eventually resulting in a decrease in the survival rate of satellite cells and a significant decrease in the proliferation rate.
Placing into incubator, culturing at 37 deg.C, preferably 5% CO 2 。
The centrifugation conditions in the step (2) and the step (4) are 4 ℃ and 1500 Xg centrifugation is carried out for 5-10 min.
And (3) when the primary satellite cells are cultured in the step (2), the liquid is not changed, and part of new complete growth culture medium for the goose skeletal muscle satellite cells is added every two days.
The goose skeletal muscle satellite cells with high differentiation potential can be obtained by in vitro separation by the method.
The goose skeletal muscle satellite cells are applied to mature myotubes obtained through differentiation.
The experiments prove that goose skeletal muscle satellite cells are obtained by dispersing and digesting goose skeletal muscle under the combined action of Dispase II and collagenase II for the first time, and filtering and separating erythrocyte lysis treatment. The differentiation potential of satellite cells was maintained during in vitro culture using DMEM/F12 growth complete medium containing 20% FBS and added bFGF. And particularly emphasizes that culture dishes involved in the culture process need to be coated by matrigel, goose skeletal muscle satellite cells with high differentiation capacity can be obtained by the method, mature myotubes can be formed by in vitro induced differentiation, and the differentiation index is as high as more than 78%.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a novel method for separating and culturing goose skeletal muscle satellite cells. The goose skeletal muscle satellite cells with higher purity can be obtained by the separation method, and the culture system can maintain the high undifferentiated state of the satellite cells in the in-vitro culture process and keep the huge differentiation potential of the satellite cells.
Drawings
FIG. 1 is a diagram showing the isolation, culture and identification of goose skeletal muscle satellite cells.
FIG. 2 is a graph of morphological changes and immunofluorescence identification of goose skeletal muscle satellite cells in the process of induced differentiation.
FIG. 3 is a diagram showing the statistics of differentiation index and the variation of gene expression before and after the induction differentiation of goose skeletal muscle satellite cells.
Detailed Description
The invention is further illustrated by the following examples. Provides a method for in vitro isolation and culture of goose skeletal muscle satellite cells. Mainly comprises the contents of in vitro separation, purification and identification of goose skeletal muscle satellite cells, in vitro culture, in vitro induced differentiation, differentiation capability evaluation and the like.
Example 1
1. Isolation of goose skeletal muscle satellite cells
(1) And preparing a digestive working solution in advance. Wherein the preparation of the digestion working solution is as follows:
dispase II stock (10X) 1g of Dispase II (Coolaber, CD4691-1 g) was dissolved in 50mL of DPBS, filtered through a 0.2 μm filter after sufficient dissolution and stored at-20 ℃.
Collagenase II stock (10X) 5g of collagenase II (Sigma, C6885-5 g) was dissolved in 125mL of DPBS, and after sufficient dissolution was filtered through a 0.2 μm filter, it was stored at-20 ℃.
The digestion working solution comprises 6.9mL of high-sugar DMEM (Gibco, C11995500 BT), 1.5mL of collagenase II stock solution (the working concentration of collagenase II in the digestion working solution is not more than 500U/mL), 1.5mL of Dispase II stock solution (the working concentration of Dispase II in the digestion working solution is not more than 2.4U/mL) and 0.1mL of penicillin streptomycin mixed solution (Solarbio, P1400) in each 10mL of digestion working solution. The digestive working solution is prepared on site as used, and the prepared digestive working solution is kept on ice for later use.
(2) Goose leg muscle tissue was taken and immediately placed into a 50mL centrifuge tube containing high-sugar DMEM medium.
(3) The muscle tissue was transferred to a clean bench, washed 3 times with DPBS (Gibco, C14190500 BT) soak, minced with sterile scissors, and transferred to multiple 1.5mL centrifuge tubes. The muscle tissue in the centrifuge tube is continuously sheared into meat paste.
(4) Transferring the minced muscle tissue to digestive working solution (generally 1 cm) 3 The muscle tissue needs 5-10 mL of digestive working solution), and shaking and digesting for 60min on a shaker at 37 ℃.
(5) After completion of digestion, digestion was terminated by adding 10mL of high-sugar DMEM medium containing 3% FBS (Gemini, 900-108); repeatedly sucking the digestive juice with 20mL syringe, transferring to 100 μm cell sieve after fully dispersing, and filtering to obtain filtrate.
