CN114606182A - Passage purification method of sheep embryo-derived myoblasts - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a passage purification method of sheep embryo-derived myoblasts. After the sheep embryo-derived myoblasts are obtained by a primary cell culture technology, a large amount of high-purity myoblasts are continuously obtained by an improved purification method in a passage process. By adopting the passage purification method provided by the invention, the cells are automatically differentiated to generate the myotube structure after being continuously cultured for 10 generations, the cells are continuously cultured for more than 35 generations at present, the growth state of the cells is good, and the cells have the characteristics of myoblasts after characteristic analysis and karyotype identification. The myoblast obtained by the invention has wide application range, can be used for gene screening and function verification, and has great application value and social and economic values.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a passage purification method of sheep embryo-derived myoblasts.
Background
Sheep are important livestock, and the growth and development of skeletal muscle directly influence the production performance of sheep. Myoblasts are precursor cells of skeletal muscle, derived from the mesoderm, have the ability to self-renew and regenerate muscle fibers, and are present in embryonic development and maturation stages. Skeletal myoblasts are fused with each other to form muscle fibers after undergoing growth and development processes such as proliferation, differentiation, migration and the like, and further form muscle tissues. Muscle is regulated by MRF family transcription factors, mainly including myoblast determinant (MYOD) and myogenic factor 5 (myoactor 5, MYF 5), Myogenin (MYOG) is a downstream factor of MYOD to activate a myoblast differentiation program, pairing box transcription factor 7 (Paired box 7, Pax 7) is a key regulatory factor for skeletal muscle development, growth and regeneration, and desmin (desmin) and the 4 regulatory factors are important markers of skeletal muscle myoblasts.
The karyotype analysis of the myoblast chromosome takes a cell chromosome entering metaphase as a research object, analyzes, compares, sorts and numbers the chromosome according to the characteristics of the chromosome length, the position of a centromere, the proportion of long and short arms, the existence of satellite and the like, and identifies the cell according to the variation condition of the chromosome structure and the number. The karyotyping analysis can provide important basis for the genetic classification of myoblasts, the genetic relationship between species, and the study of chromosome number and structural variation.
At present, the molecular mechanism of sheep muscle growth and development is not clear, and the related research is greatly limited due to the lack of necessary sheep myoblast cell lines.
Disclosure of Invention
The invention aims to provide myoblasts which are high in purity, have high gene retention and have normal chromosome morphology after multiple passages.
In the first aspect, the invention provides a passage purification method of sheep myoblasts, wherein in the passage process, purification is carried out once every 4-6 generations in the passage process, and the purification time is 20-24 h; more specifically, the invention provides a passage purification method, wherein purification is carried out once every 5 passages, and the time of each purification is 24 h.
Namely, in the passage purification method provided by the invention, differential adherent purification is carried out once every 5 generations.
The myoblasts have self-renewal capacity and proliferation potential, and can be isolated and cultured in vitro by using primary and subculture cell culture technology to obtain stable and sustainable-passage sheep myoblasts.
The prior art primary and subculture cell culture techniques can only sustain continuous culture in vitro for a limited number of passages. The invention develops a sheep primary skeletal muscle myoblast which can be passaged for a plurality of times for the first time, continuously obtains high-purity myoblasts with higher gene retention and normal chromosome morphology by using an improved purification method, is suitable for developing researches such as molecular mechanism and the like, and has higher social and economic values and scientific research values.
In the prior art, when myoblast is cultured, as myoblast and fibroblast can not be completely separated, the situation that myoblast and fibroblast are cultured at the same time can be inevitable. In particular, in order to obtain myoblasts with strong differentiation potential, myoblasts from embryos are selected, and although the myoblasts from embryos have better differentiation potential, the myoblasts from embryos are very difficult to collect from sheep embryonic skeletal muscles, and a certain proportion of fibroblasts are collected.
Therefore, in the process of passage of the myoblasts, how to remove the fibroblasts and reduce the inhibition effect of the fibroblasts on the growth of the myoblasts becomes a key step for determining the culture purity and passage frequency of the myoblasts.
