Application of chicken embryo extract in promoting lactoferrin synthesis in mammary epithelial cells

Abstract

The invention provides application of chicken embryo extract in promoting lactoferrin synthesis in mammary epithelial cells, and relates to the technical field of in vitro culture. Experiments show that adding chicken embryo extract to a culture medium for culturing mammary epithelial cells can improve the expression of lactoferrin encoding genes in mammary epithelial cells, promote the secretion of lactoferrin, and thus improve the efficiency of biosynthesis of lactoferrin, thereby solving the technical problem in the prior art that the lactoferrin synthesis cost is high and the efficiency is low, and it is difficult to meet the market demand for lactoferrin.

Description

Technical Field

[0001] The invention relates to the technical field of in vitro culture, in particular to the application of chicken embryo extract in promoting mammary epithelial cells to synthesize lactoferrin. Background Art

[0002] Chicken embryo eggs, also known as "live eggs", "hairy eggs", "happy eggs", etc., refer to edible fertilized eggs that have not yet cracked their shells. They contain a variety of proteins, amino acids, growth factors, minerals, and vitamins. Chicken embryo eggs are not only a natural dietary supplement, known as "the public's nutritional supplement", but also can provide some key growth factors for cell culture, and are widely used to supplement the growth medium of various cell types, such as neural crest stem cells, chicken embryo fibroblasts, skeletal muscle stem cells and glomerular cells. They have potential health, medicinal and biological value.

[0003] LF is a multifunctional glycoprotein widely present in milk, saliva and other biological fluids. It has important biological activities such as antibacterial, antiviral, immunomodulatory and promotion of iron absorption.

[0004] At present, most of the research directions at home and abroad focus on the functions of LF, but there are few reports on the scientific issue of how to increase the production of LF. Based on the substantial increase in the demand for LF in China, how to get rid of the constraints of LF raw materials on my country has become an imminent issue.

[0005] Although various extraction and purification methods for LF have been developed, there are still many challenges in terms of its expression and activity. Existing production methods usually rely on complex fermentation technology, which is costly and inefficient, and it is difficult to meet the market demand for LF. In addition, the source of natural LF is limited, making it difficult to produce on a large scale.

[0006] Therefore, a new method and formulation are urgently needed to increase the expression level of LF, reduce the production cost, and ensure its biological activity.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] One of the purposes of the present invention is to provide an application of chicken embryo extract in promoting the synthesis of lactoferrin by mammary epithelial cells, so as to solve the technical problem that the efficiency of lactoferrin synthesis in the prior art is low and it is difficult to meet the market demand for LF.

[0009] A second object of the present invention is to provide a composition for promoting the synthesis of lactoferrin by mammary epithelial cells.

[0010] The third object of the present invention is to provide a method for promoting lactoferrin synthesis by mammary epithelial cells in vitro.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] In a first aspect, the present invention provides the use of chicken embryo extract in promoting lactoferrin synthesis in mammary epithelial cells.

[0013] Furthermore, the promoting mammary epithelial cells to synthesize lactoferrin includes increasing the expression of lactoferrin encoding gene and / or increasing the lactoferrin content.

[0014] Furthermore, the mammary epithelial cells are from mammals.

[0015] Furthermore, the mammal includes any one of humans, cattle, sheep, camels, horses, donkeys, reindeer, alpacas or llamas.

[0016] In a second aspect, the present invention provides a composition for promoting lactoferrin synthesis by mammary epithelial cells, comprising 0.25-0.5 mL / 100 mL of culture medium of chicken embryo extract.

[0017] Further, 0.25 mL / 100 mL of the culture medium contains chicken embryo extract.

[0018] Furthermore, the culture medium is a mammary epithelial cell culture medium.

[0019] Furthermore, the mammary epithelial cell culture medium includes DMEM culture medium and fetal bovine serum.

