CN113755440A - AFB (active carbon boron)1Cell model establishing method for inducing chicken liver injury - Google Patents
AFB (active carbon boron)1Cell model establishing method for inducing chicken liver injury Download PDFInfo
- Publication number
- CN113755440A CN113755440A CN202111063902.6A CN202111063902A CN113755440A CN 113755440 A CN113755440 A CN 113755440A CN 202111063902 A CN202111063902 A CN 202111063902A CN 113755440 A CN113755440 A CN 113755440A
- Authority
- CN
- China
- Prior art keywords
- chicken liver
- cell line
- cancer cell
- liver cancer
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000287828 Gallus gallus Species 0.000 title claims abstract description 56
- 230000001939 inductive effect Effects 0.000 title claims abstract description 35
- 206010067125 Liver injury Diseases 0.000 title claims abstract description 25
- 231100000753 hepatic injury Toxicity 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 21
- PPWPWBNSKBDSPK-UHFFFAOYSA-N [B].[C] Chemical compound [B].[C] PPWPWBNSKBDSPK-UHFFFAOYSA-N 0.000 title description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 38
- 201000007270 liver cancer Diseases 0.000 claims abstract description 26
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 26
- 239000001963 growth medium Substances 0.000 claims abstract description 21
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims abstract description 14
- 239000012091 fetal bovine serum Substances 0.000 claims abstract description 12
- 239000002115 aflatoxin B1 Substances 0.000 claims abstract description 11
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 10
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 230000006698 induction Effects 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 238000007865 diluting Methods 0.000 claims abstract description 8
- 229930182555 Penicillin Natural products 0.000 claims abstract description 7
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims abstract description 7
- 229940049954 penicillin Drugs 0.000 claims abstract description 7
- 229960005322 streptomycin Drugs 0.000 claims abstract description 7
- 230000001464 adherent effect Effects 0.000 claims abstract description 6
- 239000002609 medium Substances 0.000 claims description 6
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 description 46
- 239000000243 solution Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 238000011160 research Methods 0.000 description 8
- 230000009471 action Effects 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 6
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 210000003494 hepatocyte Anatomy 0.000 description 5
- 229940118019 malondialdehyde Drugs 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 230000006378 damage Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 3
- 108010082126 Alanine transaminase Proteins 0.000 description 3
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 3
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 210000005229 liver cell Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 244000144977 poultry Species 0.000 description 3
- 239000003642 reactive oxygen metabolite Substances 0.000 description 3
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 2
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 2
- 231100000678 Mycotoxin Toxicity 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000005779 cell damage Effects 0.000 description 2
- 208000037887 cell injury Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000003908 liver function Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- 239000002636 mycotoxin Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 229930195730 Aflatoxin Natural products 0.000 description 1
- 241000228197 Aspergillus flavus Species 0.000 description 1
- 241000228230 Aspergillus parasiticus Species 0.000 description 1
- -1 Cell Counting Kit-8 Proteins 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108010040476 FITC-annexin A5 Proteins 0.000 description 1
- 206010019837 Hepatocellular injury Diseases 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 231100000439 acute liver injury Toxicity 0.000 description 1
- 231100000570 acute poisoning Toxicity 0.000 description 1
- 239000005409 aflatoxin Substances 0.000 description 1
- 210000001557 animal structure Anatomy 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 231100000739 chronic poisoning Toxicity 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 231100000334 hepatotoxic Toxicity 0.000 description 1
- 230000003082 hepatotoxic effect Effects 0.000 description 1
- 230000002625 immunotoxic effect Effects 0.000 description 1
- 231100000849 liver cell damage Toxicity 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000008816 organ damage Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
- C12N2500/62—DMSO
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Gastroenterology & Hepatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for establishing a cell model of chicken liver injury, which comprises the following steps: 1) preparing a DMEM/F12 culture medium containing 10% fetal bovine serum at a final concentration, wherein the culture medium contains penicillin and streptomycin; 2) subjecting aflatoxin B1Dissolving in dimethyl sulfoxide solution, diluting with the culture medium in step 1) to obtain inducing solution containing aflatoxin B1The final concentration is 0.05-0.25 mug/mL, and the final concentration of dimethyl sulfoxide is 0.01-0.025%; 3) culturing the chicken liver cancer cell line until the growth vigor is good and the state is stable; 4) and (3) when the chicken liver cancer cell line grows to 70-80% of the adherent wall, adding the induction liquid in the step 2), and performing induction culture for 6-24 hours to obtain the chicken liver cancer cell line.
