KR102179463B1 - Myoblast cell established from chicken pectoralis major and method for screening physiological active substance using the same - Google Patents

Abstract

The present invention relates to a method for screening a physiological active substance using a myoblast cell line established from chicken pectoralis major. The myoblast cell line established from chicken pectoralis major was treated with extracts of angelica and angelica by-products as physiologically active candidates, and then gene expression levels thereof were compared with that of untreated control myoblast cell lines. As a result, the angelica and angelica by-product extracts have been found to have physiological activity to increase or decrease the expression level of genes that regulate cell proliferation, myogenesis, adipogenesis, or glycometabolism. Therefore, the screening method using the myoblast cell line established from chicken pectoralis major may be provided as an in vitro screening method for selection of livestock feed additives or physiologically active substances.

Description

Myoblast cell established from chicken pectoralis major and method for screening physiological active substance using the same}

The present invention relates to a method for screening a substance exhibiting physiological activity using a myoblastic cell line isolated and cultured from the pectoralis major muscle of a chicken.

Livestock and the products they produce are important sources of protein necessary for human survival, and the development of breeding and use of livestock is closely related to the improvement of human health and quality of life. The development of a feed additive composition promotes the fattening and digestion of livestock, thereby maximizing feed efficiency and reducing feed consumption, as well as enabling the production of high-quality livestock products due to rapid growth and fattening, thereby improving the income level of farmers. It is very important in that it can contribute to.

Conventional feed additives have been manufactured to contain synthetic chemicals such as antibiotics or antibacterial agents for the purpose of improving the gain rate of livestock, increasing feed efficiency, and preventing diseases. The amount of antibiotics (for feed additives) used in compound feed manufacturing was 670 tons or more, 52% or more, more than half of the antibiotics used in livestock, of which 68.7% was used for pigs, 25% was used for chickens, and used for cattle. It is known that the amount produced accounts for 6.5%. However, mixing and using these feed additives in livestock feed is a drug abuse for livestock, and this results in a decrease in livestock's immunity against disease and increased resistance to antibiotics, resulting in demand for stronger antibiotics. Will result. In addition, since drugs such as antibiotics remain in the body of livestock, the health of those who consume them may also be threatened.

Recently, the European Union (EU) has banned the addition of antibiotics to livestock feed for growth purposes, and has been subject to strict controls for therapeutic purposes.

Various measures to replace the use of antibiotics are being tried in Korea, among which research is being conducted on the development of livestock feed additives using natural materials that are safe for livestock and human body, but to prove the effect of bioactive substances as feed additives. The field test for research is expensive and requires a lot of time and effort, and it is difficult to identify biological functions and mechanisms in the result analysis process.

Accordingly, there is a need for research on an in vitro efficacy evaluation system to confirm accurate biological activities such as cellular functions and regulatory mechanisms of candidate feed additives, including natural product extracts.

Republic of Korea Patent Publication No. 10-2017-0095762 (published on Aug. 23, 2017)

The present invention is a technology for the development of livestock feed additives for maximizing the profit of value-added products of the livestock industry, and in order to screen for physiologically active substances that can be substantially effective as feed additives for livestock among various natural material materials, male embryos A chicken myoblast cell line (pCM) has been newly established in the breast, and an attempt is made to provide a method for screening using it.

The present invention provides a myoblastic cell line (KCLRF-BP-00410) established from the pectoralis major muscle of a chicken.

The present invention comprises the steps of treating a candidate material for regulating physiological activity in a myoblastic cell line (KCLRF-BP-00410) established from the pectoralis muscle of a chicken; Checking the gene expression level of the myoblastic cell line treated with the candidate material; And comparing the identified gene expression level with an untreated control group.

In addition, the present invention comprises the steps of treating a candidate material for muscle differentiation in the muscle fibrous cell line (KCLRF-BP-00410) established from the pectoralis major muscle of the chicken; Checking the gene expression level of the myoblastic cell line treated with the candidate material; And it provides a method for screening a muscle differentiation regulator comprising the step of comparing the identified gene expression level with an untreated control.

According to the present invention, as a result of comparing the gene expression level with the untreated control myoblast cell line after treatment with an angelica and angelica byproduct extracts as physiologically active candidate substances in a myoblastic cell line isolated and cultured and established from the pectoralis major muscle of a chicken, angelica and As the physiological activity of increasing or decreasing the expression level of genes that regulate cell proliferation, myogenesis, adipogenesis, or glycometabolism was confirmed, the Angelicae by-product extract was established from the pectoralis muscle of the chicken. The screening method using the myoblastic cell line may be provided as an in vitro screening method for screening livestock feed additives or bioactive substances.