[0020] In a third aspect, the present invention provides a method for promoting lactoferrin synthesis by mammary epithelial cells in vitro, wherein the mammary epithelial cells are cultured using the above-mentioned composition.

[0021] In some specific embodiments, when the chicken embryo extract is added in an amount of 0.25 mL / 100 mL of culture medium, the culture time is at least 4 days;

[0022] When the chicken embryo extract is added in an amount of 0.5 mL / 100 mL of the culture medium, the culture time is at least 6 days.

[0023] The chicken embryo extract provided by the present invention is used in promoting the synthesis of lactoferrin by mammary epithelial cells. Experiments have shown that adding chicken embryo extract (CEE) to the culture medium for culturing mammary epithelial cells can increase the expression of lactoferrin encoding genes in mammary epithelial cells and promote the secretion of lactoferrin (LF), so as to improve the efficiency of biosynthesis of lactoferrin, thereby solving the technical problems in the prior art that the synthesis cost of lactoferrin is high and the efficiency is low, and it is difficult to meet the market demand for lactoferrin. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The growth curves of MAC-T cells from day 1 to day 6 under different CEE addition treatments provided in Example 2 of the present invention;

[0026] Figure 2 The MAC-T cell CK18 immunofluorescence staining images provided in Example 3 of the present invention without adding CEE and adding 0.25% CEE;

[0027] Figure 3 This is a microscopic observation diagram of MAC-T cells without adding CEE and with adding 0.25% CEE provided in Example 4 of the present invention;

[0028] Figure 4 This is a statistical graph of MAC-T cell counts without adding CEE and with adding 0.25% CEE provided in Example 4 of the present invention;

[0029] Figure 5 This is a statistical graph of LF content in MAC-T cells without CEE and with 0.25% CEE provided in Example 5 of the present invention;

[0030] Figure 6 This is a statistical graph of LF gene expression in MAC-T cells without CEE and with 0.25% CEE provided in Example 6 of the present invention;

[0031] Figure 7 This is a microscopic observation picture of MCF-10A cells without adding CEE and with adding 0.25% CEE provided in Example 8 of the present invention;

[0032] Figure 8 This is a statistical chart of MCF-10A cell counts without adding CEE and with adding 0.25% CEE provided in Example 8 of the present invention;

[0033] Fig. 9 This is a statistical graph of LF content in MCF-10A cells without CEE addition and with 0.25% CEE addition provided in Example 9 of the present invention. DETAILED DESCRIPTION

[0034] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0035] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0036] In one aspect, the present invention provides the use of chicken embryo extract in promoting lactoferrin synthesis in mammary epithelial cells.

[0037] Experiments have shown that adding chicken embryo extract (CEE) to the culture medium for culturing mammary epithelial cells can increase the expression of lactoferrin encoding genes in mammary epithelial cells and promote the secretion of lactoferrin (LF), thereby improving the efficiency of biosynthesis of lactoferrin. This solves the technical problem that the existing technology has high cost and low efficiency for lactoferrin synthesis, making it difficult to meet the market demand for lactoferrin.

[0038] In some specific embodiments, promoting the synthesis of lactoferrin by mammary epithelial cells includes increasing the expression of a lactoferrin encoding gene and / or increasing the lactoferrin content.

[0039] In some specific embodiments, the mammary epithelial cells are from a mammal.

[0040] Among them, mammals include animals that can secrete milk to feed their young.

[0041] Experiments have shown that chicken embryo extract can promote the expression of lactoferrin gene in human mammary epithelial cells and bovine mammary epithelial cells, and the mammary epithelial cell synthesis pathway includes the gene for expressing lactoferrin in mammary epithelial cells, so chicken embryo extract can promote the synthesis of lactoferrin by mammary epithelial cells from mammals. In some specific embodiments, the mammal includes any one of humans, cattle, sheep, camels, horses, donkeys, reindeer, alpacas or llamas.

[0042] According to another aspect of the present invention, there is also provided a composition for promoting lactoferrin synthesis by mammary epithelial cells, comprising 0.25-0.5 mL / 100 mL of chicken embryo extract.