Description
Technical Field
The invention belongs to a cell modelThe field of establishment, in particular to aflatoxin B1 (AFB1) A method for establishing a cell model for inducing chicken liver injury.
Background
According to the research of the Food and Agriculture Organization (FAO) of the United nations, more than about 25 percent of the grains in the whole world are polluted by Mycotoxins (Mycotoxins), and the pollution of aflatoxins (Aflataxin, AF) is the most serious. According to Chinese feed raw material research, the AF pollution ratio in Chinese corn is as high as 84%. AF is a secondary metabolite of Aspergillus flavus and Aspergillus parasiticus, and about 20 types of AF have been found so far, of which AFB1It is listed as the first grade carcinogen because of its strongest toxicity, greatest harm and widest distribution. At the same time, AFB1Is also the most potent cytotoxic substance, and enters the body to generate Reactive Oxygen Species (ROS) to cause oxidative stress, causing chronic or acute poisoning, and ultimately causing organ damage. It is reported that AFB1Has hepatotoxic, genotoxic and immunotoxic effects on animals, wherein the poultry is resistant to AFB1High susceptibility. By AFB1The poultry fed by the polluted feed can cause acute liver injury, reduced production performance, reduced disease resistance and even death, and brings great economic loss to the poultry industry.
The detoxified organ of the animal, the liver, is AFB1The major target organ of action. Although the animal itself is on AFB1Has certain detoxication metabolism ability, but the young animal organ is not completely developed, and the AFB is taken in for a long time1Can stimulate the liver cells to generate a large amount of ROS and promote the liver cells to die, thereby influencing the development and normal functions of the liver of young animals and seriously harming the healthy production of the animals.
The cell is an important model for in vitro test and molecular mechanism research, and is used for searching for preventing and treating AFB1The effect target point of inducing chicken liver injury has important effect. Most of cells commonly used for constructing a hepatocyte injury model at present are chicken primary hepatocytes, however, the chicken primary hepatocytes have the disadvantages of complicated separation and culture process, unstable cell state and the like in the application process. The prior art lacks an AFB with simple and convenient operation and stable effect1Induced chicken liver finesCellular injury model severely restricts AFB1In-vitro molecular mechanism research for inducing chicken liver injury and screening of prevention and treatment drugs. The chicken liver cancer cell Line (LMH) was established by a japanese scholar in 1981, and has the characteristics of adherent growth and epithelial cell-like morphology. LMH grows more rapidly and is easy to cultivate, and the survival rate is high, and the activity is good. Therefore, the LMH is taken as a model cell, and AFB with different mass concentrations is observed1Effect on survival and apoptosis ratio of LMH, evaluation of AFB1Method for establishing cell model for inducing chicken liver injury, and can be used for further researching AFB1Lays a foundation for inducing the action mechanism of chicken liver cell damage and also prevents and treats AFB in production1Provides an in vitro test model for chicken liver injury research.
Disclosure of Invention
In order to solve the technical problem, the invention provides an AFB1A method for establishing a cell model for inducing chicken liver injury aims at establishing a chicken liver cell injury model which is AFB and has simple operation and stable effect1The prevention and treatment research for inducing chicken liver injury provides a test model.
In order to achieve the purpose, the technical scheme of the invention is as follows: a method for establishing a cell model of chicken liver injury comprises the following steps: 1) preparing a complete culture medium; 2) subjecting AFB to1Dissolving in dimethyl sulfoxide (DMSO), and diluting with complete culture medium to obtain inducing solution; 3) Culturing a chicken liver cancer cell line; 4) adding the inducing solution into the cultured chicken liver cancer cell line to obtain the chicken liver cancer cell line.
