A method for isolating and primary culturing cardiomyocytes from adult Muscovy ducks

Abstract

The present invention belongs to the field of cell culture technology, and specifically relates to a method for isolating and primary culturing adult Muscovy duck cardiomyocytes. In the culturing method of the present invention, the steps for isolating and purifying adult Muscovy duck cardiomyocytes are simple, the cells adhere quickly, the survival rate is high, the activity is good, and the cell culture system has good stability. The method is a relatively ideal method for culturing primary Muscovy duck cardiomyocytes, which can basically meet the requirements of various research fields of medicine, biology, pharmacy and life sciences for experimental research related to Muscovy duck cardiomyocytes, and is suitable for promotion and application in general laboratories.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for separating and primary culturing adult Muscovy duck myocardial cells. Background Art

[0002] In human medicine, culturing cardiomyocytes is the main technical means for studying cardiovascular diseases. Cardiomyocytes play a vital role in the research and treatment of cardiac diseases such as myocardial hypertrophy, heart damage, and heart failure. However, studies in recent years have shown that myocardial fibroblasts are very similar to myocardial cells, which may be helpful for the treatment of heart diseases. With the development of science and technology, the research on avian heart diseases is also deepening, and projects are constantly being carried out with myocardial cells and myocardial fibroblasts as the objects. There are great differences between poultry myocardium and mammalian myocardial tissue, and compared with mammals and rodents, poultry generally have higher body temperatures. During the enzymatic digestion process, it is more difficult to control the proportion of digestive enzymes, digestion temperature and digestion time, which can easily cause tissue fragmentation but the cells fail to be successfully freed, which also increases the difficulty of primary isolation of poultry cells. The isolation and culture methods of myocardial cells and myocardial fibroblasts using chicken embryos or duck embryos as raw materials are relatively mature. It is difficult to distinguish the tissue types of those with younger age, and the isolated cells are embryonic fibroblasts; those with relatively older age can distinguish the tissue types, the degree of differentiation of embryonic tissues is low, the separation is easy, and single cells are easy to obtain. However, some poultry diseases such as fowl cholera, duck infectious serositis, and pullorum are more common in adult poultry. Therefore, using adult poultry cardiomyocytes as research objects can better reflect the relevant biological characteristics of adult poultry heart cells, more accurately reflect the development process of related diseases, and understand the pathological mechanism. However, compared with embryos, adult poultry heart tissue has a high degree of differentiation and lower cell proliferation ability than embryos. The storage, transportation, and separation of heart tissue after acquisition have a certain impact on the acquisition of single cells. Therefore, it is more difficult to obtain cardiomyocytes using adult poultry hearts as materials.

[0003] Muscovy duck is a kind of duck. From the nutritional value point of view, the protein content of duck meat is relatively high, and the fat content is not high. The niacin contained in duck meat has a protective effect on the heart. At present, there are no cardiomyocytes from the heart of adult Muscovy ducks. The inventors obtained cells from the heart tissue of 70-day-old Muscovy ducks, purified and identified them, which is of great significance to the preservation of Muscovy duck germplasm resources, the study of avian heart diseases, scientific research, etc. There is no report in the prior art on the use of poultry heart tissue as a material to obtain cardiomyocytes. Summary of the invention

[0004] The purpose of the present invention is to provide a method for culturing cardiomyocytes of adult Muscovy ducks. The method is simple to operate and the isolated cardiomyocytes have strong vitality. The method provides technical support for taking heart tissue in a field environment and isolating living cells in a short period of time. The isolated cardiomyocytes can also be induced to differentiate in a directed manner.

[0005] The technical solution of the present invention is as follows:

[0006] A method for isolating and primary culturing adult Muscovy duck cardiomyocytes comprises the following steps:

[0007] S1 Collection and pre-treatment of adult Muscovy duck heart tissue: 2 g of ventricular tissue of adult Muscovy duck heart tissue was cut with sterile scissors, soaked in 70% or 75% alcohol for 5-10 seconds, then washed three times in 1× PBS containing 2% double antibody, and minced with sterile scissors;

[0008] S2 enzyme digestion method to obtain adult Muscovy duck cardiomyocytes:

[0009] Place the chopped tissue in S1 in a 50ml centrifuge tube, add about 3-5 times the volume of mixed digestion solution, and digest in a 37°C water bath for 30min-90min. During this period, shake the centrifuge tube several times to ensure that the tissue blocks are fully digested; terminate the digestion with serum-containing α-MEM cardiomyocyte complete culture medium and let it stand for 10min; pass the digested solution through 200 and 400 mesh cell sieves in turn, centrifuge the suspension at 1000rpm for 10min, resuspend and wash once with α-MEM basal culture medium, and centrifuge at 1000rpm for 10min; then put it into 1mL α-MEM cardiomyocyte complete culture medium and resuspend it, collect all the cell suspensions and inoculate them in a T25 culture flask and culture them in a 37°C, 5% CO2 incubator for adherent culture. After 24h, collect the non-adherent cells and re-inoculate them in a new culture flask to remove fibroblasts, and then obtain adult Muscovy duck primary cardiomyocytes.