Figure 1 is a result of confirming the cell proliferation response analysis and characteristics of chicken myoblastic cell line (pCM) by Angelica root extract, Figure 1A is a result of confirming the cell proliferation of pCM cells treated with different concentrations of Angelica root extract (10 ^-7 , 10 ^-8 and 10 ^-9 ), Figure 1B is a result of confirming the morphology of pCM cells treated or not treated with a 10 ^-8 concentration of Angelicae extract (scale bar = 100 μm), Figures 1C and 1D The relative expression profiles of Pax7 and MyoD between control pCM cells and angelica-treated pCM cells are confirmed by qRT-PCR analysis results and Western blot results.
Figure 2 is a result of mRNA sequencing analysis between the control chicken myoblast cell line (pCM) and the angelica-treated chicken myoblast cell line (pCM), Figure 2A confirms the gene showing the difference in expression between the control pCM cells and the angelica-treated pCM cells As a result, FIG. 2B is a scatter plot analysis result of DEGs, and red and green dots show expression patterns of up-regulated genes and down-regulated genes in pCM cells treated with Angelicae extract.
Figure 3 is a result of confirming the mRNA sequence result and string analysis, Figure 3A is the expression profile of the upregulated genes (GCLC, PTPN6, ISL1, SLC25A13, TGFBI and YWHAH) in the chicken myoblast cell line (pCM) treated with Angelica. This is the confirmed qRT-PCR analysis result (***p <0.001), and FIG. 3B is a string analysis result of an upregulated gene set in Angelicae-treated pCM cells.
Figure 4 is a result of confirming the characteristics of the differentiation of myotubes according to the treatment of Angelicae (AGN) extract, Figure 4A is a result of confirming the morphology of the differentiated myotubes after 4 days without treatment or treatment of the Angelicae extract under differentiation conditions, control chicken The difference in morphology between the myoblastic cell line (pCM) and the Angelica-treated pCM cells was not confirmed, and the arrow indicates the differentiated myotube cells, and FIG. 4B is the percentage of the differentiated area in the control pCM cells and the Angelica-treated pCM cells 4C is a result of confirming the expression profiles of Pax7, MyoD, and Desmin genes between control pCM cells and Angelica-treated pCM cells 4 days after grouping cell differentiation.
Figure 5 is a result of confirming the differentiation characteristics of myotubes by an Angelicae (AGN) extract extracted from stem and leaf by-products, Figure 5A is a chicken myoblast cell line (pCM) concentration-dependently treated with Angelicae extract extracted from the by-product Cell proliferation analysis results (***p <0.001), FIGS. 5B and 5C are qRT-PCR analysis results and Western blot results confirming the relative expression profiles of Pax7 and MyoD between control pCM cells and pCM cells treated with angelica byproducts. (*p <0.05 and ***p <0.001), FIG. 5D is a result of comparing the percentage of differentiated regions of control pCM cells and pCM cells treated with angelica by-products, and arrows indicate differentiated myotubes, FIG. 5E And FIG. 5F is a qRT-PCR analysis result and Western blot result comparing the expression profiles of Pax7, MyoD and Desmin between control pCM cells and pCM cells treated with angelica by-products 4 days after myocyte differentiation (*p < 0.05, **p <0.05, and ***p <0.05).

Hereinafter, the present invention will be described in more detail.

The present invention is a technology for developing livestock feed additives for maximizing the profit of value-added products in the livestock industry, and the inventors of the present invention select physiologically active substances that can actually exhibit an effect as feed additives for livestock among various natural materials. The present invention was completed by confirming the physiological activity effect of the natural product extract using a novel chicken myoblast cell line (pCM) established in the breast of a male embryo while studying a screening method for it.

The present invention can provide a myoblastic cell line (KCLRF-BP-00410) established from the pectoralis muscle of chickens.

More specifically, the myoblastic fibrous cell line established from the pectoralis muscle of the chicken may be isolated and cultured from the pectoralis muscle of a 10-day-old male chick embryo.

The present invention comprises the steps of treating a candidate material for regulating physiological activity in a myoblastic cell line (Korea Cell Line Bank accession number: KCLRF-BP-00410) established from the pectoralis major muscle of the chicken; Checking the gene expression level of the myoblastic cell line treated with the candidate material; And it can provide a bioactive substance screening method comprising the step of comparing the identified gene expression level with an untreated control.