Preferably, the complete medium is DMEM/F12 medium containing 10% fetal bovine serum at final concentration.
Preferably, the AFB in the inducing solution1The final concentration of (b) is 0.05-0.25. mu.g/mL.
Preferably, the condition for adding the inducing liquid into the cultured chicken liver cancer cell line is that the inducing liquid is added when the cell line is attached to 70-80%.
Preferably, the inducing liquid is added into the cultured chicken liver cancer cell line, and the inducing culture time is 6-24 h.
Method for establishing cell model of chicken liver injury, and cell modelThe method comprises the following steps: 1) preparing a DMEM/F12 culture medium containing 10% fetal bovine serum at a final concentration, wherein the culture medium contains penicillin and streptomycin; 2) subjecting AFB to1Dissolving in DMSO, diluting with the culture medium in step 1) to obtain inducing solution, and adding AFB in the inducing solution1The final concentration is 0.05-0.25 mug/mL, and the final concentration of DMSO is 0.01-0.025%; 3) culturing the chicken liver cancer cell line until the growth vigor is good and the state is stable; 4) and (3) when the chicken liver cancer cell line cells grow to 70-80% of the adherent cells, adding the induction liquid obtained in the step 2), and performing induction culture for 6-24 hours to obtain the chicken liver cancer cell line cells.
A method for establishing a cell model of chicken liver injury comprises the following steps: 1) preparing a DMEM/F12 culture medium containing 10% fetal bovine serum at a final concentration, wherein the culture medium contains penicillin and streptomycin; 2) subjecting AFB to1Dissolving in DMSO, diluting with the culture medium in step 1) to obtain inducing solution, and adding AFB in the inducing solution1The final concentration is 0.1 mug/mL, and the final concentration of DMSO is 0.01%; 3) culturing the chicken liver cancer cell line until the growth vigor is good and the state is stable; 4) and (3) when the chicken liver cancer cell line cells grow to 70-80% of the adherent cells, adding the induction liquid obtained in the step 2), and performing induction culture for 12 hours to obtain the chicken liver cancer cell line cells.
The invention has the beneficial effects that the establishment of AFB is determined1The optimal concentration and action time of a cell model for inducing chicken liver injury are established to further deeply research AFB (acute respiratory syndrome)1The action mechanism for inducing chicken liver injury and the application of the novel medicine provide a simple and stable hepatocyte injury model, and have important significance for reducing the economic loss of the breeding industry.
Drawings
FIG. 1 shows the different concentrations of AFB1Effect of 12h stimulation on LMH growth status. Wherein AFB of FIG. 1A1 Concentration 0, but 0.02% DMSO; AFB of FIG. 1B1The concentration is 0; AFB of FIG. 1C1The concentration is 0.05 mu g/mL; AFB of FIG. 1D1The concentration is 0.1 mug/mL; AFB of FIG. 1E1The concentration is 0.15 mug/mL; AFB of FIG. 1F1The concentration was 0.2. mu.g/mL.
FIG. 2 is AFB1Effect of 12h stimulation on LMH cell survival. P compared to control group<0.05;**P<0.01;***P<0.001。
FIG. 3 is AFB1Effect of 12h stimulation on LMH apoptosis rate. P compared to control group<0.05;**P<0.01;***P<0.001。
FIG. 4 is AFB1Effect on the rate of LMH apoptosis (flow cytogram). Wherein AFB of FIG. 4A 10 concentration, containing 0.02% DMSO; AFB of FIG. 4B1The concentration is 0; AFB of FIG. 4C1The concentration is 0.05 mu g/mL; AFB of FIG. 4D1The concentration is 0.1 mug/mL; AFB of FIG. 4E1The concentration is 0.15 mug/mL; AFB of FIG. 4F1The concentration was 0.15. mu.g/mL.