[0010] Furthermore, the α-MEM complete culture medium for cardiomyocytes described in S2 is 10-20% FBS and 0.5-2% MEMNEAA.

[0011] Furthermore, the α-MEM cardiomyocyte complete culture medium described in S2 also includes 0.5-2% sodium pyruvate, 0.5-2% glutamine, 10-30ng / ml EGF, 10-30ng / ml FGF, 5-20ug / ml insulin, 0.05-0.15mM 5-Brdu, and α-MEM basic culture medium.

[0012] Furthermore, the α-MEM cardiomyocyte complete culture medium described in S2 is 15% FBS, 1% MEMNEAA, 1% sodium pyruvate, 1% glutamine, 20ng / ml EGF, 20ng / ml FGF, 10ug / ml insulin, 0.1mM 5-Brdu, and α-MEM basic culture medium.

[0013] Furthermore, the α-MEM complete culture medium for cardiomyocytes described in S2 is supplemented with 1-2% double antibody.

[0014] Furthermore, the digestion solution described in S2 is 0.1-0.2% collagenase II and 0.1-0.25% pancreatin.

[0015] Furthermore, 0.2% collagenase II and 0.25% pancreatin were mixed in a volume ratio of 1:1 in the digestion solution described in S2.

[0016] Furthermore, the digestion time described in S2 is 60 min.

[0017] Further, the acquisition and preservation of adult Muscovy duck myocardial cells described in S1: 70-day-old Muscovy duck myocardial tissue was obtained outdoors, and the disinfected and cleaned tissue was cut into 5 mm 3 The myocardial tissue was placed in AQIX tissue preservation solution containing 2% double antibody and transported back to the laboratory at 4°C. After being transported back to the laboratory, it was washed twice in 1×PBS containing 2% double antibody in a biosafety cabinet. The myocardial tissue can be transported and stored for 2-7 days.

[0018] The present invention provides a method for culturing cardiomyocytes of adult Muscovy ducks, wherein the method uses AQIX living tissue preservation solution to preserve the tissue for a short period of time and ensure the activity of cells in the tissue, uses an optimized enzyme digestion solution to digest the treated heart tissue, obtains cardiomyocytes, and uses a culture solution to which a variety of cell growth additives suitable for the growth of cardiomyocytes are added for culturing, wherein each growth factor cooperates with each other, the cell proliferation ability is strong, the activity is good, and the differentiation of cardiomyocytes into myofibroblasts can be reduced. After species identification and isozyme detection experiments, it is shown that the isolated cardiomyocytes are derived from Muscovy ducks, and after growth curves, cell activity detection and cell surface antigen identification, it is shown that the isolated cardiomyocytes have strong activity and high purity.

[0019] 1. The cardiomyocytes in the present invention are derived from adult Muscovy ducks, which solves the problem of obtaining primary cardiomyocytes from Muscovy ducks that are not derived from embryonic tissues.

[0020] 2. The present invention is simple to operate, and the obtained Muscovy duck cardiomyocytes have strong vitality and high purity, and can also be subcultured.

[0021] 3. A variety of growth factors are added to the myocardial cell culture solution used in the present invention, which avoids the growth of other cells other than myocardial cells, enhances the growth vitality of myocardial cells, and ensures the purity of adult Muscovy duck myocardial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a microscopic image showing the morphology of myocardial cells in Muscovy duck;

[0023] Figure 2 This is the growth curve of the cardiomyocytes of Muscovy duck;

[0024] Figure 3 This is the result of immunofluorescence identification of Muscovy duck cardiomyocytes;

[0025] Figure 4 is the sample electropherogram, where Figure 4 A is the electrophoresis diagram of multi-species DNA samples. Figure 4 B is the electrophoresis diagram after PCR amplification;

[0026] Figure 5 A is the electrophoresis of DNA sample of Muscovy duck cardiomyocytes. Figure 5 B is the electrophoresis diagram of cardiomyocytes of each sample after PCR amplification. Note: The order of primer amplification from left to right: human, rat, mouse, dog, pig, cattle, monkey, Muscovy duck, horse, rabbit;

[0027] Figure 6 This is the isozyme map of myocardial cells of Muscovy duck; Figure 6 A, 6B, and 6C are the spectra after adding lactate dehydrogenase, malate dehydrogenase, and 6-phosphate glucose dehydrogenase, respectively. The order of adding samples from left to right in each figure is Hela, 3T3-L1, and Muscovy duck cardiomyocytes;