The physiologically active substance may increase or decrease the expression level of a gene that regulates cell proliferation, myogenesis, adipogenesis, or glycometabolism.

The candidate material may be selected from the group consisting of natural extracts, peptides, proteins, polysaccharides, amino acids, hormones, growth factors, antibiotics, anticancer agents, antibacterial agents, antiviral agents, and anticoagulants, but is not limited thereto.

The natural product extract may be an Angelicae or Angelica by-product extract, and in more detail, the Angelicae by-product may be an Angelicae stem or leaf, but is not limited thereto. In addition, the extract may be extracted with water, C1 to C4 alcohol, or a mixed solution thereof.

The screening method may be a method of selecting a physiologically active substance for an animal feed additive, but is not limited thereto.

In addition, the present invention comprises the steps of treating a candidate material for muscle differentiation in the muscle fibrous cell line (KCLRF-BP-00410) established from the pectoralis major muscle of the chicken; Checking the gene expression level of the myoblastic cell line treated with the candidate material; And it is possible to provide a method for screening a muscle differentiation regulator comprising the step of comparing the identified gene expression level with an untreated control.

The gene may be selected from the group consisting of Pax7, MyoD, and desmin, but is not limited thereto.

Methods for determining the expression level of the gene include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA; RNase protection assay), Northern Any one selected from the group consisting of blotting (Northern blotting) and microarray may be used, but is not limited thereto.

The "bioactive substance" of the present invention is a substance that regulates genetic expression and physiological function, and plays a role in correcting abnormal conditions due to lack or excessive secretion of substances involved in function regulation in the body, It is known to delay aging or prevent various diseases through antioxidant, detoxification, immune function enhancement, hormone control, antibacterial or antiviral action.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental Example>

The following experimental examples are intended to provide experimental examples commonly applied to each of the examples according to the present invention.

1. Experimental animals

The experimental management and use of chickens was approved by the Animal Care and Use Committee of Seoul National University (SNU-170417-18-2). All experimental chickens were maintained in accordance with the standard management program at the International Animal Research Farm at Seoul National University Pyeongchang Campus, and procedures for animal management followed the standard operating method of the laboratory.

2. Preparation of Angelica gigas Nakai

The roots and by-products (stems and leaves) of Angelica Angelica (AGN) were collected and dried at 65° C. for 48 hours. The moisture content of the dry ingredients of AGN was about 15%.

After mixing dried roots (100 g) and by-products (300 g; stems and leaves) with 1 L and 0.9 L distilled water, respectively, in a sterilizer under high temperature (110°C) and high pressure (5.69 pound per square inch [PSI]) conditions. Extracted for 15 minutes, the supernatant was collected and stored at -80°C until use. Finally, the extract of Angelica Angelica was lyophilized.

3. Chicken myoblast cell line (pCM) establishment and myotube differentiation

Chicken myoblastic cell lines were established through isolation and culture from the pectoralis muscle of 10-day-old male chick embryos, and culture conditions were 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, Massachusetts), 2% chicken serum (Sigma-Aldrich, St. Louis, Missouri) and 1x antibiotic-antifungal (Invitrogen) containing 199 medium (Invitrogen, Carlsbad, California) was maintained in a 5% CO ₂ atmosphere at 37°C, 60%-70% relative humidity.

In order to induce myotube differentiation, about 80% confluent cells were washed with PBS (phosphate buffered saline) and replaced with a differentiation medium containing 0.5% FBS and 1× antibiotic-antifungal agent to differentiate. Fresh differentiation medium was replaced daily during the differentiation period.

4. Analysis of proliferation reaction using pCM cells

After diluting the Angelicae extract powder with a medium or differentiation medium with 10 ^-7 , 10 ^-8 , and 10 ^-9 (100 ng/L, 10 ng/L and 1 ng/L, respectively), pCM cells were mixed with the Angelicae extract. Treated together. For proliferative reaction analysis, pCM cells were inoculated at 2 × 10 ⁴ cells/well in a 24-well culture dish and cultured for 5 days in the presence or absence of Angelicae extract, and then using Countess II FL automated cell counter (Invitrogen). The total number of cells was counted.

5. Quantitative RT-PCR (qRT-PCR) analysis

Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA was quantified using NanoDrop 2,000 (Thermo Fisher Scientific), and 2 μg of RNA was used for cDNA synthesis under standard conditions using a random primer (Invitrogen).