[0043] The composition adds chicken embryo extract to the culture medium, and the chicken embryo extract can increase the expression of lactoferrin coding gene in mammary epithelial cells and synthesize lactoferrin.

[0044] Among them, the content of chicken embryo extract can be but is not limited to 0.25mL / 100mL culture medium, 0.27mL / 100mL culture medium, 0.3mL / 100mL culture medium, 0.33mL / 100mL culture medium, 0.35mL / 100mL culture medium, 0.38mL / 100mL culture medium, 0.4mL / 100mL culture medium, 0.43mL / 100mL culture medium, 0.45mL / 100mL culture medium, 0.48mL / 100mL culture medium or 0.5mL / 100mL culture medium, and can also be any content between 0.25~0.5mL / 100mL culture medium.

[0045] In some specific embodiments, 0.25 mL / 100 mL of culture medium is included.

[0046] In some specific embodiments, the culture medium is mammary epithelial cell culture medium.

[0047] In some specific embodiments, the mammary epithelial cell culture medium comprises DMEM medium and fetal bovine serum.

[0048] Specifically, DMEM culture medium plus fetal bovine serum can be selected, and penicillin and / or streptomycin can also be added.

[0049] According to another aspect of the present invention, a method for promoting lactoferrin synthesis by mammary epithelial cells in vitro is provided, wherein the mammary epithelial cells are cultured using the above composition.

[0050] By adding CEE to the culture medium for culturing mammary epithelial cells, the mammary epithelial cells are promoted to express the lactoferrin encoding gene and synthesize lactoferrin during the culture process.

[0051] Experimental discovery, when the addition of described chicken embryo extract is 0.25mL / 100mL culture medium, on the 4th day of cultivation, compared with not adding chicken embryo extract, cell amount has produced stable remarkable difference.On the 6th day of cultivation, the addition of chicken embryo extract is 0.5mL / 100mL culture medium compared with not adding chicken embryo extract, and cell amount has produced remarkable difference.In some specific embodiments, when the addition of described chicken embryo extract is 0.25mL / 100mL culture medium, the time of cultivation is at least 4 days.When the addition of described chicken embryo extract is 0.5mL / 100mL culture medium, the time of cultivation is at least 6 days.

[0052] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] Fetal bovine serum (FBS) and DMEM medium were purchased from Gibco (Grand Island Biological Company).

[0054] Preparation method of chicken embryo extract:

[0055] (1) Take 10-day-old specific pathogen-free chicken embryo eggs (SPF) that have been incubated in an incubator at a temperature of 37.8°C and a humidity of 60%. After cleaning, soak them in 75% alcohol for 20 minutes for disinfection;

[0056] (2) Under sterile conditions, use sterilized scissors and tweezers to place the chicken embryo egg with the air chamber facing upwards, use tweezers to break the eggshell at the air chamber, clamp the chicken embryo into a beaker, and remove the air sac, membrane sac, and chorioallantoic membrane;

[0057] (3) Rinse four times with sterile 4°C PBS solution until the solution is clear, add an equal amount of sterile 4°C Hanks solution, and then homogenize for 2 minutes using a high-speed homogenizer at 18,000 rpm.

[0058] (4) Divide the homogenized chicken embryos into several small portions and seal them tightly. Incubate them at 37°C for 30 minutes.

[0059] (5) After leaching, the samples were crushed using a non-contact ultrasonic crusher for 1 h at a temperature of 4 °C and a power of 1000 W. The treatment conditions were: treatment for 30 s, pause for 30 s, and repeat this cycle.

[0060] (6) Place the crushed chicken embryos in liquid nitrogen and thaw at room temperature. Repeat the freeze-thaw cycle twice.

[0061] (7) Centrifuge at 6000g for 10 min in a refrigerated centrifuge at 4°C, aspirate the supernatant, filter through 10um / 3um / 1um / 0.45um / 0.22um sterile filters in sequence, and store in a -80°C refrigerator for later use.

