阅读说明：本技术 一种afb1诱导鸡肝脏损伤的细胞模型建立方法 (AFB (active carbon boron)1Cell model establishing method for inducing chicken liver injury ) 是由 刘文超 郭艳 赵志辉 高振 赵越 邱盛坚 金永燕 于 2021-09-10 设计创作，主要内容包括：本发明公开了一种鸡肝脏损伤的细胞模型得建立方法,其步骤是：1)配制含有终浓度为10％胎牛血清的DMEM/F12培养基,培养基中含有青霉素和链霉素；2)将黄曲霉毒素B-(1)溶解于二甲基亚砜溶液中,并用步骤1)中所述的培养基稀释,得到诱导液,诱导液中黄曲霉毒素B-(1)终浓度为0.05～0.25μg/mL,二甲基亚砜终浓度为0.01～0.025％；3)培养鸡肝癌细胞系至长势良好且状态稳定；4)当鸡肝癌细胞系生长至贴壁70～80％时,加入步骤2)中所述诱导液,诱导培养6～24h,即得。(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 B 1 Dissolving in dimethyl sulfoxide solution, diluting with the culture medium in step 1) to obtain inducing solution containing aflatoxin B 1 The 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.)

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.

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. 1A₁Concentration 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. 4A₁0 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.