[0028] Figure 7 This is the activity diagram of the experimental group and the control group of Muscovy duck cardiomyocytes;

[0029] Figure 8 This is the apoptosis diagram of the experimental group and the control group of Muscovy duck cardiomyocytes; Figure 8 A is the flow cytometry comparison chart of the control group. Figure 8 B is a comparison chart for the experiment. Figure 8 C is an in situ fluorescence comparison image;

[0030] Fig. 9 The results of the differentiation of duck cardiomyocytes into skeletal muscle in the experimental group and the control group are shown in Figure 2. Fig. 9 A is the protein expression result, Fig. 9 B and Fig. 9 C is the mRNA expression result. DETAILED DESCRIPTION

[0031] The object of the present invention is to provide a method for culturing cardiomyocytes from adult Muscovy ducks. Adult Muscovy duck cardiomyocytes are successfully isolated and cultured by collecting and transporting the heart tissue of 70-day-old Muscovy ducks outdoors, and the cell morphology, growth curve, immunofluorescence identification, species identification, isoenzyme detection, vitality detection, apoptosis detection, and skeletal muscle induction differentiation test are described in detail, but the present invention is not limited to the following specific embodiments.

[0032] Example 1: Isolation and culture of adult Muscovy duck cardiomyocytes

[0033] (1) Collection of cardiac tissue from adult Muscovy ducks: 70-day-old adult Muscovy ducks were killed outdoors, and 2 g of cardiac tissue from the ventricular part was cut with sterile scissors. The tissue was soaked in 70% or 75% alcohol for 5-10 seconds, and then washed three times in 1× PBS containing 2% double antibody. The sterilized and washed tissue was cut into 5 mm 3 The specimens were placed in AQIX tissue preservation solution containing 2% double antibody and transported back to the laboratory at 4°C.

[0034] (2) Tissue pretreatment: The cardiac tissues were stored in the preservation solution for 2-7 days. After being transported back to the laboratory, they were washed twice in 1× PBS containing 2% double antibody in a biosafety cabinet and chopped with sterile scissors.

[0035] (3) Obtaining Muscovy duck cardiomyocytes by enzyme digestion: Place the above-mentioned chopped tissues in a 50 ml centrifuge tube, add about 3-5 times the volume of mixed digestion solution, and digest in a 37°C water bath for 30 min-90 min. During this period, shake the centrifuge tube several times to ensure that the tissue blocks are fully digested; terminate the digestion with serum-containing α-MEM complete cell culture medium and let it stand for 10 min; pass the digested solution through 200 and 400 mesh cell sieves in turn, centrifuge the suspension at 1000 rpm for 10 min, resuspend and wash once with α-MEM basal culture medium, and centrifuge at 1000 rpm for 10 min; resuspend in 1 mL of α-MEM complete cardiomyocyte culture medium, collect all the cell suspensions and inoculate them in a T25 culture flask and culture them in a 37°C, 5% CO2 incubator for adherent culture. After 24 hours, collect the non-adherent cells and re-inoculate them in a new culture flask to remove fibroblasts, and thus obtain adult Muscovy duck primary cardiomyocytes.

[0036] In this example, α-MEM culture medium, FBS, trypsin, MEM NEAA, sodium pyruvate, glutamine, and double antibodies were purchased from Gibco; collagenase II, DMSO, and 5-Brdu were purchased from Sigma; and EGF, FGF, and insulin were purchased from Peprotech.

[0037] The present invention uses repeated shaking of the centrifuge tube during the digestion process to achieve the effect of full digestion. Direct blowing in the prior art will cause mechanical damage to the freed single cells. Multiple digestions are common in cases where the amount of tissue is large and the tissue is not easy to digest. In the present invention, the amount of heart tissue of the Muscovy duck is small, and the enzyme ratio is appropriate, so multiple digestions are not required. Moreover, when the present invention obtains the myocardial cells of the Muscovy duck by the enzyme digestion method, the centrifuge tube is repeatedly shaken during the digestion process to achieve the effect of full digestion. Direct blowing is also attempted, which will cause mechanical damage to the freed single cells; the digestion time is controlled and the digestion is terminated to reduce the damage to the cells caused by excessive digestion. In the present invention, the digested solution is first sieved and then centrifuged. Sieving first will remove the tissue blocks, and the sieved ones are all single cells, which is easier to collect cells; centrifuging first in the prior art will cause the single cells and the incompletely digested tissue blocks to settle together, which is not conducive to the collection of cells. The present invention only performs differential attachment once, and attempting to use multiple differentials will lose some myocardial cells. After the digestion is terminated, the α-MEM complete cell culture fluid with serum is added, and the culture fluid is added with the drug 5-Brdu for removing fibroblasts, which is a growth factor for maintaining the vitality of myocardial cells. While ensuring cell growth, it can also meet the purpose of removing myocardial fibrosis. At the same time, studies have shown that the addition of 5-Brdu will also have a certain effect on the vitality of myocardial cells (Xia Tian et al., chicken embryo myocardial cell separation and culture study), so other growth factors such as EGF, FGF, etc. are also added to the culture fluid of the present invention. Specific experimental data are shown in Examples 8 and 9. In the present invention, by controlling the proportion of digestive enzymes, digestion temperature and digestion time, it is ensured that tissue fragmentation allows adult duck cells to be successfully freed, thereby reducing the difficulty of collecting adult duck cells.

