CN109182247B - In-vitro isolation culture and steatosis model establishment method for chick embryo hepatocytes - Google Patents
In-vitro isolation culture and steatosis model establishment method for chick embryo hepatocytes Download PDFInfo
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Abstract
The invention discloses a method for in vitro isolation culture of chick embryo hepatocytes and establishment of a steatosis model, belonging to the technical field of cytotoxicosis. The method comprises the following steps: (1) taking SPF chick embryos; (2) shearing into pieces; (3) digesting; (4) centrifuging: discarding the supernatant, and adding the precipitate into preheated Williams' E complete culture solution for resuspension to obtain a cell suspension B; (5) cell separation: performing Percoll density gradient centrifugation; (6) taking the liver cells: sucking cells at an interface, adding D-Hank's solution, blowing, uniformly mixing, placing in a centrifuge, centrifuging, discarding the supernatant, reserving the precipitate, adding D-Hank's solution, washing, re-suspending, repeatedly centrifuging for 2-3 times, discarding the supernatant, reserving the precipitate, adding Williams ' E complete culture solution, and obtaining cell suspension C; (7) counting; (8) a seed plate; (9) liquid changing; (10) induction of steatosis: and after 30-40h of plating, replacing the inducer for culturing for 10-15h to obtain the fatty degeneration liver cells. The invention can rapidly obtain primary culture cells, has high purity and short induction time.
Description
Technical Field
The invention relates to the technical field of cell toxicology, in particular to a method for in vitro isolation culture of chick embryo hepatocytes and establishment of a steatosis model.
Background
Layer chicken fatty liver is also called fatty liver syndrome or fatty liver hemorrhagic syndrome and is a common nutritional metabolic disease of layer chicken. Mainly, the fat is excessively accumulated in the liver cells, the normal functions of the liver are affected, and the liver cells are seriously broken even, and finally, the liver cells are bleeded and die. The laying peak of the chicken flocks with fatty liver is difficult to appear, and the laying rate generally rises to about 85 percent and then gradually falls. The disease occurs everywhere, the incidence rate is about 5%, and the disease brings great economic loss to the laying hen industry.
The liver is the core organ of material metabolism and plays an important role in the processes of digestion, absorption, decomposition, synthesis, transportation and the like of lipids. Factors affecting intrahepatic fat content can be broadly divided into two major categories, those that increase intrahepatic fat and those that decrease intrahepatic fat. Among the factors that increase intrahepatic fat include fatty acids (acids) that enter the liver with blood; fat produced by gluconeogenesis or proteinogenesis in the liver due to overnutrition. Factors that reduce intrahepatic fat include oxidation of fat in hepatocytes; intrahepatic fat is transported out of the liver by binding to lipoproteins. Whether fat is accumulated in the liver or not depends on the comprehensive balance of the four aspects, and abnormality in any aspect can cause accumulation of fat in the liver, so that the fatty liver is finally generated. Therefore, the exact reason for fatty liver is not clear, and there is no specific preventive treatment so far.
The development of the fatty liver prevention and control research of the laying hens has important significance for exerting the laying performance of the laying hens and reducing the economic loss. The in vitro culture of the primary chicken liver cells well maintains the biological properties of the in vivo liver cells, reflects the in vivo state of the chicken liver cells to a certain extent and shows the characteristics of original tissues. The chicken liver cell fatty degeneration model is prepared by using primary cultured chicken liver cells, and the research on the metabolic mechanism of the fatty degeneration liver cells is beneficial to developing effective medicaments for preventing and treating fatty liver syndrome.
The invention aims to establish a chicken hepatocyte separation and culture technology and provides a technical platform for chicken lipid regulation and metabolic mechanism research.
Disclosure of Invention
The invention aims to provide a method for in vitro isolation culture and steatosis model establishment of chick embryo hepatocytes, which can obtain high-purity primary culture cells, and meanwhile, adopts an inducer to successfully establish an adipocyte model through induction, so that the induction time is short.
In order to realize the aim, the method for in vitro isolation culture of chick embryo hepatocytes and establishment of a steatosis model adopts the following technical scheme:
a chicken embryo hepatocyte in-vitro separation culture and steatosis model building method comprises the following steps:
(1) taking SPF chick embryos: selecting more SPF eggs of 13-17 days old, sterilizing the surface of an eggshell by using alcohol, breaking the eggshell and taking an eggshell;
(2) shearing: opening abdominal cavity of chick embryo, taking out liver, and shearing into D-Hank's solution to 0.5-1.5mm3The small blocks are cleaned by D-Hank's solution after being trimmed;
(3) digestion: adding trypsin into the liver cleaned in the step (2) for digestion, stirring in a water bath for 8-12min, keeping the temperature of the water bath at 37 ℃, repeatedly blowing and beating the liver with a suction pipe until the cut liver disappears after digestion is finished, and filtering to obtain a cell suspension A; the cells are digested into a single cell form by adding trypsin for digestion, so that primary culture cells are quickly obtained, and the establishment of a cell model is facilitated;
(4) centrifuging: placing the cell suspension A in the step (3) in a centrifuge for centrifugation, controlling the rotation speed to 800-; removing cell debris with smaller particles in the cell suspension A through centrifugation to obtain uniform cells, and meanwhile, repeatedly centrifuging for many times to improve the cell purity;
(5) cell separation: performing Percoll density gradient centrifugation, suspending the cell suspension B on the Percoll separating medium, controlling the rotation speed of 2500-; adopting Percoll density gradient centrifugation to screen out cells with smaller density difference;
(6) taking the liver cells: after centrifugation in the step (5), sucking off the middle and upper layer cells and liquid, reserving cells at an interface, adding D-Hank's liquid to blow, uniformly mixing, then placing the mixture into a centrifuge for centrifugation, controlling the rotation speed to 800 plus 1300rpm, centrifuging for 5-8min, discarding the supernatant, reserving the precipitate, adding D-Hank's liquid to wash and resuspend, continuously centrifuging after resuspending the cells, controlling the rotation speed to 800 plus 1300rpm, centrifuging for 5-8min, repeatedly centrifuging for 2-3 times, then discarding the supernatant, reserving the precipitate, adding Williams ' E complete culture solution, and uniformly mixing to obtain a cell suspension C; after Percoll density gradient centrifugation, sucking cells at the interface for continuous centrifugation, further removing cell debris to obtain uniform cells, and centrifuging for multiple times to obtain separated cells with high purity;
(7) counting: adding trypan blue into the cell suspension C obtained in the step (6), uniformly mixing, adding a cell counting plate, and counting through the cell counting plate;
(8) plate preparation: according to the counting result of the cell counting plate in the step (7), Williams' E complete culture solution is added for dilution, and the cell density is kept to be 1X 105-5×105Inoculating to cell culture plate, mixing, culturing in cell culture box at 37 deg.C under CO2The concentration is 5%;
(9) liquid changing: changing Williams' E complete culture solution once every 20-26 h;
the high-purity hepatic cells are separated and cultured by the steps, the hepatic tissue is directly digested by trypsin to form single cell suspension, the problem that the hepatic tissue is directly cultured in the prior art so as to be easily polluted by fiber cells is solved, meanwhile, the trypsin is low in price and cost, then, the digested single cell suspension is centrifugally separated for multiple times, a common small centrifugal machine is low in price and can be used for multiple times, the cost is low, in addition, a Percoll density gradient centrifugation method is adopted in the separation process to improve the cell purity, and the price of a commercially available Percoll separation solution is not high;
(10) induction of steatosis: preparing an inducer, wherein the inducer is a Williams' E complete culture solution containing oleic acid, and the concentration of the oleic acid is 0-1 mmol/L; and (4) after 30-40h of plating, replacing the culture solution, sucking off the Williams' E complete culture solution added in the step (9), replacing the inducer, culturing for 10-15h, and obtaining the fatty degeneration liver cells.