(6) The filtrate was filtered again through a 40 μm cell sieve. After filtration was complete, centrifugation at 1500 Xg was carried out for 5min at 4 ℃. The upper solution was discarded.
(7) Resuspending cells with 3mL of erythrocyte lysate (Solarbio, R1010), gently blowing and beating for multiple times, standing for 2min, adding 5mL of DPBS buffer solution, centrifuging at 4 ℃ and 1500g for 5min, discarding the supernatant, and collecting precipitate;
(8) The pellet contained a large number of isolated goose skeletal muscle satellite cells. Suspending and precipitating with 5mL of growth completion culture medium, gently blowing and beating uniformly, transferring into culture dish coated with matrigel, placing into incubator at 37 deg.C, and 5% CO 2 And (5) culturing. Generally, primary cells can grow over a culture dish in 5-7 days, liquid change is not needed in the culture process, and part of new complete culture medium for growth of the goose skeletal muscle satellite cells is added every two days.
2. Culture and purification of goose skeletal muscle satellite cells
Because goose skeletal muscle satellite cells have weak natural adherence capability, the culture dish needs to be coated with matrigel firstly in the culture process and then used for culturing the cells.
(1) Coating a culture dish: adding the matrigel coating solution into a culture dish, covering the bottom of the culture dish, placing the culture dish into an incubator, standing for more than 3min, and recovering the matrigel coating solution. The matrigel coating solution is prepared as follows:
matrigel coating solution: matrigel (BD Biosciences, 356231) and high-sugar DMEM were mixed at a volume ratio of 1.
(2) Culturing satellite cells: satellite cells were cultured in growth-complete medium. In the process of primary cell culture, in order to reduce cell loss, liquid does not need to be changed in the culture process, and part of new complete growth culture medium is added every two days. The preparation of the complete culture medium for goose skeletal muscle satellite cell growth comprises the following steps:
to 39.5mL of DMEM/F12 (Hyclone, SH30023.01B) medium was added 10mL of FBS (Gemini, 900-108), 0.5mL of penicillin streptomycin mixture (Solarbio, P1400) and bFGF (Recombinant Human FGF-basic, peproTech, 100-18B) at a final concentration of 5ng/mL, and the mixture was mixed and stored at 4 ℃ until the prepared medium was used within one week.
(3) Differential adherent purification of satellite cells: the confluence of the primary satellite cells in a culture dish coated by matrigel needs 5-7 days to reach more than 90%, and at the moment, passage is needed. Digesting with pancreatin, collecting cells into a 15mL centrifuge tube, centrifuging for 5min at 1500g, suspending and precipitating with growth complete culture medium, transferring to a 10cm culture dish which is not coated with matrigel, placing into an incubator, standing for 1h, allowing fibroblasts to adhere to the wall, allowing satellite cells to not adhere to the wall, sucking the upper medium layer of the 10cm culture dish into a new 10cm dish coated with matrigel, mixing uniformly in a cross manner, placing into the incubator at 37 ℃, and 5 percent CO 2 And (5) continuing normal culture until the confluence reaches 90 percent, and then passaging. And (4) separating in vitro to obtain goose skeletal muscle satellite cells with high differentiation potential.
Example 2
1. Isolation and identification of goose skeletal muscle satellite cells
1. Isolation of goose skeletal muscle satellite cells
Skeletal muscle of 10-week old \28294ugoose was collected, digested with Dispase II and collagenase II according to the method of example 1, isolated and purified by differential adherence to obtain skeletal muscle satellite cells. The isolated primary satellite cells were spherical and small in size (see fig. 1A, red arrow). Satellite cells were cultured in a complete growth medium for goose skeletal muscle satellite cells, and after 12h culture, the satellite cells began to adhere slowly to form a fusiform structure with needles at both ends expanded in the middle (see fig. 1A, yellow arrow, bar =20 μm). The satellite cells were fully adherent after 24h, in the shape of a spindle, and mass proliferation of satellite cells after adherence began (fig. 1b, bar =200 μm).
2. Identification of goose skeletal muscle satellite cells
PAX7 is a marker gene of satellite cells, and the purified primary satellite cells highly express PAX7 (FIG. 1C, bar =20 μm) through immunofluorescence identification, thereby showing that the obtained satellite cells have high purity and are in a highly undifferentiated state, and can be used for late stage differentiation research. In addition, after being cultured in a complete growth medium, RNA and protein of the cells are respectively extracted, and the expression levels of PAX7 mRNA and protein are detected by utilizing qRT-PCR and Western blotting, and the result shows that the cultured satellite cells still highly express PAX7, which indicates that the culture system can maintain the satellite cells in a higher undifferentiated state (FIGS. 3B and C).