[0038] Example 2: Morphological observation of cardiomyocytes in adult Muscovy ducks

[0039] In Example 1, when the cell density reached about 80%, the cells were observed and photographed under an inverted microscope. Adult Muscovy duck cardiomyocytes were relatively short and thick, and grew in a short columnar shape. Figure 1 Cell morphology shown.

[0040] Example 3: Plotting of the Growth Curve of Adult Muscovy Duck Cardiomyocytes

[0041] F1 cells were obtained from Example 1, the density was about 80%, washed twice with 1×PBS, digested with 0.25% trypsin for 3 min, digestion was terminated, centrifuged at 1000 rpm for 3 min, and the cells were resuspended in 1 ml culture medium; 100 ul of the cell suspension was taken, mixed with 100 ul of 0.4% trypan blue solution, and counted on a counting plate; 0.5×10 4 / ml cells were plated on a 24-well culture plate, with 1ml per well, and cultured in an incubator; cells were randomly selected from 3 wells every 24 hours, digested with trypsin to form a cell suspension, and counted, with each well counted three times to take the average value; the counting was continued for 7 days, with cell density as the ordinate and time as the abscissa, and the plot was as follows: Figure 2 The cell growth curve is shown in Figure 2. The 0.4% trypan blue solution in this example was purchased from Gibco.

[0042] The results showed that adult Muscovy duck cells all showed S-shaped growth, which was consistent with the basic biological characteristics of cells.

[0043] Example 4: Identification of adult Muscovy duck cardiomyocytes by immunofluorescence

[0044] F2 cells were obtained from Example 1, inoculated into a 24-well culture plate and cultured for 48 h; the culture medium was discarded, the cells were washed 3 times with 1×PBS, and fixed with 4% paraformaldehyde at room temperature for 30 min; the fixative was discarded, the cells were washed 3 times with 1×PBS, and the cells were incubated with a cell immunofluorescence permeabilization solution (0.1% 100×Triton) at room temperature for 20 min; the permeabilization solution was discarded, the cells were washed 3 times with 1×PBS, and the cell immunofluorescence blocking solution was added, and the cells were blocked and incubated at 37°C for 1 h; the blocking solution was discarded, the cells were washed 3 times with 1×PBS, and the primary antibodies Vimentin and Troponin T were added respectively (dilution ratio 1:200), and the cells were incubated at 4°C in the dark overnight; the primary antibodies were recovered, the cells were washed 3 times with 1×PBS, and Alexa Fluor 488, Cy3 secondary antibody (dilution ratio 1:500), incubate at room temperature in the dark for 1 hour; recover the secondary antibody, wash 3 times with 1×PBS, add DAPI solution for counterstaining, incubate at 37℃ in the dark for 30 minutes; recover DAPI, wash 3 times with 1×PBS, add anti-fluorescence quencher, observe the staining results under a fluorescent inverted microscope and take pictures. Figure 3 The immunofluorescence identification result is shown in the figure. In this example, 4% paraformaldehyde, cell immunofluorescence permeabilization solution, cell immunofluorescence blocking solution, and DAPI were purchased from Beyotime; Vimentin was purchased from CST; Troponin T was purchased from abcam; Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, AlexaFluor 488 was purchased from Invitrogen; Cy3 Goat Anti-Mouse IgG (H+L) was purchased from Abclonal. The results showed that adult Muscovy duck cardiomyocytes positively expressed Troponin T and weakly expressed Vimentin, indicating that the adult Muscovy duck cardiomyocytes were successfully isolated by the present invention.

[0045] Example 5: Species-specific PCR amplification and identification

[0046] Human Hela cells, rat H9C2 (2-1) cells, mouse C2C12 cells, dog MDCK cells, pig PK15 cells, bovine MACT cells, monkey cos-7 cells were cultured, and domestic horse kidney tissue and rabbit muscle tissue were obtained; DNA of each species was extracted using the full-form gold DNA extraction kit; nucleic acid electrophoresis was performed on 2% agarose gel, and the gel imaging system was used to take pictures to obtain the following results: Figure 4 A shows the electrophoresis diagram of DNA samples of various species; DNA of various species was amplified with specific primers of the corresponding species, and after amplification, it was detected by electrophoresis on 2% agarose gel and photographed to obtain the following Figure 4 B shows the PCR amplification electrophoresis diagram; the primer information is as follows, and some primer sequences are cited from the prior art (Establishment and analysis of chicken immortalized preadipocyte cell line, Wang Wei). The DNA extraction kit of this example was purchased from Quanshijin Company; 2×Vazyme LAmp MasterMix was purchased from Vazyme Company; and the primers were synthesized at Qingke Biotechnology Company.