Preferably, the inducer in step (10) is prepared by the following steps:
1) preparing an oleic acid storage solution: weighing 50mg of sodium oleate, placing the sodium oleate into a test tube, adding 1080 mu L of 0.1mmol/L NaOH solution into the test tube, placing the test tube in a water bath at the temperature of more than 90 ℃, heating the test tube, continuously shaking the test tube for 10min, placing the test tube in ultrasonic waves at intervals, carrying out ultrasound for 3 times for 30 seconds each time, then adding BSA solution into the test tube while the test tube is hot to ensure that the final volume is 10.8ml, and the final concentration of an oleic acid storage solution is 15 mmol/L;
2) preparing an inducer: the oleic acid stock was added to Williams 'E complete medium and the oleic acid stock was diluted to 0-1mmol/L by Williams' E complete medium.
Preferably, the oleic acid-containing Williams' E complete medium in step (10) has an oleic acid concentration of 0-0.5 mmol/L.
Preferably, the filtering in the step (3) comprises two-stage filtering, wherein the first-stage filtering is 50-mesh screen filtering, and the second-stage filtering is 200-mesh screen filtering.
Preferably, the Williams 'E complete culture solution contains penicillin 10 per 100mL of Williams' E culture medium4U, streptomycin 10mg, glutamine 0.2mmol, 15ml fetal bovine serum.
Preferably, the amount of the trypsin is determined according to the number of the chick embryos, and 800 mu L to 1mL of 0.25% trypsin is added into 15 chick embryos.
Preferably, the amount of Williams 'E complete medium added in step (6) is determined by the number of chick embryos, and the hepatocytes collected from 10 chick embryos are added to 35-45ml of Williams' E complete medium.
Compared with the prior art, the invention has the beneficial effects that:
1. the culture method of the invention overcomes the problems of high cost and easy pollution of fibroblasts in the prior art for culturing the chick embryo hepatocytes, can quickly obtain primary culture cells with the purity of more than 95 percent, is beneficial to establishing a steatosis model by the in vitro culture method of the liver cells, and is correlated with the steatosis model;
2. according to the invention, the single cell suspension after trypsinization is subjected to centrifugal separation for multiple times, differential centrifugation and density gradient centrifugation are combined to remove cell debris, and the purity of the separated cells is ensured to be high; meanwhile, in the separation process, low-rotation-speed repeated centrifugation, high-rotation-speed density gradient centrifugation and low-rotation-speed repeated centrifugation are adopted in sequence, so that cell fragments are gradually removed, the cell separation effect is ensured, and the purity is improved;
3. according to the invention, oleic acid is dissolved in a weakly alkaline BSA solution, so that oleic acid in a Williams' E complete culture solution is uniformly distributed and fully contacted with cells, the oleic acid can fully induce hepatocyte degeneration, the induction time is short, the hepatocyte steatosis is uniform, a chick embryo hepatocyte steatosis model is successfully established, and the obtained cell model can be used for toxicology tests of poultry and prevention and treatment research of laying hen fatty liver syndrome;
4. the inducer is a Williams' E complete culture solution containing oleic acid, the concentration of the oleic acid is 0-1mmol/L, the concentration of the oleic acid directly influences the steatosis of the cells, the more preferable concentration of the oleic acid adopted by the invention is 0-0.5mmol/L, the steatosis of the liver cells is successfully induced, and the lipotropy rate of the cells is high.
Drawings
FIG. 1 is an observation view of chick embryo hepatocytes at different culture times;
FIG. 2 is a graph comparing glycogen staining of hepatocytes of a control group and glycogen staining of hepatocytes of a model group in accordance with the present invention;
FIG. 3 is a microscopic view of intracellular lipid droplets after oil red staining;
FIG. 4 is the transmission electron microscope observation picture of the ultrastructure of chick embryo liver cells.
Detailed Description
The present invention is further illustrated by the following detailed description, which is to be construed as merely illustrative and not limitative of the remainder of the disclosure, and modifications and variations such as those ordinarily skilled in the art are intended to be included within the scope of the present invention as defined in the appended claims.