FIG. 5 is AFB1The influence of 12h stimulation on liver function indexes in LMH cell culture solution. P compared to control group<0.05;**P<0.01;***P<0.001。
Detailed Description
In order to make the objects, embodiments and advantages of the present invention more clear, the present invention will be further described in detail with reference to the accompanying drawings and examples. The following examples are merely illustrative of preferred embodiments of the present invention and are not intended to limit the scope of the invention.
Materials and methods
1. Principal material
Chicken liver cancer cell Line (LMH), aflatoxin B1(AFB1) Dimethyl sulfoxide (DMSO), 50mL of double antibody complete medium: 44.5mL DMEM/F12 medium +5mL fetal bovine serum FBS +0.5mL diabody (penicillin and streptomycin), DPBS at pH 7.2, 0.25% Trypsin-EDTA, 0.25% Trypsin, Cell Counting Kit-8, Annexin V-FITC/PI Apoptosis Detection Kit.
Wherein DMEM/F12 basal medium, polyclonal antibody, Australian fetal bovine serum, 0.25% Trypsin-EDTA, and 0.25% Trypsin were purchased from Gibco; DPBS was purchased from Hyclone; cell Counting Kit-8, aspartate Aminotransferase (AST) test Kit, alanine Aminotransferase (ALT) test Kit and Malondialdehyde (MDA) test Kit are all purchased from Nanjing to build bioengineering research institute Co., Ltd; AFB1Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Nyjinomo Zan Biotech, Inc.
2. Main instrument
5%CO2Incubator, inverted fluorescence microscope, water bath, super clean bench, common pipettor, Synergy HTX type multifunctional microplate reader, etc. Among them, Synergy HTX model multifunctional microplate readers were purchased from BioTek Instruments, inc.
Example 1
Establishing a cell model of chicken liver injury:
(1) the complete culture solution was prepared as follows (50mL system): 44.5mL DMEM/F12 medium +5mL fetal bovine serum FBS +0.5mL diabody (penicillin and streptomycin).
(2) Subjecting AFB to1Dissolving in DMSO, and diluting with complete culture medium to obtain AFB1The final concentration is 0.05-0.25 mug/mL, and the final concentration of DMSO is 0.01-0.025%, namely the inducing solution.
(3) And (3) culturing the chicken LMH, and obtaining cells with good growth vigor and stable state for subsequent experiments.
(4) And (3) inoculating the cells to a cell culture plate, keeping the cell amount of each hole consistent, enabling the cells to be 70-80% of the bottom of each hole after the cells are attached to the wall, and adding induction liquid with different concentrations into the culture holes for culture for 6-24 hours.
Example 2
And (3) cell survival rate detection:
the cell viability of each group was examined by the CCK-8 method, and the other 6 test groups were statistically analyzed with group 1 as a control group. Finding the lowest AFB with significantly reduced cell viability compared to the control group1The concentration in the test group and the appropriate action time period. The cell survival rate is calculated by the formula:
the results are shown in the following table:
TABLE 1 influence of LMH on cell viability (%)
Note: the difference of different shoulder marked letters is obvious, and P is less than 0.05.
Example 3
The apoptosis rate was measured by flow cytometry and the results are shown in the following table:
note: the difference of different shoulder marked letters is obvious, and P is less than 0.05.
Example 4
The AST and ALT test boxes are used for detecting the enzyme activity in the culture solution, and the results are as follows:
TABLE 3 influence of LMH cell liver function index
Note: the difference of different shoulder marked letters is obvious, and P is less than 0.05.
Example 5
Treating with ultrasonic disruptor for 5-10min to disrupt cells, with ultrasonic time of 2s, gap time of 3s, and power of 50%. Detecting the content of MDA in the cells by using an MDA test box:
TABLE 4 influence of MDA content of LMH cells
Note: the difference of different shoulder marked letters is obvious, and P is less than 0.05.
The results show that AFB was determined by a series of experiments1LMH can be acted upon explicitly. By establishing the cell damage model, the AFB can be further studied1The action mechanism for inducing chicken liver injury and the application of the novel medicine provide a simple and stable hepatocyte model, and have important significance for reducing the economic loss of the breeding industry.