[0047] Table 1 Primer information

[0048]

[0049]

[0050] Table 2 PCR reaction system

[0051] Reagents Volume(ul) DNA (100ng) x Forward Primer (10uM) 1.25 Reverse Primer (10uM) 1.25 2 × Vazyme L Amp Master Mix 12.5 <![CDATA[ddH2O]]> 10-x Total 25

[0052] PCR procedure:

[0053] The results showed that the DNA of each species had a clear main band, without tailing and RNA contamination, and the DNA quality was ideal; the DNA was amplified by PCR, and the electrophoresis pattern was clear. Each species had a single band only when amplified with the corresponding specific primers, indicating that the primers of each species had good specificity and there was no cross-contamination between species. It can be used for subsequent species identification of adult Muscovy duck cells.

[0054] Example 6: Species Identification of Adult Muscovy Duck Cardiomyocytes

[0055] The F2 cells from Example 1 were inoculated in a 6-well plate and cultured until the cell density reached about 90%. The cells were digested with 0.25% trypsin for 3-5 minutes, and the digestion was terminated by centrifugation at 1000 rpm for 3 minutes, and the supernatant was discarded. The cells were washed once with 1×PBS and DNA was extracted with a full-form gold DNA extraction kit. The nucleic acid was electrophoresed on a 2% agarose gel and photographed with a gel imaging system to obtain the following results: Figure 5 A shows the electrophoresis diagram of the DNA sample; the sample DNA was amplified with the specific primers of the 10 common species in Example 5, and after the amplification, it was detected by electrophoresis using 2% agarose gel, and the gel imaging system was photographed to obtain the following Figure 5 B shows the electrophoresis diagram of the sample after PCR amplification; the PCR amplification product was sequenced to obtain the sequence result comparison table shown in Table 3.

[0056] Table 3 Sequence alignment of PCR amplification products of Muscovy duck cardiomyocytes

[0057]

[0058]

[0059] The results showed that the DNA extracted from F2 adult Muscovy duck cardiomyocytes had a clear main band without tailing and RNA contamination; the DNA was amplified by PCR, and the electrophoresis pattern was clear, with a single band only when amplified with adult Muscovy duck-specific primers, indicating that the cell species originated from Muscovy duck; sequence comparison analysis showed that the PCR-specific amplification product was consistent with the 7091-7564bp of the Muscovy duck mitochondrial DNA sequence NC-010965.1 published on NCBI, which was consistent with the PCR results.

[0060] Example 7: Detection of isozyme in myocardial cells of adult Muscovy ducks

[0061] Take F2 cells from Example 1, and use human Hela and mouse 3T3-L1 cells as standard references; inoculate in a 60 mm culture dish and culture until the cell density reaches about 90%, digest with trypsin, centrifuge at 1000 r / min for 3 min, and discard the supernatant; wash once with 1×PBS, centrifuge at 1000 r / min for 3 min, discard the supernatant, add 2 times the volume of cell lysis buffer according to the volume of the precipitate, blow thoroughly, and lyse at 4°C for 30 min; centrifuge at 4°C, 12000 r / min for 10 min, collect the supernatant, and store it at -20°C; prepare 6% non-denaturing PAGE protein gel; 5× non-denaturing PAGE protein loading buffer was mixed at a ratio of 4:1, and 10ul of sample was loaded. The order of loading from left to right was Hela, 3T3-L1, and Muscovy duck cardiomyocytes. Lactate dehydrogenase was electrophoresed at 100V for 4 hours, malic enzyme was electrophoresed at 100V for 3 hours, and 6-phosphate glucose dehydrogenase was electrophoresed at 100V for 3 hours. After the electrophoresis, the gel was gently peeled off and stained in the corresponding enzyme staining solution in the dark for 30 minutes for lactate dehydrogenase, 60 minutes for malic enzyme, and 120 minutes for 6-phosphate glucose dehydrogenase until a band appeared. The staining solution was discarded, and the sample was washed with ddH2O for 3-5 times. The gel imaging system was used to take pictures as shown below. Figure 6 Isozyme spectra shown in A, 6B, and 6C.