Example 1:
a chicken embryo hepatocyte in-vitro separation culture and steatosis model building method comprises the following steps:
(1) taking SPF chick embryos: selecting more SPF eggs with the age of 13 days, sterilizing the surface of an eggshell by using 75% alcohol, breaking the eggshell with big heads, taking chick embryos, and killing the chick embryos after breaking the heads;
(2) shearing: opening abdominal cavity of chick embryo, taking out liver, placing into culture dish containing D-Hank's solution, and trimming liver to 0.5mm in culture dish with ophthalmic scissors3The small blocks are cut for a period of time, the D-Hank's solution is changed to remove blood cells as much as possible, after the small blocks are cut, the cut liver is transferred to a sterile centrifuge tube and washed for 2 times by the D-Hank's solution;
(3) digestion: transferring the liver cleaned in the step (2) into a 50mL beaker with a rotor, placing the beaker on a water bath constant-temperature magnetic stirrer, adding trypsin for digestion, wherein the using amount of the trypsin is determined according to the number of chicken embryos, adding 800 mu L of 0.25% trypsin into 15 chicken embryos, keeping the water bath temperature at 37 ℃, digesting for 8min, repeatedly blowing and beating the chicken embryos by using a suction pipe after digestion is completed until the cut liver disappears, wherein in order to prevent over digestion, the whole liver blocks are not required to be digested, a little amount of the trypsin can be remained, the edge of the liver adsorbed on the wall is in a dispersed state, and then respectively filtering the liver by a 50-mesh sieve for the first time and filtering the liver by a 200-mesh sieve for the second time to obtain a cell suspension A;
(4) centrifuging: subpackaging the cell suspension A filtered in the step (3) into a plurality of first centrifuge tubes, adding 10mL of cell suspension A into each first centrifuge tube, placing the first centrifuge tube into a centrifuge, controlling the rotation speed to be 800rpm, centrifuging for 5min, removing the supernatant, reserving the precipitate, adding 8mL of D-Hank's solution into the first centrifuge tube, washing and resuspending, re-suspending the cells, continuing to centrifuge, controlling the rotation speed to be 800rpm, centrifuging for 5min, repeatedly centrifuging for 3 times, washing and resuspending the D-Hank's solution for 2 times, then removing the supernatant, reserving the precipitate, adding 2mL of Williams ' E complete culture solution preheated at 37 ℃ into the first centrifuge tube, and re-suspending to obtain cell suspension B;
(5) cell separation: adopting Percoll density gradient centrifugation, preparing a plurality of second centrifuge tubes, paving 4mL of Percoll separating liquid in each second centrifuge tube, suspending 1mL of cell suspension B on the Percoll separating liquid, adding the cell suspension slowly, attaching a dropper to the wall of the centrifuge tube, adding dropwise while rotating the second centrifuge tube to form an interface between a cell layer and the centrifuging liquid, placing the second centrifuge tube in a centrifuge for centrifugation, controlling the rotation speed to be 2500rpm, and centrifuging for 10 min; wherein, the preparation step of the Percoll separating medium is as follows, 3mL of 100 percent Peroll is taken and added with 2mL of PBS to obtain the Percoll separating medium;
(6) taking the liver cells: after centrifugation in the step (5), sucking off upper cells and liquid in a second centrifuge tube by using a pipette gun, sucking cells at an interface, adding the cells into a third centrifuge tube, adding 8mL of D-Hank's liquid, blowing, beating and uniformly mixing, adding the D-Hank's liquid to reduce the concentration of the Percoll separating liquid and reduce the influence of the Percoll separating liquid on the cells as much as possible, then placing the mixture into a centrifuge for centrifugation, controlling the rotation speed to be 800rpm, centrifuging for 5min, discarding supernatant, reserving precipitate, adding 8mL of D-Hank's liquid into the third centrifuge tube for washing and resuspending, continuing centrifugation after cell resuspension, controlling the rotation speed to be 1000rpm, centrifuging for 5min, repeatedly centrifuging for 2 times, discarding supernatant, reserving precipitate, uniformly mixing and adding Williams' E complete culture solution, and obtaining cell suspension C in a 100mL beaker; the addition amount of Williams 'E complete culture solution is determined according to the number of chick embryos, and 35ml of Williams' E complete culture solution can be used for collecting hepatocytes from 10 chick embryos;
(7) counting: adding 6 mu L of the cell suspension C obtained in the step (6) into a fourth centrifuge tube, continuously adding 6 mu L of trypan blue into the fourth centrifuge tube, uniformly mixing, adding into a cell counting plate, and counting through the cell counting plate;
(8) plate preparation: according to the counting result of the cell counting plate in the step (7), the cell density is 1 multiplied by 106Per mL, then add Williams' E complete medium to dilute the cell suspension C concentration to 1X 105Inoculating the cell suspension C on a cell culture plate, uniformly mixing, and putting into a cell culture box for culture, wherein the culture temperature of the cell culture box is 37 ℃, and the culture temperature is CO2The concentration is 5%;
(9) liquid changing: changing the culture solution once every 20h, sucking off blood cells and cell fragments containing non-adherent blood cells and cell fragments during the culture solution changing, sucking off old Williams ' E complete culture solution, washing the cell culture plate twice by using D-Hank's solution, adding fresh Williams ' E complete culture solution again, slightly inclining the cell culture plate during the culture solution changing, sucking waste liquid, and slowly dripping from the middle of the cell culture plate during the liquid adding to prevent the cells from being washed;
(10) induction of steatosis: preparing an inducer, wherein the inducer is a Williams' E complete culture solution containing oleic acid, and the concentration of the oleic acid is 0.2 mmol/L; and (5) after 30h of plating, replacing the culture solution, sucking off the Williams' E complete culture solution added in the step (9), replacing the inducer, culturing for 10h, and obtaining the fatty degeneration liver cells.
The preparation steps of the inducer in the step (10) are as follows:
1) preparing an oleic acid storage solution: weighing 50mg of sodium oleate, placing the sodium oleate into a test tube, adding 1080 mu L of 0.1mmol/L NaOH solution into the test tube, placing the test tube in a water bath at the temperature of more than 90 ℃, heating the test tube, continuously shaking the test tube for 10min, placing the test tube into ultrasonic waves at intervals, carrying out ultrasonic treatment for 3 times for 30 seconds each time, transferring the test tube into a 15ml centrifugal tube while the test tube is hot, adding 1% BSA solution into the test tube to ensure that the final volume is 10.8ml, and the final concentration of an oleic acid storage solution is 15 mmol/L;
2) preparing an inducer: the oleic acid stock solution was added to Williams 'E complete medium, and the concentration of the oleic acid stock solution was diluted to 0.2mmol/L by Williams' E complete medium, specifically, 80. mu.L of 15mmol/L oleic acid stock solution was added to 6mL of Williams 'E complete medium to prepare 0.2mmol/L oleic acid Williams' E complete medium.
The Williams 'E complete medium contains penicillin 10 per 100mL of Williams' E medium4U, streptomycin 10mg, glutamine 0.2mmol, 15ml fetal bovine serum.