The differential adherence method adopted by the invention is a common purification method in the primary culture process, but is rare in the passage process. Meanwhile, the purification time and passage interval before the culture dish is replaced by the differential adherence method are the key points for the success of the method, adherent fibroblasts cannot be effectively removed if the purification time is too short or the passage interval is too long, and myoblasts are lost if the purification time is too long or the passage interval is too short. The traditional differential adherent purification method mostly adopts culture time of 2-4 h. The invention adopts an improved method of purifying every 5 generations for 1 time and purifying and culturing for 24 hours every time, and can obtain more pure myoblasts with more cell amount as far as possible.
In a second aspect, the present invention provides a method for culturing a ovine myoblast, which self-differentiates to generate a myotube structure after continuous culture for 10 generations, the method comprising: adopting sheep embryo myoblast to carry out primary culture, carrying out digestion passage when the adherent cell proportion reaches 75-80%, carrying out differential adherent purification once every 5 generations in the passage process, wherein the purification time is 24h each time.
In one embodiment of the present invention, the present invention provides a culture method comprising:
(1) sterilizing sheep embryo skeletal muscle tissue, soaking in PBS buffer solution containing double antibodies, removing sarcolemma and fibrous tissue, and then cutting muscle tissue into pieces;
(2) adding collagenase type I into the muscle tissue cut in the step (1) for digestion, centrifuging to remove supernatant, adding DMEM/F12 into the muscle tissue, and carrying out full-culture basic suspension precipitation to obtain a mixed solution;
(3) grinding and filtering the mixed solution obtained in the step (2) by using a cell sieve, standing, removing the upper layer of single cell suspension, keeping the lower layer of precipitate, repeatedly adding a whole culture medium for resuspension, standing to remove the single cell suspension and impurities to the maximum extent, resuspending the finally obtained precipitate by using a DMEM/F12 culture medium containing fetal calf serum, and lightly blowing and uniformly to obtain a precipitate suspension;
(4) inoculating the uniformly mixed precipitate suspension in the step (3) to a cell culture dish, and then placing the cell culture dish in an incubator for culturing for 5d-7 d;
(5) and when the proportion of adherent cells reaches 75-80%, carrying out digestion passage, purifying and removing fibroblasts by using a differential adherence method every 5 generations in the passage process, wherein the purification time is 24h, and transferring the supernatant to a new culture dish for culture after purification.
In the culture method provided by the invention, the sheep embryo skeletal muscle tissue in the step (1) is derived from a 72-day-old embryo of Hu sheep.
In the culture method provided by the invention, in the step (2), the concentration of the collagenase type I used is 0.1%.
In the culture method provided by the invention, in the step (3), the fetal bovine serum is 10% of fetal bovine serum.
When the prior art carries out myoblast in-vitro culture, the cultured cells can spontaneously form an infinite cell line in the continuous culture process; or the cultured cells are chemically or virally induced to form an unlimited cell line with cancerous properties. Therefore, the prior art can not achieve the normal cell state after 30 passages by culturing the sheep myoblasts. However, the cultured cells have stable cell morphology and proliferation level after 30 passages, have good gene retention amount and can be normally passaged.
In the prior art, horse serum is generally adopted to induce myotube differentiation, and low-concentration horse serum has stronger induced differentiation. The horse serum does not induce differentiation, and after the subculture method provided by the invention is adopted to continuously culture the myoblasts to 10 generations, a large number of myotubes appear due to the extremely high purity of the obtained myoblasts.
According to the understanding of the skilled person, the expression level of mRNA of the myoblast specific gene can be increased by using the subculture purification method or the culture method provided by the present invention.
Specifically, in the application provided by the invention, the myoblast specific gene isPax7Gene, gene,MYF5Gene, gene,MYODGene, gene,MYOGGenes anddesmina gene.
In the third aspect, it was found during the experiment of the present invention,MYF5the up-regulation of the expression level is related to myotube formation, andMYF5the expression quantity is greatly different from the expression quantity of other 4 genes in different growth and development stages of myoblasts. Previous studies have considered myotubes to be characterized by the expression of myosin heavy chain (MyHC), however, experimental results of the present invention have shown thatMYF5The expression of the gene may alternatively be a characteristic marker specific to the new myotube.