2. In vitro induced differentiation of goose skeletal muscle satellite cells
And carrying out in-vitro induced differentiation on the goose skeletal muscle satellite cells obtained by separation and purification. When the degree of cell confluence reached 80% to 90%, the complete growth medium was replaced with the differentiation-inducing medium (DMEM high-glucose medium containing 2% hs). Morphological observation of satellite cells induced and differentiated for 0 to 6 days revealed that the cells began to elongate, enlarge and initially develop small myotubes 2 days after induction. After 4 days of induction, clear myotube formation began. Thereafter, these myotubes continued to fuse to form mature larger myotubes (fig. 2a, bar =200 μm). Continuous observation shows that the large myotube can be maintained for 0.5 to 1 day after being formed, and then gradually falls off due to the limitation of culture conditions.
Myosin heavy chain MyHC is a constitutive protein of muscle fibers. The mature myotubes were labeled with MyHC antibody (MF 20) and immunofluorescent staining showed that, at the late stage of differentiation, the myotubes fused with myotubes to form larger myotubes in length and breadth, which may contain nearly a hundred nuclei (fig. 2c, bar =200 μm). Further, it was statistically found that the differentiation index (number of nuclei in myotubes containing 2 or more nuclei/total number of nuclei) of the cells was as high as 78% or more (FIG. 3A, bar =200 μm). In addition, crystal violet staining also showed massive mature myotube production (fig. 2b, bar =200 μm). The results show that the satellite cells obtained by the separation method have the capacity of high differentiation in vitro and keep higher differentiation potential.
Respectively collecting cell samples before induced differentiation (GM) and after induced Differentiation (DM), extracting RNA, and detecting the expression levels of differentiation marker genes MYOD and MYOG through qRT-PCR. The results show that expression of MYOD, MYOG was both significantly increased after induced differentiation (fig. 3B). In addition, western blotting detection results also show that the PAX7 protein expression level is reduced and the MyHC (MF 20) protein expression level is obviously increased after induced differentiation (FIG. 3C). The satellite cells separated in the research have good differentiation potential and can be used as a cell in-vitro differentiation model of subsequent experiments.
In conclusion, the goose skeletal muscle satellite cells with higher purity can be obtained by the separation method, and the culture system can maintain the high undifferentiated state of the satellite cells in the in-vitro culture process and keep the huge differentiation potential of the satellite cells.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for in vitro separation and culture of goose skeletal muscle satellite cells, which comprises the following steps:
(1) Digestion: cutting the muscle tissue of the washed goose into a minced meat shape, and adding a digestive working solution for digestion for 50-60 min;
(2) Separation: adding high-glucose DMEM medium containing 3% fetal calf serum to stop digestion; after the digestive juice is fully dispersed, filtering the digestive juice by using a 100 mu m cell sieve and a 40 mu m cell sieve in sequence, centrifuging and removing the upper solution; resuspending the centrifuged cells with erythrocyte lysate, gently blowing and beating the cells for multiple times, standing the cells for 2 to 5min, adding DPBS buffer solution, centrifuging the cells, discarding the upper solution, and collecting the precipitate;
(3) Culturing satellite cells: suspending and precipitating by using a goose skeletal muscle satellite cell growth complete culture medium, gently blowing and beating uniformly, transferring to a culture dish coated by matrix glue, and culturing primary satellite cells in an incubator by using a goose skeletal muscle satellite cell growth complete culture medium;
(4) Differential adherent purification of satellite cells: the confluence of the primary satellite cells in a culture dish coated by matrigel reaches over 90 percent after 5 to 7 days, and passage is carried out; digesting with pancreatin, collecting cells into a centrifuge tube, centrifuging, collecting precipitates, suspending the precipitates by using a goose skeletal muscle satellite cell growth complete culture medium, transferring the precipitates to a culture dish which is not coated with matrix glue, placing the culture dish into an incubator for standing for 45-60 min, absorbing an upper culture medium in the culture dish into a new culture dish coated with the matrix glue, mixing uniformly in a cross shape, and placing the culture dish into the incubator for continuous culture.