Example 2:
a chicken embryo hepatocyte in-vitro separation culture and steatosis model building method comprises the following steps:
(1) taking SPF chick embryos: selecting more SPF eggs with the age of 15 days, sterilizing the surface of an eggshell by using 75% alcohol, breaking the eggshell with big heads, taking chick embryos, and killing the chick embryos after breaking the heads;
(2) shearing: opening abdominal cavity of chick embryo, taking out liver, placing into culture dish containing D-Hank's solution, and trimming liver to 1mm with ophthalmic scissors3The small blocks are cut for a period of time, the D-Hank's solution is changed to remove blood cells as much as possible, after the small blocks are cut, the cut liver is transferred to a sterile centrifuge tube and washed for 2 times by the D-Hank's solution;
(3) digestion: transferring the liver cleaned in the step (2) into a 50mL beaker with a rotor, placing the beaker on a water bath constant-temperature magnetic stirrer, adding trypsin for digestion, wherein the using amount of the trypsin is determined according to the number of chicken embryos, adding 900 mu L of 0.25% trypsin into 15 chicken embryos, keeping the water bath temperature at 37 ℃, digesting for 10min, repeatedly blowing and beating the chicken embryos by using a suction pipe after digestion is completed until the cut liver disappears, wherein in order to prevent over digestion, the whole liver blocks are not required to be digested, a little amount of the trypsin can be remained, the edge of the liver adsorbed on the wall is in a dispersed state, and then respectively filtering the liver by a 50-mesh sieve for the first time and filtering the liver by a 200-mesh sieve for the second time to obtain a cell suspension A;
(4) centrifuging: subpackaging the cell suspension A filtered in the step (3) into a plurality of first centrifuge tubes, adding 10mL of cell suspension A into each first centrifuge tube, placing the first centrifuge tube into a centrifuge, controlling the rotation speed to be 1000rpm, centrifuging for 6min, removing the supernatant, reserving the precipitate, adding 10mL of D-Hank's solution into the first centrifuge tube, washing and resuspending, continuously centrifuging after resuspending the cells, controlling the rotation speed to be 1000rpm, centrifuging for 6min, repeatedly centrifuging for 3 times, washing and resuspending the D-Hank's solution for 2 times, then removing the supernatant, reserving the precipitate, adding 2mL of Williams ' E complete culture solution preheated at 37 ℃ into the first centrifuge tube, and resuspending to obtain cell suspension B;
(5) cell separation: adopting Percoll density gradient centrifugation, preparing a plurality of second centrifuge tubes, paving 5mL of Percoll separating liquid in each second centrifuge tube, suspending 2mL of cell suspension B on the Percoll separating liquid, adding the cell suspension slowly, attaching a dropper to the wall of the centrifuge tube, dropwise adding while rotating the second centrifuge tube to enable the cell layer and the centrifuging liquid to form an interface, placing the second centrifuge tube in a centrifuge for centrifugation, controlling the rotating speed to be 3000rpm, and centrifuging for 15 min; wherein, the preparation step of the Percoll separating medium is as follows, 3mL of 100 percent Peroll is taken and added with 2mL of PBS to obtain the Percoll separating medium;
(6) taking the liver cells: after centrifugation in the step (5), sucking off upper cells and liquid in a second centrifuge tube by using a pipette gun, sucking cells at an interface, adding the cells into a third centrifuge tube, adding 10mL of D-Hank's liquid, blowing, beating and uniformly mixing, adding the D-Hank's liquid to reduce the concentration of the Percoll separating liquid and reduce the influence of the Percoll separating liquid on the cells as much as possible, then placing the mixture into a centrifuge for centrifugation, controlling the rotation speed to be 1000rpm, centrifuging for 6min, discarding supernatant, reserving precipitate, adding 10mL of D-Hank's liquid into the third centrifuge tube for washing and resuspending, continuing centrifugation after cell resuspension, controlling the rotation speed to be 1000rpm, centrifuging for 6min, repeatedly centrifuging for 2 times, discarding supernatant, reserving precipitate, uniformly mixing and adding Williams' E complete culture solution, and obtaining cell suspension C in a 100mL beaker; the addition amount of Williams 'E complete culture solution is determined according to the number of chick embryos, and 40ml of Williams' E complete culture solution can be used for collecting hepatocytes from 10 chick embryos;
(7) counting: adding 10 mu L of the cell suspension C obtained in the step (6) into a fourth centrifuge tube, continuously adding 10 mu L of trypan blue into the fourth centrifuge tube, uniformly mixing, adding into a cell counting plate, and counting through the cell counting plate;
(8) plate preparation: according to the counting result of the cell counting plate in the step (7), the cell density is 1.2 multiplied by 106Per mL, then add Williams' E complete medium to dilute the cell suspension C concentration to 2X 105Inoculating the cell suspension C on a cell culture plate, uniformly mixing, and putting into a cell culture box for culture, wherein the culture temperature of the cell culture box is 37 ℃, and the culture temperature is CO2The concentration is 5%;
(9) liquid changing: changing the culture solution once every 22h, sucking off blood cells and cell fragments containing non-adherent blood cells and cell fragments during the culture solution changing, sucking off old Williams ' E complete culture solution, washing the cell culture plate twice by using D-Hank's solution, adding fresh Williams ' E complete culture solution again, slightly inclining the cell culture plate during the culture solution changing, sucking waste liquid, and slowly dripping from the middle of the cell culture plate during the liquid adding to prevent the cells from being washed;
(10) induction of steatosis: preparing an inducer, wherein the inducer is a Williams' E complete culture solution containing oleic acid, and the concentration of the oleic acid is 0.4 mmol/L; and (5) after 35h of plating, replacing the culture solution, sucking off the Williams' E complete culture solution added in the step (9), replacing the inducer, culturing for 12h, and obtaining the fatty degeneration liver cells.
The preparation steps of the inducer in the step (10) are as follows:
1) preparing oleic acid storage liquid
Weighing 50mg of sodium oleate, placing the sodium oleate into a test tube, adding 1080 mu L of 0.1mmol/L NaOH solution into the test tube, placing the test tube into a water bath at the temperature of more than 90 ℃, heating the test tube, continuously shaking the test tube for 10min, placing the test tube into ultrasonic waves at intervals, carrying out ultrasonic treatment for 3 times for 30 seconds each time, transferring the test tube into a 15ml centrifugal tube while the test tube is hot, adding 2% BSA aqueous solution into the centrifugal tube to ensure that the final volume is 10.8ml, and the final concentration of an oleic acid storage solution is 15 mmol/L;
2) preparing an inducer: the oleic acid stock was added to Williams 'E complete medium and diluted to 0.4mmol/L by Williams' E complete medium, specifically, 6mL of Williams 'E complete medium was added to 160. mu.L of 15mmol/L oleic acid stock to obtain 0.4mmol/L oleic acid Williams' E complete medium.
The Williams 'E complete medium contains penicillin 10 per 100mL of Williams' E medium4U, streptomycin 10mg, glutamine 0.2mmol, 15ml fetal bovine serum.