The invention therefore also claimsMYF5The expression quantity of the gene or the coded protein thereof is applied to judging the myotube formation of the myoblast in vitro culture.
The invention has the beneficial effects that:
(1) the invention can obtain more pure myoblasts with more cell amount as far as possible by optimizing a passage purification method, adopting an improved method of purifying every 5 generations for 1 time and carrying out purification culture for 24 hours each time.
(2) The method does not have the step of induced differentiation, and by adopting the subculture method provided by the invention, the obtained myoblasts have extremely high purity, and after continuous culture for 10 generations, self-differentiation occurs to form a large number of myotube structures.
(3) The myoblast obtained by the passage purification method provided by the invention has stable cell morphology and proliferation level after passage for 30 times, has good gene retention amount and can be normally passed. The prolonged culture time provides a longer experimental period for subsequent mechanism research on the cell level, and provides a richer cell population for the experiment. Is an important experimental material and a research platform for partial experiments before acquiring the sheep immortalized cell line.
(4) The method for culturing the sheep myoblasts can obtain the myoblasts with high purity, higher gene retention and normal chromosome morphology, is suitable for developing researches such as molecular mechanism and the like, and has higher social and economic values and scientific research values.
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FIG. 1 shows the results of myoblast counts in sheep subjected to serial passages, wherein the abscissa "X" represents the number of passages.
FIG. 2 shows the results of myoblast observation of different generations of sheep, where "X1" is the myoblast generation 1, "X10" is the myoblast generation 10, "X15" is the myoblast generation 15, and "X30" is the myoblast generation 30.
FIG. 3 shows the mRNA expression levels of myoblast-specific genes of different generations of sheep myoblasts and the C2C12 mouse myoblast cell line, wherein the letters a, b, C and d are different and significant (P < 0.05), and the letters are the same and significant (P > 0.05).
FIG. 4 shows the karyotype analysis results of myoblasts from different generations of sheep, where "X1" is the myoblast of generation 1 and "X15" is the myoblast of generation 15.
FIG. 5 is a diagram of the results of conventional purification method, phase contrast microscopy observation of cultured Hu sheep embryo-derived skeletal muscle myoblasts for 24 h.
FIG. 6 is a phase contrast microscope observation result diagram of the purified Hu sheep embryo-derived skeletal muscle myoblasts after 24h culture.
FIG. 7 is a diagram showing the results of conventional purification method, phase contrast microscope observation of cultured Hu sheep embryo-derived skeletal muscle myoblasts for 72 h.
FIG. 8 is a diagram of the observation results of the purified Hu sheep embryo-derived skeletal muscle myoblasts under a phase contrast microscope after being cultured for 72 h.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
Unless otherwise specified, the experimental materials, reagents, instruments and the like used in the examples of the present invention are commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.
Example 1 Hu sheep 72 day old embryo myoblast continuous culture
The embodiment provides a method for continuously culturing Hu sheep 72-day-old embryonic myoblasts, which comprises the following specific steps of:
1. disinfecting collected sheep embryo skeletal muscle tissue, soaking in PBS buffer solution containing double antibodies, removing sarcolemma and fibrous tissue, shearing muscle tissue and rinsing;
2. adding type I collagenase with the concentration of 0.1% for digestion, centrifuging the type I collagenase mixed solution containing muscle tissues after digestion is finished, removing supernatant, and adding DMEM/F12 full culture medium for resuspension;
3. grinding and filtering the re-suspended mixed solution by using a 40-mesh cell sieve, standing, removing the upper layer of single cell suspension, retaining the lower layer of sediment, repeatedly adding a whole culture medium for re-suspension, standing to remove the single cell suspension and impurities to the maximum extent, re-suspending the finally obtained sediment by using a DMEM/F12 culture medium containing 10% fetal calf serum, and lightly blowing and uniformly beating;
4. inoculating the uniformly mixed precipitate suspension to a cell culture dish, and then placing the cell culture dish in an incubator for culturing for 5d-7 d;
5. when the proportion of adherent cells reaches 80%, the cells can be digested and passaged, and the fibroblasts are purified and removed by using a differential adherence method every 5 generations in the passage process, namely, the cells after passage are cultured in an incubator for 24 hours, and then the supernatant is transferred to a new culture dish for culture.