Each 20 μl RT-PCR reaction mixture contains 2 μl cDNA, 2.5 μl PCR buffer, 1 μl dNTP mixture (2.5 mM), 0.2 μl of 1 unit Taq DNA polymerase, 1 μl of each 10 pmol forward and reverse primer as shown in Table 1, and It contained 12.3 μl of ddH2O.

Quantitative RT-PCR analysis was performed using the iCycler iQ Real-time PCR detection system (Bio-Rad, Hercules, CA) and EvaGreen (Biotium, Fremont, CA).

The PCR parameters are as follows: 40 cycles were performed under each condition after the initial incubation at 94°C for 5 minutes. The reaction was terminated by a final incubation at 72°C for 10 minutes, and the melting curve profile for an amplicon was analyzed.

6. Western blotting

The total protein was extracted with 1× radioimmunoprecipitation dissolution buffer, separated on a 10% polyacrylamide gel, and transferred to a nitrocellulose membrane (Bio-Rad).

Mouse anti-β-actin (Santa Cruz Biotechnology, Dallas, Texas), anti-Foxo3 (Invitrogen), anti-Pax7 (R&D Systems, Minneapoils, Minnesota), anti-MyoD (Santa Cruz Biotechnology), and anti -Desmin (Novus Biologicals, Littleton, Colorado) was used, and HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Bio-Rad) was used as the secondary antibody.

For detection and quantification of chemiluminescence, a blot treated with an enhanced chemiluminescence (ECL) substrate solution was exposed to a ChemiDoc XRS System (Bio-Rad).

7. Next generation sequencing (NGS) and association analysis

Using mRNA next-generation sequencing (NGS) data, differentially expressed genes (DEGs) from pCM and Angelica-treated pCM cells were identified at 0.05 p-value cutoff and 1.5 fold change cutoff.

To confirm all functional interactions of DEGs, protein-protein association was analyzed using STRING (https://string-db.org).

<Example 1> Confirmation of the effect of proliferation reaction of chicken myoblast cell line (pCM) according to carrot root extract

The pCM was treated with a different concentration of Angelica root extract and the cell proliferation efficacy was confirmed. As a result, cell proliferation was significantly reduced in a dose-dependent manner as shown in FIG. 1A. However, referring to FIG. 1B, since cell damage or morphological modification was hardly observed during the cell culture period in the presence of the Angelica root extract, the Angelica root extract is highly likely to regulate the cell cycle of chicken myoblasts.

As Angelicae extract significantly reduced cell proliferation at 10 ^-7 (100 ng/L), a medium concentration of Angelicae extract diluted to 10 ^-8 (10 ng/L) was applied from the next experiment.

<Example 2> Characterization of undifferentiated chicken myoblast cell line (pCM) treated with Angelicae extract

Pax7 acts as a transcription factor for the regulation of muscle progenitor cell status (Kim, Lee, Park, & Park, 2017), and MyoD is known to act as a muscle regulator for inducing muscle differentiation (Kim et al., 2017).

In order to confirm the action effect of the Angelicae extract, the non-differentiated pCM cells were treated with the Angelicae extract, and the expression of Pax7 and MyoD was confirmed.

As a result, referring to FIGS. 1C and 1D, it was confirmed that the mRNA and protein expression levels of the angelica-treated pCM cells were significantly up-regulated in Pax7, while the MyoD was significantly decreased as compared to the control pCM cells.

<Example 3> Analysis of total gene expression of chicken myoblast cell line (pCM) treated with Angelicae

Next, through NGS and bioinformatics analysis, the whole gene expression pattern of the pCM cells treated with Angelicae was analyzed.

As a result, compared to the control pCM cells as shown in FIG. 2A, it was confirmed that the total of 26,640 DEGs were classified in the angelica-treated pCM cells, 9,225 DEGs were upregulated, and 11,146 DEGs were downregulated.

In addition, referring to FIG. 2B, as a result of comparing DEG between the control group and the angelica-treated pCM cells, it was confirmed that the results of scatter plot analysis systematically represented by increasing or decreasing gene expression values were confirmed.

Based on the GSEA database (http://software.broadinstitute.org/gsea/index.jsp), 4 gene sets (three up-regulated genes and one down-regulated gene) in pCM cells treated with Angelicae as shown in Table 2 ) Was confirmed.

Referring to Table 2, pancreatic beta cells, fatty acid metabolism, and glycolysis-related gene sets were up-regulated, while adipogenesis-related gene sets were down-regulated.

From the above results, it was confirmed that the Angelicae extract exhibits an effect on metabolic processes related to the use of fat and glucose in chicken muscle cells.

Meanwhile, qRT-PCR analysis was performed to confirm the expression pattern of the gene selected from the RNA sequence analysis result.