Example 3:
a chicken embryo hepatocyte in-vitro separation culture and steatosis model building method comprises the following steps:
(1) taking SPF chick embryos: selecting more SPF eggs with the age of 17 days, sterilizing the surface of an eggshell by using 75% alcohol, breaking the eggshell with big heads, taking chick embryos, and killing the chick embryos after breaking the heads;
(2) shearing: opening abdominal cavity of chick embryo, taking out liver, placing into culture dish containing D-Hank's solution, and shearing liver into 1.5mm with ophthalmic scissors3The small blocks are cut for a period of time, the D-Hank's solution is changed to remove blood cells as much as possible, after the small blocks are cut, the cut liver is transferred to a sterile centrifuge tube, and the small blocks are washed for 3 times by the D-Hank's solution;
(3) digestion: transferring the liver cleaned in the step (2) into a 50mL beaker with a rotor, placing the beaker on a water bath constant-temperature magnetic stirrer, adding trypsin for digestion, wherein the using amount of the trypsin is determined according to the number of chicken embryos, adding 1mL of 0.25% trypsin into 15 chicken embryos, keeping the water bath temperature at 37 ℃, digesting for 12min, repeatedly blowing and beating the chicken embryos by using a suction pipe until the cut liver disappears after digestion is finished, wherein in order to prevent over digestion, the liver blocks are not required to be completely digested, a little amount of the trypsin can be remained, the edge of the liver adsorbed on the wall is in a chyle state, and then respectively filtering the liver by a 50-mesh screen for the first time and filtering the liver by a 200-mesh screen for the second time to obtain a cell suspension A;
(4) centrifuging: subpackaging the cell suspension A filtered in the step (3) into a plurality of first centrifuge tubes, adding 10mL of cell suspension A into each first centrifuge tube, placing the first centrifuge tube into a centrifuge, controlling the rotation speed to 1300rpm, centrifuging for 8min, removing the supernatant, reserving the precipitate, adding 12mL of D-Hank's solution into the first centrifuge tube, washing and resuspending, re-suspending the cells, continuing to centrifuge, controlling the rotation speed to 1300rpm, centrifuging for 8min, repeatedly centrifuging for 4 times, washing and resuspending the D-Hank's solution for 3 times, then removing the supernatant, reserving the precipitate, adding 2mL of Williams ' E complete culture solution preheated at 37 ℃ into the first centrifuge tube, and re-suspending to obtain cell suspension B;
(5) cell separation: adopting Percoll density gradient centrifugation, preparing a plurality of second centrifuge tubes, paving 6mL of Percoll separating liquid in each second centrifuge tube, suspending 3mL of cell suspension B on the Percoll separating liquid, adding the cell suspension slowly, attaching a dropper to the wall of the centrifuge tube, dropwise adding while rotating the second centrifuge tube to enable the cell layer and the centrifuging liquid to form an interface, placing the second centrifuge tube in a centrifuge for centrifugation, controlling the rotation speed to be 3500rpm, and centrifuging for 20 min; wherein, the preparation step of the Percoll separating medium is as follows, 3mL of 100 percent Peroll is taken and added with 2mL of PBS to obtain the Percoll separating medium;
(6) taking the liver cells: after centrifugation in the step (5), sucking off upper cells and liquid in a second centrifuge tube by using a pipette gun, sucking cells at an interface, adding the cells into a third centrifuge tube, adding 12mL of D-Hank's liquid, blowing, beating and uniformly mixing, adding the D-Hank's liquid to reduce the concentration of the Percoll separating liquid and reduce the influence of the Percoll separating liquid on the cells as much as possible, then placing the mixture into a centrifuge for centrifugation, controlling the rotation speed to be 1300rpm and the centrifugation to be 8min, discarding the supernatant, leaving the precipitate, adding 12mL of D-Hank's liquid into the third centrifuge tube for washing and resuspending, continuing centrifugation after resuspending the cells, controlling the rotation speed to be 1000rpm and the centrifugation to be 5-8min, repeatedly centrifuging for 3 times, then discarding the supernatant, leaving the precipitate, adding Williams' E complete culture solution, and uniformly mixing to a 100mL beaker to obtain cell suspension C; the addition amount of Williams 'E complete culture solution is determined according to the number of chick embryos, and 45ml of Williams' E complete culture solution can be used for collecting hepatocytes from 10 chick embryos;
(7) counting: adding 15 mu L of the cell suspension C obtained in the step (6) into a fourth centrifuge tube, continuously adding 15 mu L of trypan blue into the fourth centrifuge tube, uniformly mixing, adding into a cell counting plate, and counting through the cell counting plate;
(8) plate preparation: according to the counting result of the cell counting plate in the step (7), the cell density is 1.5 multiplied by 106Per mL, then adding Williams' E complete medium to dilute the cell suspension C concentration to4×105Inoculating the cell suspension C on a cell culture plate, uniformly mixing, and putting into a cell culture box for culture, wherein the culture temperature of the cell culture box is 37 ℃, and the culture temperature is CO2The purity is 5%;
(9) liquid changing: changing the culture solution once every 26h, sucking off blood cells and cell fragments containing non-adherent blood cells and cell fragments during the culture solution changing, sucking off old Williams ' E complete culture solution, washing the cell culture plate twice by using D-Hank's solution, adding fresh Williams ' E complete culture solution again, slightly inclining the cell culture plate during the culture solution changing, sucking waste liquid, and slowly dripping from the middle of the cell culture plate during the liquid adding to prevent the cells from being washed;
(10) induction of steatosis: preparing an inducer, wherein the inducer is a Williams' E complete culture solution containing oleic acid, and the concentration of the oleic acid is 0.5 mmol/L; and (4) after 30-40h of plating, replacing the culture solution, sucking off the Williams' E complete culture solution added in the step (9), replacing the inducer, culturing for 15h, and obtaining the fatty degeneration liver cells.
The preparation steps of the inducer in the step (10) are as follows:
1) preparing oleic acid storage liquid
Weighing 50mg of sodium oleate, placing the sodium oleate into a test tube, adding 1080 mu L of 0.1mmol/L NaOH solution into the test tube, placing the test tube in a water bath at the temperature of more than 90 ℃, heating the test tube, continuously shaking the test tube for 10min, placing the test tube into ultrasonic waves at intervals, carrying out ultrasonic treatment for 3 times for 30 seconds each time, transferring the test tube into a 15ml centrifugal tube while the test tube is hot, adding 3% BSA solution into the test tube to ensure that the final volume is 10.8ml, and the final concentration of an oleic acid storage solution is 15 mmol/L;
2) preparing an inducer: the oleic acid stock was added to Williams 'E complete medium and diluted to 0.5mmol/L by Williams' E complete medium, specifically, 6mL of Williams 'E complete medium was added to 200. mu.L of 15mmol/L oleic acid stock to obtain 0.5mmol/L oleic acid Williams' E complete medium.
The Williams 'E complete medium contains penicillin 10 per 100mL of Williams' E medium4U, streptomycin 10mg, glutamine 0.2mmol, 15ml fetal bovine serum.