The invention adopts the Hu sheep 72-day-old embryonic myoblasts for continuous culture, and the embryonic myoblasts are taken as a pluripotent stem cell and have the advantages of large proliferation and differentiation potential, easy culture and the like.The sheep myoblast can be continuously cultured for 30 generations in limited generations, and the cell yield can reach 2 multiplied by 10 at the early stage in the whole generation process7The cell count per mL shows a slow decline with the increase of the number of passages, and the cell count can still reach the passage threshold after 30 passages (FIG. 1).
The cells are clear in form and typical in fibrous form at the early stage of passage, the cells are regularly arranged after being flaked, and the confluence rate reaches over 90 percent. After continuous culture for 10 generations, a large number of myotubes appeared and the refractive index of the cells was enhanced, and the morphology of the cells remained in a fibrous arrangement until 30 generations (FIG. 2). The results show that the myoblasts obtained by the culture method can be continuously cultured for more than 30 generations, the cell morphology at the later stage is normal, the cell quantity can reach the passage threshold value, and the myoblasts can be normally cultured within limited generations and used for subsequent molecular mechanism exploration.
The reason why a large number of myotubes are differentiated by themselves after continuous culture up to 10 generations in the present invention is presumed to be: on the premise of extremely high purity of myoblasts, the concentration of fetal calf serum in the culture medium is gradually reduced along with the increase of the culture time, and the fetal calf serum with lower concentration can have a certain induced differentiation effect. Therefore, the self-differentiation of myotubes resulted in a large number of myotubes, and it was confirmed that the passage purification method used in the present invention enables to obtain myoblasts with extremely high purity.
Example 2 mRNA expression level of specific Gene of myoblast
This example examined the mRNA expression level of the myoblast-specific gene obtained in example 1.
MYF5、MYOD、MYOG 3Each transcription factor belongs to the myogenic regulatory factor family (MRF), which is considered a myogenic determinant that directs progenitor cells to establish skeletal muscle and activate the myogenic differentiation program. Desmin, a species of intermediate filament protein, is involved in the construction of the muscle tissue cytoskeleton. The Pax7 regulatory factor is involved in regulating myoblast proliferation and growth during the embryonic period. Significant difference exists in mRNA expression quantity of different generation submuscular cell specific genes (P)<0.05). WhereinPax7、MYOD、MYOGAnddesminthe gene expression amount is obviously reduced along with the increase of the passage number, and X10 myoblastsMYF5The gene expression level is significantly higher than that of X1, X15 and X30, and the expression levels of 5 specific genes in X1, X10, X15 and X30 myoblasts are all higher than that of C2C12 mouse myoblast cell line (FIG. 3). Shows that the number proportion and the proliferation and differentiation capacity of the myoblasts are gradually reduced in the continuous passage process, but the myoblasts cultured to 30 generations still have good gene retention. The gene retention was slightly higher than that of C2C12 mouse myoblast cell line, although lower than that of the prophase cells. The cells obtained by primary culture are confirmed to be myoblasts, and the cells have myoblasts in 30 generations and can be normally proliferated and passaged for subsequent mechanism research experiments.
Example 3 myoblast morphological characterization by multiple passages
Myotubes are special structures formed by high differentiation of myoblasts, are also called multinucleated muscle fibers and are formed by contacting and fusing a plurality of myoblasts. Under-mirror observation results and morphological characteristics show that the X10 myoblasts have obvious myotube structures different from other generations (figure 2), and the X10 myoblasts are analyzed by combining fluorescence quantitative resultsMYF5The gene expression level was significantly higher than other generations including X1 (fig. 3). Show thatMYF5The gene expression level is closely related to myotube formation. During the proliferation and differentiation of myoblast, the expression level of myoblast marker gene is down regulated with the generation increase, and once myotube is formedMYF5The gene expression level can be obviously up-regulated without being influenced by the generation increase. Furthermore, all of the 5 genes are closely related to the proliferation and differentiation of skeletal muscle, butMYOD、MYOG、desminAndPax7the gene expression level was down-regulated at passage 10, probably because other specific gene expression was suppressed at the end of myoblast differentiation to prevent unwanted gene overexpression affecting myotube formation.