[0062] Example 1 Composition for promoting lactoferrin synthesis in mammary epithelial cells

[0063] The formula of the composition is shown in Table 1.

[0064] Table 1

[0065]

[0066] Among them, 0.125% CEE means adding 0.125 ml CEE to every 100 ml culture medium, 0.25% CEE means adding 0.25 ml CEE to every 100 ml culture medium, 0.5% CEE means adding 0.5 ml CEE to every 100 ml culture medium, 1% CEE means adding 1 ml CEE to every 100 ml culture medium, and 2% CEE means adding 2 ml CEE to every 100 ml culture medium.

[0067] Example 2 CCK8 method to measure the growth curve of MAC-T cells

[0068] Using the CCK8 method, 100ul of 2000 MAC-T cells were added to each well of a 96-well plate. After the cells were attached to the plate in complete medium without CEE for 12 hours, the control group was replaced with complete medium without CEE as shown in Table 1 (control group 1), and the experimental group was replaced with complete medium with 0.125-2% CEE (experimental groups 1-5). After every 1 day, 10ul of CCK-8 solution was added to each well. Incubate in the cell culture incubator for 2 hours, and then measure the absorbance at 450nm for 6 consecutive days.

[0069] The results are as follows Figure 1 As shown, the cell growth curves of the experimental group and the control group conform to the growth law of general cells. After adding different proportions of CEE of the present invention to the complete medium, the cell proliferation rate of 0.25% CEE added was significantly higher than that of the control group on the 2nd, 4th, 5th and 6th days, and the cell proliferation rate of 0.5% CEE added was significantly higher than that of the control group on the 6th day.

[0070] Example 3 Comparison of MAC-T cell CK18 immunofluorescence staining

[0071] After 36 h of adherence culture of MAC-T cells in the control group and experimental group 2, immunofluorescence staining was performed.

[0072] Preparation of PBSTx: Add Triton X-100 to PBS to a final concentration of 0.1% and mix well to obtain TBSTx.

[0073] Preparation of blocking solution BSA-PBSTx: Add 5g BSA into 100ml PBSTx, dissolve and mix to obtain BSA-PBSTx.

[0074] Preparation of primary antibody diluent: dilute CK18 antibody 300 times with blocking solution BSA-PBSTx.

[0075] Preparation of secondary antibody dilution: dilute the secondary antibody 200 times with blocking solution BSA-PBSTx.

[0076] 1. Take a clean cover glass and soak it in 70% ethanol for 5 minutes, wash it three times with cell culture grade PBS solution, and then wash it once with cell culture medium. Place the cover glass in a six-well plate and inoculate 2.5×10 5 After 12 hours, the control group was replaced with complete medium without chicken embryos and the control group was replaced with complete medium containing 0.25% chicken embryos. After 24 hours of continuous culture, the cell density was about 50%-80%.

[0077] 2. Discard the culture medium and wash three times with sterile PBS solution, 5 minutes each time. Add 1 ml of 4% paraformaldehyde fixative to each well and fix at 37°C for 10 minutes.

[0078] 3. Discard the fixative solution and wash three times with PBS solution, shaking gently by hand for 5 minutes each time, and absorb all the liquid.

[0079] 4. Block with blocking solution BSA-PBSTx at 37℃ for 60 minutes.

[0080] 5. Remove the blocking solution and add 1 ml of diluted primary antibody to each well and incubate at 4°C overnight.

[0081] 6. After overnight, remove the primary antibody and wash three times with PBS solution, 5 minutes each time, gently shaking during each wash.

[0082] 7. Remove the washing solution, add 1 ml of diluted fluorescent-labeled secondary antibody and incubate at 37°C in the dark for 1 hour.

[0083] 8. Drop a drop of anti-fluorescence quenching sealing solution on the slide, cover it with the cover slip with cells, and try to avoid bubbles. Make the cells contact with the sealing solution.