1. Identification of chick embryo hepatocyte purity by glycogen staining
Model group hepatocyte glycogen staining: inoculating the hepatocytes separated in the step (6) in the embodiment into a 6-well culture plate paved with cover slips, continuously culturing for different time including 0h, 6h, 24h and 36h, then observing cell morphology by taking pictures with a microscope, replacing complete Williams' E culture solution containing oleic acid as an inducer after respectively culturing for 24h, continuously culturing for 12h, then taking out cell climbing sheets, fixing for 10-15min with PAS (PAS) fixing solution, washing with water, drying in the air, adding an oxidant, oxidizing for 15-20min at room temperature, washing for 2 times with tap water, washing for 2 times with distilled water, then adding a Schiff reagent and covering, performing dip dyeing for 10-20min at room temperature in the dark, washing for 2 times with sodium sulfite solution after dip dyeing, washing for 2min with running water, adding Mayer hematoxylin dyeing solution, re-dyeing for 1-2min, washing with water, drying in the air, and performing microscopic examination; microscopic observation shows that the survival rate of the cells is more than 90%, the primary chick embryo hepatocytes which are just separated are transparent and spherical, adhere to the walls after being inoculated for 6 hours, most of the cells adhere to the walls after being cultured for 24 hours, the hepatocytes are mutually adhered and are connected in an island shape, individual cells are stretched and deformed, and the hepatocytes with poor vitality are in a suspension state due to no adherence, as shown in the attached drawing 1, the primary cultured cells can be rapidly obtained by the embodiment of the invention, and the cell activity is strong;
glycogen staining of hepatocytes of control group: inoculating the hepatocytes separated in the step (6) in the embodiment into a 6-well culture plate paved with cover slips, culturing for 24h, taking out cell slide, fixing for 10-15min by PAS (PAS) fixing solution, washing with water, drying in the air, adding an oxidant, oxidizing for 15-20min at room temperature, washing for 2 times by tap water, washing for 2 times by distilled water, adding a Schiff reagent and covering, performing dip dyeing for 10-20min at room temperature in the shade, washing for 2 times by using a sodium sulfite solution after dip dyeing, washing for 2min by running water, adding a Mayer hematoxylin dyeing solution, re-dyeing for 1-2min, washing with water, drying in the air, and performing microscopic examination;
the glycogen staining results of the model group and the control group are shown in figure 2, wherein A in figure 2 represents glycogen staining of hepatocytes of the control group, B represents glycogen staining of hepatocytes of the model group, after the glycogen staining, dense pink glycogen particles can be seen in cytoplasm of hepatocytes of the control group and hepatocytes of the model group, the dense pink glycogen particles are distributed in a homogeneous or granular manner, and the purity of hepatocytes is high, so that primary cultured cells obtained by the culture method of the embodiment are high in purity.
2. Determination of the acting concentration of oleic acid
Oleic acid group: adding complete Williams' E culture solution of 0mmol/L, 0.1mmol/L, 0.2mmol/L, 0.4mmol/L, 0.5mmol/L, 0.8mmol/L and 1.0mmol/L of oleic acid into the chick embryo hepatocytes in the step (6) of the embodiment of the invention, culturing in a 96-well plate, and measuring the cell proliferation by an MTT colorimetric method, wherein the value is expressed as absorbance A;
group BSA: firstly, preparing 0.5mmol/L oleic acid, weighing 50mg sodium oleate, adding 1080 mu L of 0.1mmol/L NaOH solution, placing in a water bath above 90 ℃ for heating and continuously shaking for 10min, placing in ultrasonic waves at intervals for 3 times, every 30 seconds, transferring into a 15mL centrifuge tube while the solution is hot, adding BSA solutions with different concentrations to make the final volume be 10.8mL, wherein the BSA solutions with different concentrations are 0% BSA solution, 0.5% BSA solution, 1% BSA solution, 2% BSA solution, 3% BSA solution, 4% BSA solution and 5% BSA solution, at the moment, the final concentration of the oleic acid storage solution is 15mmol/L, weighing 200 mu L of 15mmol/L oleic acid storage solution, adding 6mL Williams' E complete culture solution to prepare 0.5mmol/L oleic acid, finally, culturing the chick embryo hepatocytes in a 96 well plate in the invention example (6), then adding 0.5mmol/L oleic acid, measuring the cell proliferation by MTT colorimetry, and expressing the value of absorbance A;
the results are given in table 1 below:
TABLE 1
The following results were obtained from the data in table 1 above: the observation of the morphology of the cultured cells shows that the cells grow normally when the oleic acid concentration is 0.1-0.5mmol/L, the number of floating necrotic cells is small, and MTT (methyl thiazolyl tetrazolium) detection indicates that the absorbance A value is obviously reduced when the oleic acid concentration exceeds 0.5mmol/L, and the influence on cell proliferation is large, so that the oleic acid concentration is preferably 0-0.5mmol/L in the embodiment of the invention; moreover, the BSA solution concentration of less than 3% has no influence on the proliferation of hepatocytes, so that the BSA solution concentration of less than 3% is preferred in the preparation of oleic acid in the embodiment of the present invention, and oleic acid is dissolved in a weakly alkaline BSA solution, so that oleic acid in a cell culture solution is uniformly distributed, and cells are sufficiently contacted.
3. AST, ALT and TG assays
(1) AST and ALT measurement
1) AST measurement, which is divided into two groups, namely a control group and an oleic acid group;
oleic acid group: in the step (10) of this embodiment, a Williams' E complete culture solution containing oleic acid is added as an inducer to induce steatosis, then the steatosis hepatocytes are obtained by culturing for 0h, 6h, 12h and 24h, respectively, and then the steatosis hepatocytes are planted in a 24-well culture plate, each well contains 1 sample, 6 samples are set for each group, the cells and the supernatant are collected after culturing for 24-72 h and are counted, the supernatant is subjected to AST measurement, the AST measurement is performed according to the kit procedures, and the AST measurement results are as shown in table 2 below; AST measurement is carried out according to the kit steps, and the AST measurement results are shown in the following table 2;
control group: adopting hepatic cells which are not added with an inducer to generate steatosis, respectively culturing for 0h, 6h, 12h and 24h to obtain the steatosis hepatic cells, then planting the steatosis hepatic cells in a 24-hole culture plate, wherein each hole is provided with 1 sample, each group is provided with 6 samples, after culturing for 24-72 h, collecting cells and supernatant, counting the cells, and performing AST determination on the supernatant, wherein the AST determination is operated according to the kit steps, and the AST determination result is shown in the following table 2;
2) ALT determination, which is divided into two groups for determination, namely a group of control group and a group of oleic acid group;
oleic acid group: in the step (10) of the embodiment, a Williams' E complete culture solution containing oleic acid is added as an inducer to induce steatosis, then the steatosis hepatocytes are obtained by culturing for 0h, 6h, 12h and 24h respectively, then the steatosis hepatocytes are planted in a 24-hole culture plate, each hole contains 1 sample, each group contains 6 samples, after 24-72 h of culture, the cells and the supernatant are collected and counted, the supernatant is subjected to ALT determination, the ALT determination is performed according to the kit steps, and the ALT determination results are shown in the following table 2;
control group: the method comprises the steps of adopting hepatic cells which are not added with an inducer to generate steatosis, respectively culturing for 0h, 6h, 12h and 24h to obtain the steatosis hepatic cells, then planting the steatosis hepatic cells in a 24-hole culture plate, wherein each hole is provided with 1 sample, each group is provided with 6 samples, after culturing for 24-72 h, collecting cells and supernatant, counting the cells, and carrying out ALT determination on the supernatant, wherein the ALT determination is operated according to the steps of a kit, and the ALT determination result is shown in the following table 2.