[0062] Table 4 Non-denaturing PAGE protein gel preparation system

[0063]

[0064]

[0065] The reagents in the sample embodiment are prepared as follows:

[0066] Cell lysis solution: weigh 0.33g Tris, 0.0186g EDTA, 2% Tritonx-10050ul, quantitate in 50mlddH2O, store at 4℃ for later use; lactate dehydrogenase (LD) staining solution: weigh 50mg NAD, 30mg NBT, 4mg PMS, measure 10ml 1M sodium lactate, 5ml 0.1M NaCl, 15ml 0.5M Tris-HCl (PH8.0), quantitate in 100ml ddH2O, store at 4℃ in dark for later use; malic enzyme (MD) staining solution: weigh 50mg NAD, 30mg NBT, 2mg PMS, measure 10ml 1MDL-malic acid sodium salt, 5ml 0.1M NaCl, 20ml 0.5M Tris-HCl (PH8.0), quantitate in 100ml ddH2O, stored at 4°C in the dark for later use; preparation of 6-phosphate glucose dehydrogenase (G6PD) staining solution: weigh 40mg G6PD, 20mg MTT, 20mgNBT, 4mg PMS, 20ml Tris buffer, 2ml 1% NADP, 4ml 0.5M Mgcl2, quantitate in 100ml ddH2O, store at 4°C in the dark for later use; the PAGE gel preparation kit, 5× non-denaturing PAGE protein loading buffer, and Tritonx-100 solution of this embodiment were purchased from Biyuntian Company, Tris, EDTA, NaCl, 60% sodium lactate, NAD, NBT, PMS, and Mgcl2 were purchased from Shengong Company, Tris-hcl (PH8.0) and G6PD were purchased from Solebao Company, and DL-malic acid sodium salt was purchased from McLean Company.

[0067] The results showed that in the F2 generation of adult Muscovy duck cardiomyocytes, the LD isozyme banding pattern had one strong band, the MD isozyme had two bands, one strong band, one weak band, and G6PD had one weak band, which were significantly different from the banding patterns and migration distances of human Hela cells and mouse 3T3-L1 cells, indicating that the isozyme banding patterns of isolated adult Muscovy duck cardiomyocytes were distinct and there was no cross-contamination with human and mouse cells.

[0068] Example 8: Comparison of cardiomyocyte activity in adult Muscovy ducks

[0069] The culture medium without any growth factor addition (α-MEM basic culture medium + 15% FBS + 2% S / P) was used as the experimental group, and the culture medium added with multiple growth factors of the present invention (α-MEM basic culture medium + 15% FBS + 1% MEM NEAA + 1% sodium pyruvate + 1% glutamine + 20ng / ml EGF + 20ng / ml FGF + 10ug / ml insulin + 0.1mM 5-Brdu + 2% S / P) was used as the control group to compare the advantages of the complete growth culture medium of the present invention on the growth of duck cardiomyocytes; the F2 cells of Example 1 were taken, 3×10 4 / ml cells were plated in a 96-well plate, 100ul per well, 6 replicate wells were set up, and cultured for 24h. The group without cells but only with culture medium was used as a blank reference. 10ul CCK-8 reagent was added to each well and cultured for 2h. The absorbance value at 450nm was detected by an ELISA reader, and the following was obtained: Figure 7 The cell viability graph is shown in Figure 2. In this example, CCK-8 was purchased from Dojindo Company.

[0070] The results showed that the cell viability of the group with added growth factors was significantly higher than that of the group without added growth factors, indicating that the cell culture medium optimized by the present invention can effectively improve the viability of adult Muscovy duck cardiomyocytes.

[0071] Example 9: Detection of apoptosis in myocardial cells of Muscovy duck

[0072] The advantages of the complete growth medium of the present invention on the growth of duck cardiomyocytes were compared by taking the culture medium without any growth factor added (α-MEM basic culture medium + 15% FBS + 2% S / P) as the experimental group and the culture medium with multiple growth factors added (α-MEM basic culture medium + 15% FBS + 1% MEM NEAA + 1% sodium pyruvate + 1% glutamine + 20ng / ml EGF + 20ng / ml FGF + 10ug / ml insulin + 0.1mM 5-Brdu + 2% S / P) as the control group. The F2 cells were taken from Example 1, 5×10 4 / ml cells were plated in a 6-well plate and cultured for 72h, with 3 replicate wells. Digested with trypsin, centrifuged at 1000r / min for 3min; resuspended in 1×PBS, centrifuged at 1000r / min for 5min, and the supernatant was discarded; resuspended in 100ul Bindingbuffer, added with 10ul Annexin V-FITC, incubated at room temperature in the dark for 15min; added with 200ul Bindingbuffer, passed through a 70uM cell sieve; added with 10ulPI, and subjected to analytical flow cytometry, and the results were as follows: Figure 8 Figures A and B show cell apoptosis. F2 cells were taken from Example 1, 1×10 4 / ml cells were plated in a 24-well plate and cultured for 72 hours. 1×PBS was gently washed once, 150ul Binding buffer containing 5ul Annexin V-FITC was added to each well, and incubated at room temperature in the dark for 15 minutes; 100ul Binding buffer containing 5ul PI was added to each well, and incubated at room temperature in the dark for 10 minutes; photos were taken under a fluorescent inverted microscope to obtain the following results: Figure 8 Cell apoptosis diagram shown in C. The AnnexinV-FITC apoptosis detection kit in this example was purchased from Sigma.