In conclusion, the expression level of MYF5 is up-regulated and related to myotube formation, and the expression level of MYF5 is greatly different from the expression levels of other 4 genes in different growth and development stages of myoblasts. The myotube is considered to be characterized by expression of myosin heavy chain (MyHC) in previous researches, and the experimental result of the invention shows that MYF5 can be a characteristic marker specific to the new myotube.
Example 4 karyotyping of myoblasts by multiple passages
The 1 st generation and 15 th generation sheep myoblasts are diploid karyotypes detected by G banding chromosome karyotype analysis (figure 4), the chromosome number is 2n =54, no other karyotypes exist, the chromosome morphology, structure and number are normal, the sheep chromosome biological characteristics are met, and no chromosome mutation occurs. The shapes, structures and numbers of the chromosomes of the two generations of cells are kept consistent, which shows that the genetic characteristics of the cultured sheep myoblasts are stable and can be used for subsequent gene function verification and molecular mechanism research.
The prolonged culture time provides a longer experimental period for subsequent mechanism research on the cell level, and provides a richer cell population for the experiment. Is an important experimental material and a research platform for partial experiments before acquiring the sheep immortalized cell line. In conclusion, the cells cultured by the invention can be used as experimental materials in the 30 generations and applied to scientific research and other fields.
Comparative example 1 Observation under the microscope for different purification times and interval culture to passage 10
This comparative example provides a conventional purification method, the same as example 1 except that the conventional purification method in this comparative example uses purification time without intervals, purification is performed for each generation, and the purification time is 2 to 3 hours. This purification method is a method conventionally used in the art, and the principle of this invention is to obtain myoblasts of higher purity by a plurality of short-time purifications, whereas the principle of this invention is to obtain myoblasts of higher purity more easily by a plurality of long-time purifications.
When the purification time is 2-3 h, fibroblasts cannot be effectively removed due to too short purification time in each generation. Although the number of purification times is increased, myoblasts of higher purity cannot be obtained. When the cells are cultured for 24 hours, more adherent cells can be observed in the conventional purification method, but when the cells are cultured for 72 hours, the cells can not be self-differentiated to form myotubes. It shows that there are more fibroblasts doped with the same, but the purity is lower although the amount of the cells is larger.
When the purification time is 24h, and the purification is carried out every 5 generations, the purification time is long, the fibroblasts can be effectively removed, so the cell amount is less when the cells are cultured for 24h, but obvious myotube structures can be observed when the cells are cultured for 72 h, which shows that the purity is higher, and the purification interval is longer, so the loss of the cell amount and the cell activity caused by the purification on the myoblasts can be effectively reduced.
The results of observation under a phase contrast microscope after culturing the Hu sheep embryo-derived skeletal muscle myoblasts purified by the conventional purification method provided by the comparative example for 24 hours are shown in FIG. 5. The purification method provided by the embodiment 1 of the invention is used for observing the results under a phase contrast microscope after the Hu sheep embryo-derived skeletal muscle myoblasts are cultured for 24 hours after purification, and the results are shown in figure 6.
The results of observation under a phase contrast microscope after culturing the Hu sheep embryo-derived skeletal muscle myoblasts purified by the conventional purification method provided by the comparative example for 72 hours are shown in FIG. 7. The comparative example also provides the results of observation under a phase contrast microscope after culturing the Hu sheep embryo-derived skeletal muscle myoblasts for 72 h by the purification method provided in example 1 of the invention, and the results are shown in FIG. 8.