In table 2, indicates significant difference (P <0.05), and indicates significant difference (P <0.01)
TABLE 2
The results are given from the data in table 2 above: compared with ALT and AST of a control group, the difference of the oleic acid group and ALT and AST of the control group has statistical significance (P <0.05 and P <0.01), compared differences of all time phase points have statistical significance (P <0.01), when the oleic acid group and the control group are cultured for 6 hours, the release of liver cell enzymes of the oleic acid group is increased, and the release of liver cell enzymes is gradually increased along with the increase of time, so that the embodiment of the invention successfully induces the steatosis of liver cells through oleic acid, and the induction time is short.
(2) TG determination: dividing into two groups, one group is a control group, and the other group is an oleic acid group;
oleic acid group: in the step (10) of the embodiment, a Williams' E complete culture solution containing oleic acid is added as an inducer to induce steatosis, the steatosis is cultured for 0h, 6h, 12h and 24h respectively, then a freeze-thaw method is adopted to detect TG, cells are repeatedly frozen and lysed, then a centrifuge is controlled to rotate at a speed of 3000 r/min for centrifugation, then supernatant is taken to carry out TG detection, the content of triglyceride in the supernatant is detected, and the operation is carried out according to the instruction of a kit;
control group: adopting hepatic cells without adding an inducer to generate steatosis, respectively culturing for 0h, 6h, 12h and 24h, then adopting a freezing and thawing method to detect TG, repeatedly freezing and thawing the lysed cells, then controlling the rotating speed of a centrifuge to 3000 r/min for centrifugation, then taking the supernatant for TG detection, detecting the content of triglyceride in the supernatant, and operating according to the kit specification;
culturing the triglyceride content (mg/10) in the liver cells7cell, n ═ 6) is shown in table 3 below:
TABLE 3
The following results were obtained from the data in table 3 above: when the chicken embryo hepatocyte liver induced by oleic acid is cultured for 6 hours, the content of triglyceride in the cell is obviously increased (P <0.01) compared with that of a control group, and is obviously increased along with the time prolongation, and the comparison difference of each time phase point has statistical significance (P <0.01), so that the embodiment of the invention successfully induces the steatosis by adding the oleic acid, and the induction time is short.
4. Observation of intracellular lipid droplet formation and determination of oleic acid-induced incubation time
1) Determination of the oleic acid concentration: culturing in four groups, and observing with light microscope, wherein the group comprises a control group, an experimental group 1, an experimental group 2 and an experimental group 3;
experimental group 1: planting the hepatocytes separated in the step (6) in the embodiment of the invention in a 24-well culture plate, adding a Williams 'E complete culture solution with oleic acid concentration of 0.25mmol/L, then placing a 1cm multiplied by 1cm slide in the culture plate for cell climbing, culturing for 24-36 h, taking out, rinsing with PBS, changing the solution, adding a Williams' E complete culture solution with oleic acid concentration of 0.25mmol/L as an inducer again, culturing for 10h, rinsing with PBS, fixing with alcohol, and observing lipid droplets in the cells by adopting oil red O staining;
experimental group 2: planting the hepatocytes separated in the step (6) in the embodiment of the invention in a 24-well culture plate, adding a Williams 'E complete culture solution with oleic acid concentration of 0.5mmol/L, then placing a 1cm multiplied by 1cm slide in the culture plate for cell climbing, culturing for 24-36 h, taking out, rinsing with PBS, changing the solution, adding a Williams' E complete culture solution with oleic acid concentration of 0.5mmol/L as an inducer again, culturing for 10h, rinsing with PBS, fixing with alcohol, and observing lipid droplets in the cells by adopting oil red O staining;
experimental group 3: planting the hepatocytes separated in the step (6) in the embodiment of the invention in a 24-well culture plate, adding a Williams 'E complete culture solution with oleic acid concentration of 1mmol/L, then placing a 1cm multiplied by 1cm slide in the culture plate for cell climbing, culturing for 24-36 h, taking out, rinsing with PBS, changing the solution, adding a Williams' E complete culture solution with oleic acid concentration of 1mmol/L as an inducer again, culturing for 10h, rinsing with PBS, fixing with alcohol, and observing lipid droplets in the cells by adopting oil red O staining;
control group: planting the hepatocytes separated in the step (6) in the embodiment of the invention in a 24-hole culture plate, then placing a 1cm × 1cm slide in the culture plate for cell climbing, culturing for 24-36 h, taking out, rinsing with PBS, fixing with alcohol, and observing lipid droplets in the cells by adopting oil red O staining;
the observation result after staining with oil red O is shown in fig. 3, fat droplets with orange red color in cytoplasm are used as the steatosis cells, meanwhile, in fig. 3, a represents a control group, B represents an experimental group 1, C represents an experimental group 2, D represents an experimental group 3, and the following conclusion is obtained according to the observation of fig. 3: the cells are observed by an optical microscope after being dyed by oil red O, the edges of the cells of a control group are clear, cytoplasm is rich, a nuclear membrane is complete, and a small amount of red lipid drops are in the cells; observing the experiment groups 1, 2 and 3, the intracellular lipid drops are in an increasing trend along with the increase of the concentration of the oleic acid, and when the concentration of the oleic acid is more than 0.5mmol/L, the number of cells is greatly reduced, so that the concentration of the oleic acid in the embodiment of the invention is preferably 0-0.5 mmol/L;
2) determination of incubation time
The hepatocytes separated in the step (6) of the embodiment of the invention are planted in a 24-well culture plate, Williams 'E complete culture solution with the oleic acid concentration of 0.5mmol/L is added, then a 1cm × 1cm slide is placed in the culture plate for cell climbing, the cells are taken out after being cultured for 24-36 h, the cells are rinsed by PBS and changed in solution, Williams' E complete culture solution with the oleic acid concentration of 0.5mmol/L is added again as an inducer for continuous culture, after 6h, 8h, 10h, 12h, 15h, 18h, 20h and 24h are respectively cultured, the cells are respectively rinsed by PBS and fixed by alcohol, then lipid drops in the cells are respectively observed by adopting oil red O staining and the fatty degeneration cells are counted, and the record is carried out according to the following standard: randomly selecting 6 high-power fields, counting 100 cells in each field, and calculating the average lipid variability of the cells, wherein the lipid variability of the chicken embryo hepatocytes at different times is shown in the following table 4:
TABLE 4
The following results were obtained according to table 4 above: the cell lipid variability is increased along with the increase of the induction time, the cell lipid variability value is larger in 10-15h, the lipid variability is larger than 80%, the cell lipid variability is reduced in 24h, and the cell lipid variability is (77.6 +/-4.36)%, so that 10-15h is selected as the optimal time for inducing the chicken embryo hepatocyte steatosis by using oleic acid.