[0073] The results showed that: the flow cytometry results showed that the proportion of live cells in the group with added growth factors was higher than that in the group without added growth factors, indicating that the addition of growth factors helped the growth of the duck myocardium and reduced cell apoptosis. The fluorescence in situ results showed that the number of positive cells labeled by Annexin V-FITC and PI in the group with added growth factors was less than that in the group without added growth factors, indicating that the addition of the cell culture medium optimized by the present invention can reduce the number of apoptotic cells in adult duck myocardial cells.

[0074] Example 10: Induction of differentiation of duck cardiomyocytes

[0075] During the cell proliferation period, the culture medium without any growth factor addition (α-MEM basic culture medium + 15% FBS + 2% S / P) was used as the experimental group, the culture medium added with multiple growth factors in the present invention (α-MEM basic culture medium + 15% FBS + 1% MEMNEAA + 1% sodium pyruvate + 1% glutamine + 20ng / ml EGF + 20ng / ml FGF + 10ug / ml insulin + 0.1mM 5-Brdu + 2% S / P) was used as the control group, and the cells not induced to differentiate were used as the blank group to compare the effect of the complete growth culture medium in the present invention on the induction and differentiation of duck cardiomyocytes into skeletal muscle cells; F2 generation cells were taken from Example 1 and inoculated into a 6-well culture plate until the cell density converged to about 80%; the complete growth culture medium was discarded, 2ml of skeletal muscle was added to each well for differentiation induction, and the culture medium was changed every 24h for 5 days for differentiation induction; after the induction and differentiation, RIPA was used to extract total protein, and western blot was used to detect myosin heavy in duck cardiomyocytes and cardiac fibroblasts and cells after differentiation induction. chain and myogenin expression, based on Pierce TM BCA Protein Assay Kit was used to determine protein concentration, and 20ug of protein was loaded for detection. 10% separation gel was used for protein electrophoresis, 60V for 30min; 120V for 120min, and the protein gel preparation system was as follows:

[0076] Table 5 10% SDS-PAGE protein gel preparation system

[0077] 5% stacking gel Volume(ml) 10% separation gel Volume(ml) 30% Acr-Bis (29:1) 0.5 30% Acr-Bis (29:1) 5 1M Tris-HCl, pH 6.8 0.38 1M Tris-HCl, pH 8.8 5.7 10% APS 0.03 10% APS 0.15 TEMED 0.003 TEMED 0.006 10% SDS 0.03 10% SDS 0.15 <![CDATA[ddH2O]]> 2.1 <![CDATA[ddH2O]]> 4 Total 3 Total 15

[0078] After electrophoresis, the gel was cut according to the protein maker, and the bands on the gel were transferred to the PVDF membrane at 100V for 60min; 5% BSA was blocked for 2h, and the primary antibodies β-actin (dilution ratio 1:1000), myosin heavy chain (dilution ratio 1:250), and myogenin (dilution ratio 1:250) were incubated at 4℃ overnight; the primary antibodies were recovered, washed 3 times with 1×TBST, and the secondary antibodies Anti-mouse IgG (dilution ratio 1:2000) were added, and incubated at room temperature for 1h; the secondary antibodies were recovered, washed 3 times with 1×TBST, and the exposure developer was added, and incubated in the dark for 10min; the protein gel imaging system was used to take pictures as shown below. Fig. 9 The result is shown in Figure A. After the induction of differentiation, total RNA was extracted with the full-form gold RNA extraction kit, and the changes in the expression of myosin heavy chain and myogenin mRNA before and after differentiation were detected by fluorescence quantitative PCR after reverse transcription. Fig. 9 B and Fig. 9 The result is shown in Figure C. In this example, HS was purchased from BIOIND, β-actin and Anti-mouse IgG were purchased from CST, MyoG was purchased from Santa Cruz, MF-20 was purchased from DSHB, RIPA Lysis and Extraction Buffer, Pierce TM BCA Protein Assay Kit was purchased from Thermo Scientific, SDS-PAGE gel preparation kit, SDS-PAGE protein loading buffer (5X), BeyoColor TM Color pre-stained protein molecular weight standards, BSA, and Western antibody diluent were purchased from Beyotime, and Clarity TM Western ECL Substrate was purchased from BIO-RAD, and RNA extraction kit was purchased from Quanshijin. RT reagentKit with gDNAEraser reverse transcription kit was purchased from Dalian Baosheng Biotechnology Co., Ltd.; SYBR select Master Mix was purchased from ABI Company, and Tris and other conventional reagents were purchased from Sangon Company.