2. The method for separating and culturing goose skeletal muscle satellite cells according to claim 1, wherein the working solution of step (1) comprises 6.9mL of high-sugar DMEM, 1.5mL of collagenase II stock, 1.5mL of Dispase II stock and 0.1mL of penicillin streptomycin mixture per 10mL of the working solution.
3. The method for isolating and culturing goose skeletal muscle satellite cells according to claim 2, wherein the solution of Dispase II is prepared by dissolving 1g of Dispase II in 50mL of DPBS, filtering the solution with a 0.2 μm filter after sufficient dissolution, and storing the solution at-20 ℃; the collagenase II stock was prepared by dissolving 5g of collagenase II in 125mL of DPBS, filtering the solution through a 0.2 μm filter after sufficient dissolution, and storing the solution at-20 ℃.
4. The method for isolating and culturing goose skeletal muscle satellite cells, according to claim 1, wherein the muscle tissue of the washed goose in step (1) is washed by soaking in DPBS for 3 times.
5. The method for isolating and culturing goose skeletal muscle satellite cells according to claim 1, wherein the concentration of the satellite in step (1) is 1cm 3 The muscle tissue is digested with 5-10 mL of working solution under the condition of shaking on a shaker at 37 ℃.
6. The method for isolating and culturing goose skeletal muscle satellite cells according to claim 1, wherein the goose skeletal muscle satellite cell growth completion medium of step (3) is DMEM/F12 containing 20% FBS and added bFGF.
7. The method for isolating and culturing goose skeletal muscle satellite cells according to claim 1, wherein the coating step of the culture dish coated with matrigel in step (3) is as follows: adding the matrigel coating solution into a culture dish, covering the bottom of the culture dish, placing the culture dish into an incubator, standing for more than 3min, and recovering the matrigel coating solution.
8. The method for separating and culturing goose skeletal muscle satellite cells according to claim 7, wherein the matrigel coating solution used in the goose skeletal muscle satellite cell culture is prepared as follows: the matrigel and high-sugar DMEM were mixed at a volume ratio of 1.
9. The method for isolating and culturing goose skeletal muscle satellite cells according to claim 1, wherein the centrifugation in step (2) and step (4) is performed at 1500 Xg at 4 ℃ for 5-10 min.
10. The method for isolating and culturing goose skeletal muscle satellite cells according to claim 1, wherein a portion of the culture medium for complete growth of goose skeletal muscle satellite cells is added every two days during the culturing of primary satellite cells in step (2).
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105854005A (en) * | 2016-04-29 | 2016-08-17 | 黑龙江八农垦大学 | New application of beta-N-acetylglucosaminyl transferase |
| CN112512595A (en) * | 2018-05-03 | 2021-03-16 | 哈佛学院校长同事会 | In vivo homology-directed repair in cardiac, skeletal muscle and muscle stem cells |
| CN114206327A (en) * | 2019-06-10 | 2022-03-18 | 华盛顿大学 | SWELL1-LRRC8 complex modulators |
-
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- 2022-08-30 CN CN202211057034.5A patent/CN115181722A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105854005A (en) * | 2016-04-29 | 2016-08-17 | 黑龙江八农垦大学 | New application of beta-N-acetylglucosaminyl transferase |
| CN112512595A (en) * | 2018-05-03 | 2021-03-16 | 哈佛学院校长同事会 | In vivo homology-directed repair in cardiac, skeletal muscle and muscle stem cells |
| CN114206327A (en) * | 2019-06-10 | 2022-03-18 | 华盛顿大学 | SWELL1-LRRC8 complex modulators |
Non-Patent Citations (5)
| Title |
|---|
| 余慕雪等: "胎鼠骨骼肌卫星细胞的原代培养、纯化与鉴定", 中国生育健康杂志, vol. 27, no. 4, pages 335 - 339 * |
| 张亚慧: "鹅CAPN3编码区克隆、表达及其在肌肉生长发育中的作用研究", 中国优秀硕士学位论文全文数据库 农业科技辑, no. 01, pages 050 - 377 * |
| 张力等: "鹅骨骼肌卫星细胞的分离培养与鉴定", 畜牧兽医学报, pages 1 - 11 * |
| 曾雪花: "CTRP3对鹅骨骼肌细胞成肌分化的调控作用", 中国优秀硕士学位论文全文数据库 农业科技辑, no. 01, pages 050 - 379 * |
| 陈从波等: "骨骼肌卫星细胞纯化及培养的实验研究", 实用医学杂志, vol. 22, no. 7, pages 760 - 762 * |
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