5. Ultrastructural observation of steatosis chick embryo liver cell
Observing the ultrastructure of the liver cell by an electron microscope, and dividing the ultrastructure into two groups for observation, namely a control group and a model group; wherein the control group is observed by using the hepatocytes separated in the step (6) of the embodiment, the model group is obtained by culturing the fatty degeneration hepatocytes in the embodiment of the present invention in a 40ml culture bottle, the cells are digested and collected by using trypsin after 12 hours of oleic acid action, and the cells are used as the model group, and the transmission electron microscopy specimen of the control group and the model group are respectively prepared for observation by using a traveling electron microscope, as shown in fig. 4, a represents the control group, and B represents the model group; the following results were obtained by observation: the size of the liver cell of the control group is small, the inner mitochondria are small and many, the ridge is complete and clear, the gap is uniform, lipid drops are occasionally seen in cytoplasm, and the nucleus is positioned near the center of the cell; the model group has large hepatocyte volume, a large amount of homogeneous lipid droplets with medium electron density are diffused and distributed, the nuclear membrane is unclear, the number of mitochondria in the cell is reduced, the cell is swollen, the ridge is rare, the gap is enlarged, and the endoplasmic reticulum is expanded to form a vesicular sample.
In conclusion of experiments, in the embodiment of the invention, 13-17-day-old chick embryos are selected as test materials, trypsinase is used for digestion, the liver cells are separated and purified by a gradient density centrifugation method, oleic acid induces steatosis of the liver cells, after 10-15 hours, oil red staining and a transmission electron microscope are used for verifying that a large number of lipid droplets are accumulated in the cells, the content of TG in the detected cells is obviously increased compared with that of a control group, and the fact that oleic acid can successfully induce steatosis of the chick embryo liver cells is proved, and the optimal concentration of the oleic acid is 0-0.5 mmol/L.
Claims (7)
1. A chicken embryo hepatocyte in-vitro separation culture and steatosis model building method is characterized by comprising the following steps:
(1) taking SPF chick embryos: selecting more SPF eggs of 13-17 days old, sterilizing the surface of an eggshell by using alcohol, breaking the eggshell and taking an eggshell;
(2) shearing: opening abdominal cavity of chick embryo, taking out liver, and shearing into D-Hank's solution to 0.5-1.5mm3The small blocks are cleaned by D-Hank's solution after being trimmed;
(3) digestion: adding trypsin into the liver cleaned in the step (2) for digestion, stirring in a water bath for 8-12min, keeping the temperature of the water bath at 37 ℃, repeatedly blowing and beating the liver with a suction pipe until the cut liver disappears after digestion is finished, and filtering to obtain a cell suspension A;
(4) centrifuging: placing the cell suspension A in the step (3) in a centrifuge for centrifugation, controlling the rotation speed to 800-;
(5) cell separation: performing Percoll density gradient centrifugation, suspending the cell suspension B on the Percoll separating medium, controlling the rotation speed of 2500-;
(6) taking the liver cells: after centrifugation in the step (5), sucking off the middle and upper layer cells and liquid, reserving cells at an interface, adding D-Hank's liquid to blow, uniformly mixing, then placing the mixture into a centrifuge for centrifugation, controlling the rotation speed to 800 plus 1300rpm, centrifuging for 5-8min, discarding the supernatant, reserving the precipitate, adding D-Hank's liquid to wash and resuspend, continuously centrifuging after resuspending the cells, controlling the rotation speed to 800 plus 1300rpm, centrifuging for 5-8min, repeatedly centrifuging for 2-3 times, then discarding the supernatant, reserving the precipitate, adding Williams ' E complete culture solution, and uniformly mixing to obtain a cell suspension C;
(7) counting: adding trypan blue into the cell suspension C obtained in the step (6), uniformly mixing, adding a cell counting plate, and counting through the cell counting plate;
(8) plate preparation: according to the counting result of the cell counting plate in the step (7), Williams' E complete culture solution is added for dilution, and the cell density is kept to be 1X 105-5×105Inoculating to cell culture plate, mixing, culturing in cell culture box at 37 deg.C under CO2The concentration is 5%;
(9) liquid changing: changing Williams' E complete culture solution once every 20-26 h;
(10) induction of steatosis: preparing an inducer, wherein the inducer is a Williams' E complete culture solution containing oleic acid, and the concentration of the oleic acid is 0.1-1 mmol/L; and (5) after 30-40h of plating, replacing the culture solution, sucking off the Williams' E complete culture solution added in the step (9), replacing the inducer, culturing for 10-15h, and obtaining the fatty degeneration liver cells.
2. The method for in vitro isolation and culture of chicken embryo hepatocytes and establishment of a steatosis model according to claim 1, wherein the preparation of the inducer in step (10) comprises the following steps:
1) preparing an oleic acid storage solution: weighing 50mg of sodium oleate, placing the sodium oleate into a test tube, adding 1080 mu L of 0.1mmol/L NaOH solution into the test tube, placing the test tube in a water bath at the temperature of more than 90 ℃, heating the test tube, continuously shaking the test tube for 10min, placing the test tube in ultrasonic waves at intervals, carrying out ultrasound for 3 times for 30 seconds each time, then adding BSA solution into the test tube while the test tube is hot to ensure that the final volume is 10.8ml, and the final concentration of an oleic acid storage solution is 15 mmol/L;
2) preparing an inducer: the oleic acid stock was added to Williams 'E complete medium and the oleic acid stock was diluted to a concentration of 0.1-1mmol/L by Williams' E complete medium.
3. The method for in vitro isolation culture and steatosis model establishment of chick embryo hepatocytes as claimed in claim 1, wherein the method comprises the following steps: in the step (10), the complete culture solution of Williams' E containing oleic acid has the concentration of 0.1-0.5 mmol/L.
4. The method for in vitro isolation culture and steatosis model establishment of chick embryo hepatocytes as claimed in claim 1, wherein the method comprises the following steps: the filtering in the step (3) comprises two-stage filtering, wherein the first-stage filtering is 50-mesh screen filtering, and the second-stage filtering is 200-mesh screen filtering.
5. The method of claim 1, wherein the Williams 'E complete medium contains penicillin 10 per 100mL of Williams' E medium4U, streptomycin 10mg, glutamine 0.2mmol, 15ml fetal bovine serum.
6. The method for in vitro isolation culture and steatosis model establishment of chick embryo hepatocytes as claimed in claim 1, wherein the method comprises the following steps: the dosage of the trypsin is determined according to the number of the chick embryos, and 800 mu L to 1mL of 0.25 percent trypsin is added into 15 chick embryos.
7. The method for in vitro isolation culture and steatosis model establishment of chick embryo hepatocytes as claimed in claim 1, wherein the method comprises the following steps: the addition amount of the Williams 'E complete culture solution in the step (6) is determined according to the number of the chick embryos, and 35-45ml of Williams' E complete culture solution is added to the liver cells collected from 10 chick embryos.
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