The results of this comparative example show that: the cells purified by the conventional purification method have a high proliferation speed in the initial stage, but cannot be automatically differentiated to form myotubes in the later stage. Although the number of the adherent cells at the early stage is small, the purity of the myoblasts is high, and myotubes can be formed automatically at the later stage.
Myoblasts are precursor cells of skeletal muscle, are derived from muscle-derived stem cells of mesoderm, have self-renewal and muscle fiber regeneration capabilities, are mainly present in embryos and mature muscle tissues, are proliferated and differentiated in the embryos to be fused to form multinuclear muscle fibers, namely myotubes, and are fused to form hollow tubular structures, so that cell nuclei move to the edges of the myotubes and are difficult to observe. Has cell polarity, irregular distribution and no directionality.
The results of this comparative example show that: the proportion of myoblasts in the cells purified by the conventional purification method is low, and the cells are difficult to differentiate by self to form a myoblast specific myotube structure; the cells purified by the purification method of the invention have higher proportion of myoblasts and are easy to differentiate to form myoblast specific myotube structures.
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 passage purification method of sheep myoblasts is characterized in that purification is carried out every 4-6 generations in the passage process, and the purification time is 20-24 h.
2. The method of subculture purification according to claim 1, wherein differential adherent purification is performed every 5 passages.
3. A method for culturing sheep myoblasts, which are self-differentiated to generate myotube structures after the sheep myoblasts are continuously cultured for 10 generations, and the method comprises the following steps: performing primary culture by adopting sheep embryo myoblasts, performing digestion passage when the proportion of adherent cells reaches 75-80%, and performing differential adherent purification once every 5 generations in the passage, wherein the purification time is 24 h.
4. The culture method according to claim 3, comprising:
(1) disinfecting sheep embryo skeletal muscle tissues, soaking the sheep embryo skeletal muscle tissues in a PBS (phosphate buffer solution) containing double antibodies, removing sarcolemma and fibrous tissues, and shearing the muscle tissues;
(2) adding collagenase type I into the muscle tissue cut in the step (1) for digestion, centrifuging to remove supernatant, adding DMEM/F12 into the muscle tissue, and carrying out full-culture basic suspension precipitation to obtain a mixed solution;
(3) grinding and filtering the mixed solution obtained in the step (2) by using a cell sieve, standing, removing the upper layer of single cell suspension, keeping the lower layer of precipitate, repeatedly adding a whole culture medium for resuspension, standing to remove the single cell suspension and impurities to the maximum extent, resuspending the finally obtained precipitate by using a DMEM/F12 culture medium containing fetal calf serum, and lightly blowing and uniformly to obtain a precipitate suspension;
(4) uniformly mixing the precipitate suspension obtained in the step (3), inoculating the mixture to a cell culture dish, and then placing the cell culture dish in an incubator for culturing for 5d-7 d;
(5) and when the ratio of the adherent cells in the cell culture dish reaches 75-80%, carrying out digestion passage, purifying every 5 generations in the passage by using a differential adherence method to remove fibroblasts, wherein the purification time is 24h, and transferring the supernatant to a new culture dish for culture after purification.
5. The culture method according to claim 4, wherein the ovine embryonic skeletal muscle tissue in step (1) is derived from a 72-day-old embryo of Hu sheep.
6. The culture method according to claim 4, wherein the collagenase type I is used in the step (2) at a concentration of 0.1%.
7. The culture method according to claim 4, wherein in the step (3), the fetal bovine serum is 10% fetal bovine serum.
8. Use of the subculture purification method according to any one of claims 1-2 or the culture method according to any one of claims 3-7 for increasing the expression level of mRNA of a myoblast-specific gene.
9. Use according to claim 8, wherein the myoblast-specific gene isPax7Gene, gene,MYF5Gene, gene,MYODGene, gene,MYOGGenes anddesmina gene.
10.MYF5The expression quantity of the gene or the coded protein thereof is applied to judging the myotube formation of the myoblast in vitro culture.
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| 王红娜等: "绵羊成肌细胞的纯化、培养、鉴定及其成肌诱导分化研究", 《河北农业大学学报》 * |
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| CN114606182B (en) | 2022-10-04 |
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