[0084] 9. Observe the fluorescence under a fluorescence microscope.

[0085] The results are as follows Figure 2 As shown in the figure, it can be seen from the CK18 immunofluorescence staining pictures that the addition of CEE did not cause obvious changes in the morphology and structure of the cells.

[0086] Example 4 Microscopic observation and cell counting of MAC-T cells

[0087] MAC-T cells were seeded in six-well plates at 2.5×10 5 The cells were cultured in complete medium without CEE for 12 h. The control group was replaced with complete medium without CEE (control group 1), and the experimental group was replaced with complete medium supplemented with 0.25% CEE (experimental group 2). Microscope photography and cell counting were performed the next day.

[0088] The results are as follows Figure 3As shown in the figure, after culturing MAC-T cells overexpressing LF by trypsin digestion for 48 hours, the cells grew well. Figure 4 The cell counting results showed that the number of cells cultured with 0.25% CEE was greater than that of cells in the control group without CEE.

[0089] Example 5 Elisa kit was used to detect the expression of LF in MAC-T cells.

[0090] MAC-T cells were seeded in six-well plates at 2.5×10 5 After culturing for 12 hours with complete medium without CEE, the control group was replaced with complete medium without CEE (control group 1), and the experimental group was replaced with complete medium with 0.25% CEE (experimental group 2). After culturing for another 12 hours, 20ul of 100ug / ml doxycycline (DOX) induction solution was added to each well of the six-well plate. On the third day of culturing, the cell culture medium was aspirated and centrifuged at 1000×g for 20 minutes, and the supernatant was taken to detect the expression of bovine LF.

[0091] 1. Sample addition: Set up standard wells, test sample wells, and blank wells. Set up 7 standard wells, and add 100μL of standards of different concentrations in sequence. Add 100μL of standard diluent to the blank wells, and 100μL of the test sample to the remaining wells. Cover the ELISA plate with a film and incubate at 37℃ for 1 hour.

[0092] 2. Discard the liquid, spin dry, and do not wash.

[0093] 3. Add 100 μL of detection solution A working solution to each well (prepared before use), cover the ELISA plate, and incubate at 37°C for 1 hour.

[0094] 4. Discard the liquid in the wells, wash each well with 350μL of washing buffer, soak for 1-2 minutes, and tap the plate on absorbent paper to remove all the liquid in the wells. Repeat the plate wash 3 times. After the last wash, absorb or pour out the remaining washing buffer, turn the plate upside down on absorbent paper, and absorb all the liquid remaining in the wells.

[0095] 5. Add 100 μL of detection solution B working solution (prepared before use) to each well, cover the ELISA plate, and incubate at 37°C for 30 min.

[0096] 6. Discard the liquid in the wells, spin dry, and wash the plate 5 times using the same method as step 4.

[0097] 7. Add 90 μL of TMB substrate solution to each well, cover the ELISA plate, and color develop at 37°C in the dark for 10 minutes. When the first 3-4 wells of the standard wells have a clear gradient of blue, and the gradient of the last 3-4 wells is not obvious, the process can be terminated.

[0098] 8. Add 50 μL of stop solution to each well to stop the reaction. The blue color will turn yellow immediately. The order of adding the stop solution should be the same as that of adding the substrate solution.

[0099] 9. After ensuring that there are no water drops on the bottom of the ELISA plate and no bubbles in the wells, immediately measure the absorbance of each well at a wavelength of 450nm using an ELISA reader, and then convert the absorbance into LF concentration.

[0100] The results are as follows Figure 5 As shown, it can be seen that the LF content of cells in the experimental group supplemented with 0.25% CEE was higher than that in the control group.