Claims (7)
1. A method for establishing a cell model of chicken liver injury comprises the following steps: 1) preparing a complete culture medium; 2) subjecting aflatoxin B1Dissolving in dimethyl sulfoxide solution, and diluting with complete culture medium to obtain inducing solution; 3) culturing a chicken liver cancer cell line; 4) adding the inducing solution into the cultured chicken liver cancer cell line to obtain the chicken liver cancer cell line.
2. The method for establishing the chicken liver injury cell model of claim 1, wherein the complete culture medium is DMEM/F12 medium containing 10% fetal bovine serum at final concentration.
3. The method for establishing the chicken liver injury cell model as claimed in claim 1, wherein the aflatoxin B in the inducing liquid1The final concentration of (b) is 0.05-0.25. mu.g/mL.
4. The method for establishing the chicken liver injury cell model according to claim 1, wherein the inducing solution is added to the cultured chicken liver cancer cell line under the condition that the inducing solution is added when the cell line adherence is 70-80%.
5. The method for establishing the chicken liver injury cell model according to claim 1, wherein an inducing solution is added into the cultured chicken liver cancer cell line, and the inducing culture time is 6-24 h.
6. A method for establishing a cell model of chicken liver injury comprises the following steps: 1) preparing a DMEM/F12 culture medium containing 10% fetal bovine serum at a final concentration, wherein the culture medium contains penicillin and streptomycin; 2) subjecting aflatoxin B1Dissolving in dimethyl sulfoxide, diluting with the culture medium in step 1) to obtain inducing solution containing aflatoxin B1The final concentration is 0.05-0.25 mug/mL, and the final concentration of dimethyl sulfoxide is 0.01-0.025%; 3) culturing the chicken liver cancer cell line until the growth vigor is good and the state is stable; 4) when the chicken liver cancer cell line cells grow to 70-80% of the adherent cellsAdding the inducing liquid obtained in the step 2), and performing induced culture for 6-24 hours to obtain the product.
7. A method for establishing a cell model of chicken liver injury comprises the following steps: 1) preparing a DMEM/F12 culture medium containing 10% fetal bovine serum at a final concentration, wherein the culture medium contains penicillin and streptomycin; 2) subjecting aflatoxin B1Dissolving in dimethyl sulfoxide solution, diluting with the culture medium in step 1) to obtain inducing solution containing aflatoxin B1The final concentration is 0.1 mu g/mL, and the final concentration of dimethyl sulfoxide is 0.01 percent; 3) culturing the chicken liver cancer cell line until the growth vigor is good and the state is stable; 4) and (3) when the chicken liver cancer cell line cells grow to 70-80% of the adherent cells, adding the induction liquid obtained in the step 2), and performing induction culture for 12 hours to obtain the chicken liver cancer cell line cells.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111063902.6A CN113755440A (en) | 2021-09-10 | 2021-09-10 | AFB (active carbon boron)1Cell model establishing method for inducing chicken liver injury |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111063902.6A CN113755440A (en) | 2021-09-10 | 2021-09-10 | AFB (active carbon boron)1Cell model establishing method for inducing chicken liver injury |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113755440A true CN113755440A (en) | 2021-12-07 |
Family
ID=78794868
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111063902.6A Pending CN113755440A (en) | 2021-09-10 | 2021-09-10 | AFB (active carbon boron)1Cell model establishing method for inducing chicken liver injury |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113755440A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114717212A (en) * | 2022-04-01 | 2022-07-08 | 华中农业大学 | Glutathione mercaptotransferases as agents for controlling AFB1Application of detoxification enzyme causing duck liver injury |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110136163A1 (en) * | 2008-06-25 | 2011-06-09 | University College Cork, National University Of Ireland, Cork | Method of Toxicological Assessment |