[0079] Table 6 Fluorescence quantitative PCR primer sequence information

[0080]

[0081]

[0082] Table 7 Reverse transcription reaction system

[0083] Reagents Volume(ul) RNA(1ug) x 5×gDNAEraserBuffer 2 gDNAEraser 1 RNase-free ddH2O 7-x Total 10

[0084] Reverse transcription program: 42°C, 2 min; 4°C, Forever

[0085] Reagents Volume(ul) Upper reaction solution 10 PrimeScriptRTEnzymemixΙ 1 RTPrimemix 1 5×PrimescriptBuffer Ι 4 RNase-free ddH2O 4 Total 20

[0086] Reverse transcription procedure:

[0087] Table 8 Fluorescence quantitative PCR reaction system

[0088] Reagents Volume(ul) cDNA 2 Forward Primer (10uM) 0.8 Reverse Primer (10uM) 0.8 SYBRselectMasterMix 10 RNase-free ddH2O 6.4 Total 20

[0089] Fluorescence quantitative PCR procedure:

[0090] The results showed that after inducing differentiation, the mRNA and protein expression levels of myogenin and myosin heavy chain in the group without growth factor addition were significantly higher than those in the group with growth factor addition and the blank group, and there was no significant difference between the blank group and the control group, indicating that the addition of growth factors helps to maintain the growth of original cardiomyocytes of adult Muscovy ducks, and the conventional culture method (using α-MEM basal culture medium + 15% FBS + 2% S / P) can transform adult Muscovy duck cardiomyocytes into myoblasts.

Claims

1. A method for isolating and primary culturing adult Muscovy duck cardiomyocytes, comprising the following steps: S1 Adult Muscovy Duck Heart Tissue Collection and Pre-treatment Use sterile scissors to cut 2 g of ventricular tissue from the heart of an adult Muscovy duck, soak it in 70% or 75% alcohol for 5-10 seconds, wash it three times in 1× PBS containing 2% double antibody, and cut it into pieces with sterile scissors; Obtaining adult Muscovy duck cardiomyocytes by S2 enzyme digestion Place the chopped tissue in S1 in a 50ml centrifuge tube, add 3-5 times the volume of digestion solution, and digest in a 37℃ water bath for 30min-90min. During this period, shake the centrifuge tube several times to fully digest the tissue block; terminate the digestion with α-MEM cardiomyocyte complete culture medium and let it stand for 10min; pass the digested solution through 200 and 400 mesh cell sieves in turn, centrifuge the suspension at 1000rpm for 10min, resuspend and wash once with α-MEM basal culture medium, and centrifuge at 1000rpm for 10min; resuspend in 1mL α-MEM cardiomyocyte complete culture medium, collect all the cell suspensions and inoculate them in a T25 culture flask and culture them in a 37℃, 5% CO2 incubator for adherent culture. After 24h, collect the non-adherent cells and re-inoculate them in a new culture flask to remove fibroblasts, and then obtain adult Muscovy duck primary cardiomyocytes; The digestion solution described in S2 is 0.1-0.2% collagenase II and 0.1-0.25% trypsin, and the components of the α-MEM cardiomyocyte complete culture medium include 10-20% FBS, 0.5-2% MEM NEAA, 0.5-2% sodium pyruvate, 0.5-2% glutamine, 10-30ng / mL EGF, 10-30 ng / mL FGF, 5-20 μg / mL insulin, 0.05-0.15 mM 5-Brdu, and α-MEM basic culture medium.

2. The method for isolating and primary culturing adult Muscovy duck cardiomyocytes according to claim 1, characterized in that: The α-MEM complete cardiomyocyte culture medium described in S2 also contains 1-2% double antibody.

3. The method for isolating and primary culturing adult Muscovy duck cardiomyocytes according to claim 1, characterized in that: The digestion solution described in S2 was mixed with 0.2% collagenase II and 0.25% trypsin in a 1:1 volume ratio.

4. The method for isolating and primary culturing adult Muscovy duck cardiomyocytes according to claim 1, characterized in that: The digestion time described in S2 was 60 min.

5. The method for isolating and primary culturing adult Muscovy duck cardiomyocytes according to claim 1, characterized in that: Acquisition and preservation of adult Muscovy duck cardiomyocytes as described in S1: The myocardial tissue of 70-day-old Muscovy duck was obtained outdoors, and the disinfected and cleaned tissue was cut into a size of 5 mm3, placed in AQIX tissue preservation solution containing 2% double antibody, and transported back to the laboratory at 4°C. After being transported back to the laboratory, it was washed twice in 1×PBS containing 2% double antibody in a biological safety cabinet. The myocardial tissue was transported and stored for 2-7 days.

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