[0101] Example 6 RT-qPCR detection of LF gene expression in MAC-T cells

[0102] MAC-T cells were seeded in six-well plates at 2.5×10 5 After culturing for 12 h with complete medium without CEE, the control group was replaced with complete medium without CEE (control group 1), and the experimental group was replaced with complete medium with 0.25% CEE (experimental group 2). After culturing for another 12 h, 20 ul of 100 ug / ml DOX induction solution was added to each well of the six-well plate. The next day, RNA was extracted, reverse transcribed into cDNA, and then qPCR amplification was performed. The target fragment length was 774 bp. The amplification primer information is shown in Table 2, the reaction system is shown in Table 3, and the reaction conditions are shown in Table 4.

[0103] Table 2

[0104]

[0105] Table 3

[0106]

[0107] The PCR mixture was 2× Rapid Taq Master Mix (Vazyme, Code No: P222).

[0108] Table 4

[0109]

[0110] The results are as follows Figure 6 As shown, the expression of LF gene in the experimental group was significantly higher than that in the control group.

[0111] Example 7 Configuration of MCF-10A cell culture medium

[0112] The formula of the MCF-10A cell proliferation culture medium in this experiment is shown in Table 5.

[0113] Table 5

[0114]

[0115] Example 8 Microscopic observation and cell counting of MCF-10A cells

[0116] MCF-10A cells were seeded in six-well plates at 2.5 × 10 5 The cells were cultured in complete medium without CEE for 12 h. The control group was replaced with complete medium without CEE (control group 2), and the experimental group was replaced with complete medium supplemented with 0.25% CEE (experimental group 6). 20 ul of 0.25 mg / ml prolactin induction solution was added to each well of the six-well plate, and microscope photography and cell counting were performed on the third day.

[0117] The results are as follows Figure 7 As shown in the figure, after culturing MCF-10A cells with trypsin digestion for 72 hours, the cells grew well. Figure 8 The cell counting results showed that there was no significant difference in the number of cells cultured with 0.25% CEE and those in the control group without CEE.

[0118] Example 9 Elisa was used to detect the expression of LF in MCF-10A cells.

[0119] MCF-10A cells were seeded in six-well plates at 2.5 × 10 5 After culturing for 12 hours with complete medium without CEE, the control group was replaced with complete medium without CEE (control group 2), and the experimental group was replaced with complete medium with 0.25% CEE (experimental group 6). 20ul of 0.25mg / ml prolactin induction solution was added to each well of the six-well plate. After culturing for the third day, the cell culture medium was aspirated and centrifuged at 1000×g for 20min, and the supernatant was taken to detect the expression of bovine LF. (The detection steps are consistent with the detection of LF expression in Example 5).

[0120] The results are as follows Fig. 9 As shown in the figure, it can be seen that the LF content of cells in the experimental group with 0.25% CEE was significantly higher than that in the control group. Figure 8 The data showed that the addition of CEE could increase LF production even when the cells did not undergo significant proliferation.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of chicken embryo extract in promoting lactoferrin synthesis in mammary epithelial cells; The concentration of the chicken embryo extract is 0.25-0.5 mL / 100 mL culture medium; The mammary epithelial cells are from mammals, including any one of humans, cattle, sheep, camels, reindeers or alpacas.

2. The use according to claim 1, characterized in that: The method of promoting the synthesis of lactoferrin by mammary epithelial cells includes increasing the expression level of lactoferrin encoding gene and / or increasing the lactoferrin content.

3. A method for promoting lactoferrin synthesis by mammary epithelial cells in vitro, characterized in that: Mammary epithelial cells were cultured using chicken embryo extract; The concentration of the chicken embryo extract is 0.25-0.5 mL / 100 mL culture medium; The mammary epithelial cells are from mammals, including any one of humans, cattle, sheep, camels, reindeers or alpacas.

4. The method according to claim 3, characterized in that: When the chicken embryo extract is added in an amount of 0.25 mL / 100 mL of the culture medium, the culture time is at least 4 days; When the chicken embryo extract is added in an amount of 0.5 mL / 100 mL of the culture medium, the culture time is at least 6 days.

Images