CN111297859A (en) * | 2020-02-18 | 2020-06-19 | 中国人民解放军海军军医大学 | Application of koumine in preparation of medicine for treating liver cell injury |
-
2021
- 2021-09-10 CN CN202111063902.6A patent/CN113755440A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110136163A1 (en) * | 2008-06-25 | 2011-06-09 | University College Cork, National University Of Ireland, Cork | Method of Toxicological Assessment |
CN111297859A (en) * | 2020-02-18 | 2020-06-19 | 中国人民解放军海军军医大学 | Application of koumine in preparation of medicine for treating liver cell injury |
Non-Patent Citations (2)
Title |
---|
CHOI等: "Transcriptomic alterations induced by aflatoxin B1 and ochratoxin A in LMH cell line", 《POULT SCI.》 * |
张婷婷: "T-2 毒素调控鸡 CYP1A5 表达的分子机制研究", 《万方学位论文数据库》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114717212A (en) * | 2022-04-01 | 2022-07-08 | 华中农业大学 | Glutathione mercaptotransferases as agents for controlling AFB1Application of detoxification enzyme causing duck liver injury |
CN114717212B (en) * | 2022-04-01 | 2023-08-01 | 华中农业大学 | Glutathione sulfhydryl transferase as AFB control agent 1 Application of detoxication enzyme for duck liver injury |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN105540867B (en) | Cultivate floc sedimentation nutrients formula and preparation method and the application of biological flocculation and beneficial bacterium | |
CN102533654B (en) | Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof | |
Dunuweera et al. | Fruit Waste Substrates to Produce Single‐Cell Proteins as Alternative Human Food Supplements and Animal Feeds Using Baker’s Yeast (Saccharomyces cerevisiae) | |
CN104164388B (en) | One strain bacillus megaterium and the application in aquaculture thereof | |
CN113755440A (en) | AFB (active carbon boron)1Cell model establishing method for inducing chicken liver injury | |
Liu et al. | Elicitation of alkaloids in in vitro PLB (protocorm-like body) cultures of Pinellia ternata | |
CN103421740B (en) | In-vitro culture and proliferation method for human mesenchymal stem cells | |
CN108669382B (en) | Application of bupleurum extract as feed additive for grouper | |
Wang et al. | Plant hormones promote growth in lichen-forming fungi | |
CN115644066B (en) | Method for improving growth quantity and flavonoid content of longan embryo callus by using exogenous polyamine | |
CN113455502B (en) | Application of water-soluble naphthylacetic acid and indolebutyric acid in oocyst algae culture | |
CN102140439A (en) | In-vitro culture method of adipocytes of large yellow croaker | |
CN109266600A (en) | A kind of fibroblastic culture medium and fibroblastic method is cultivated using its | |
CN112891333B (en) | Application of all-trans retinoic acid in preparation of anti-transmissible gastroenteritis virus medicine | |
CN108865995A (en) | Promote Fiber differentiation composition and its application of the CD8 positive T cell proliferation in spleen source | |
CN115053935A (en) | Application of lactobacillus salivarius in breeding of litopenaeus vannamei larvae | |
Erkinugli et al. | The Effect of Chlorella Suspension on the Growth, Development and Blood Parameters of Broiler Chickens | |
CN104082134A (en) | Dendrobium embryoid suspension culture process | |
CN109423451A (en) | A kind of Wine brewing yeast strain KSA01 and application thereof | |
CN105969725B (en) | The purposes of fructus lycii red pigment | |
TWI521058B (en) | A method for culturing antrodia cinnamomea and a method for manufacturing an antrodia cinnamomea alcoholic extract | |
CN105567569A (en) | Culture method for mycosphaerella arachidicola of dendrobium sw. | |
CN105420118A (en) | Culture medium which is used for cultivating dendrobium candidum brown patch pathogen and contains special amino acid | |
CN105420117A (en) | Culture medium which is used for cultivating dendrobium candidum brown patch pathogen and contains special sugar source | |
CN110881589A (en) | Enhancer for improving immunity of aquatic animals and preparation method and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211207 |
|
RJ01 | Rejection of invention patent application after publication |