As a result, all the genes (GCLC, PTPN6, ISL1, SLC25A13, TGFBI and YWHAH) upregulated in the Angelicae-treated pCM cells as shown in FIG. 3A showed the same qRT-PCR analysis results.

In addition, as a result of string analysis confirming the correlation between the set of genes upregulated in the Angelicae-treated cells, as shown in FIG. 3B, three major regulatory networks were identified that showed a very close correlation between different genes.

<Example 4> Confirmation of muscle differentiation effect of chicken myoblastic cell line (pCM) by Angelica Angelica extract

In order to confirm the effect of Angelicae extract on myoplastic differentiation of pCM cells, the ability to form root canals between control pCM cells and angelica-treated pCM cells was induced and compared.

As a result, as shown in Fig. 4A, myogenic differentiation in the presence of Angelicae extract was significantly reduced compared to undifferentiated pCM cells (19.44 ± 0.01% vs. 18.44 ± 0.01%, p <0.05). At the same time, 4 days after the induction of myotube differentiation, Pax7 was significantly increased in angelica-treated pCM cells, while myoD and desmin, myoD and desmin, were down-regulated as shown in FIG. 4C.

From the above results, it was confirmed the effect of the extract of Angelica Angelica on maintaining the undifferentiated state of the chicken myoblast cell line.

<Example 5> Analysis of the biofunctionality of an Angelica by-product extract in chicken myoblastic cell line (pCM)

The effect of the Angelicae extract extracted from by-products (stems and leaves) was confirmed.

Referring to FIG. 5, it was confirmed that the proliferation of pCM cells was decreased in a dose-dependent manner without any cytotoxicity. In addition, in order to investigate the functional effect of the Angelicae by-product extract, the undifferentiated pCM cells were treated with the Angelicae by-product extract, and the expression effects of Pax7 and MyoD were confirmed.

As a result, similar to the Angelica root extract, it was confirmed that Pax7 expression was significantly increased in both mRNA and protein expression levels compared to the control pCM cells as shown in FIGS. 5B and 5C, whereas MyoD was significantly decreased.

In addition, as shown in Figs. 5D to 5F, it was confirmed that the effect of the Angelicae by-product extract during the myotropic differentiation period of pCM cells showed a pattern similar to that of the pCM cells treated with Angelica roots.

From the above results, it was confirmed that the biofunctional activity between the extract of Angelica root and by-product was similar.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Name of donation institution: Korea Cell Line Research Foundation

Accession number: KCLRFBP00410

Consignment date: 20171010

Claims (11)

Myoblastic cell line established from the pectoralis muscle of chicken (KCLRF-BP-00410). The myoblastic cell line according to claim 1, wherein the myoblastic cell line established from the pectoralis muscle of the chicken is isolated and cultured from the pectoralis muscle of a 10-day-old male chick embryo. Treating a candidate material for regulating physiological activity in a myoblastic cell line (KCLRF-BP-00410) established from the pectoralis major muscle of the chicken;
Checking the gene expression level of the myoblastic cell line treated with the candidate material; And
Bioactive substance screening method comprising the step of comparing the identified gene expression level with an untreated control. The method of claim 3, wherein the muscle fibrous cell line established from the pectoralis muscle of the chicken is isolated and cultured from the pectoralis muscle of a 10-day-old male chick embryo. The bioactive substance screening according to claim 3, wherein the physiologically active substance increases or decreases the expression level of a gene that regulates cell proliferation, myogenesis, adipogenesis, or glycometabolism. Way. The bioactive substance screening according to claim 3, wherein the candidate substance is selected from the group consisting of natural product extracts, peptides, proteins, polysaccharides, amino acids, hormones, growth factors, antibiotics, anticancer agents, antibacterial agents, antiviral agents, and anticoagulants. Way. The method of claim 6, wherein the natural product extract is an angelica angelica or angelica byproduct extract. The method of claim 7, wherein the Angelicae by-product is an Angelicae stem or leaf. The method of claim 3, wherein the screening method is a method of selecting a bioactive substance for livestock feed additives. Treating a candidate material for muscle differentiation in a myoblastic cell line (KCLRF-BP-00410) established from the pectoralis major muscle of the chicken;
Checking the gene expression level of the myoblastic cell line treated with the candidate material; And
A method for screening a muscle differentiation regulator comprising the step of comparing the identified gene expression level with an untreated control. The method of claim 10, wherein the gene is selected from the group consisting of Pax7, MyoD, and desmin.

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