CN114149959B - Establishment method of cell model for researching autoimmune hepatitis - Google Patents
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Abstract
The invention belongs to the technical field of medical experimental model research, and discloses a method for establishing a cell model for researching autoimmune hepatitis, aiming at the severity of the autoimmune hepatitis and the importance of the cell model on the autoimmune hepatitis research at present. The macrophage/hepatocyte co-culture model is established by adopting Canavalia gladiata to induce a mouse macrophage-like cell line RAW 264.7 macrophage strain and co-culturing the RAW 264.7 macrophage strain and BRL3a mouse hepatocytes, and simulating the inflammatory injury process of macrophages on hepatocytes in the autoimmune hepatitis morbidity in vitro. The method fills the blank of autoimmune hepatitis cell model research, decomposes the pathogenesis of autoimmune hepatitis, focuses on the mechanism of hepatic cell injury induced by macrophages, and is beneficial to autoimmune hepatitis pathogenesis research and therapeutic drug screening.
Description
Technical Field
The invention belongs to the technical field of medical experimental model research, and particularly relates to a method for establishing a cell model for researching autoimmune hepatitis.
Background
Autoimmune hepatitis (autoimmune hepatitis, AIH) is a liver parenchymal inflammatory disease mediated by abnormal autoimmune reactions, which can cause cirrhosis and liver failure, and the global total prevalence is 4.0-42.9 cases/10 ten thousand people per year, and people of different complexion and ages can suffer from. The cumulative mortality rate of AIH patients over 10 years was 2.29 times that of the normal population (32.3% vs 14.1%); if cirrhosis or portal hypertension is combined, the risk of death of the patient is significantly increased. Patients with severe AIH require liver transplantation to sustain life. AIH patients undergoing liver transplantation in the united states and uk reportedly account for 3.2% and 3.6% of all liver transplantation patients between 1995 and 2014. Thus, AIH is considered to be a liver inflammatory disease that seriously jeopardizes human health in addition to viral hepatitis. The etiology and pathogenesis of the disease are not clear, and the disease is considered to be autoimmune tolerance deficiency caused by the combined action of genetic susceptibility, environmental induction factors and the like, so that immune response mainly mediated by T cells and macrophages aiming at liver antigens is induced to cause the destruction of liver tissues and the formation of inflammation.
Designing and building experimental models (including animal models for in vivo studies and cellular models for in vitro studies) that are consistent with human pathogenesis are the basis for intensive studies of autoimmune hepatitis pathogenesis. Related studies on animal models of autoimmune hepatitis have been continued for nearly half a century, and some animal models have been accepted by the academy. However, since the immunoinflammatory reaction of autoimmune hepatitis mainly occurs in the liver, the liver immune microenvironment is complex, and many factors are involved in inducing autoimmune hepatitis. The research on the pathogenesis of the disease still needs to be deeply discussed at the cellular level, for example, the research on the pathogenesis of autoimmune hepatitis of a certain gene needs to be performed with silencing or over-expression, and whether the gene participates in the occurrence and development of the disease is confirmed by a reversion experiment; for another example, it is necessary to investigate the action target of a drug when it is examined whether or not the drug has a regulatory effect on autoimmune hepatitis liver injury, and it is also necessary to support in vitro cell experimental data.
It has been found that macrophages can be involved in the induction of T cell activation as antigen presenting cells in addition to the inflammatory injury of the liver during the onset of autoimmune hepatitis. Thus, if a stable, easily-reconstituted cell model which is consistent with the pathogenesis of autoimmune hepatitis and which mediates hepatocyte damage by inducing macrophage activation could be established, the progress of the research on the pathogenesis of autoimmune hepatitis would be greatly advanced.
Disclosure of Invention
Aiming at the severity of the autoimmune hepatitis and the importance of the cell model in the research of the autoimmune hepatitis at present, the invention provides a method for establishing the cell model for researching the autoimmune hepatitis.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method of establishing a cellular model for studying autoimmune hepatitis, comprising the steps of:
Step 1, induction activation of macrophages;
And 2, establishing a macrophage and liver cell co-culture model.
Further, the macrophage is a mouse RAW 264.7 macrophage, and the liver cell is a mouse BRL3a liver cell.
Further, the macrophage and hepatocyte co-culture cell model comprises an indirect macrophage and hepatocyte co-culture cell model and a direct macrophage and hepatocyte co-culture cell model.
Further, the macrophage is activated by canavalin a induction in step 1.
Further, the specific process of inducing activated macrophages by canavalin a is as follows: cell seeding was performed on 1X10 6/RAW 264.7 macrophages per well in 24 well plates, and after adding 800. Mu.L of high-sugar DMEM medium per well, 0-320. Mu.g/mL of fluorescein isothiocyanate-labeled Canavalia gladiata was added to induce macrophages at 37℃for 0-24h, respectively.
The optimal concentration of the canavalin A-induced macrophages marked with fluorescein isothiocyanate is 160 mug/mL, and the optimal time is 12h.
The specific method for establishing the macrophage and hepatocyte indirect co-culture cell model comprises the following steps: RAW264.7 macrophages were seeded into the upper chamber of LABSELECT Transwell cells at a density of 2X 10 5, BRL3a hepatocytes were seeded into the lower chamber of Transwell cells at a density of 2X 10 5, then the Transwell cells were incubated overnight or 12h at 37℃and 5% CO 2 incubator, 160. Mu.g/mL of fluorescein isothiocyanate-labeled Canavanin A was added to the upper chamber of the Transwell cells, and RAW264.7 macrophages were induced for 12h.
The specific method for directly co-culturing the macrophage and the liver cells in the cell model comprises the following steps: BRL3a hepatocytes were first seeded into 24-well plates at a density of 2.5×10 5 and incubated for 6h at 37 ℃ with a 5% co 2 incubator; RAW264.7 macrophages were then seeded into the same culture well at a density of 2.5X10 5, incubated with BRL3a hepatocytes, and induced by the addition of 160. Mu.g/mL fluorescein isothiocyanate labeled Canavalia protein A for 12h.
Compared with the prior art, the invention has the following advantages:
1. The invention utilizes the in vitro induction of macrophage activation of the canavalin A to mediate liver cell injury, simulates partial pathogenesis of autoimmune hepatitis in vitro and simulates pathogenesis of autoimmune hepatitis mouse model induced by the canavalin A, fills up the blank of autoimmune hepatitis cell model research, and is beneficial to autoimmune hepatitis pathogenesis research and therapeutic drug screening.
2. The invention decomposes the pathogenesis of autoimmune hepatitis, focuses on the mechanism of macrophage induced hepatic cell injury, and discovers the mechanism of macrophage induced activation and the target thereof through the establishment of a model.
3. According to the invention, through screening experimental conditions, the model establishment method is optimized, so that the established model is more stable and effective.
4. The model is built by adopting a cell line, and is easy to reconstruct.
Drawings
FIG. 1 is a flow chart of a method according to an embodiment of the invention.
FIG. 2 shows the concentration-dependent assay of binding of Con A to RAW264.7 macrophages. (a) is a flow cytometer; (b) is the binding rate of Con a to RAW264.7 cells; (c) is the activity of RAW264.7 cells.
FIG. 3 shows the time-dependent assay of binding of Con A to RAW264.7 macrophages. (a) flow cytometry at 80 μg/mL ConA-FITC effect; (b) a binding rate of 80 μg/mL Con a to RAW264.7 cells; (c) flow cytometry at 160 μg/mL ConA-FITC effect; (d) a binding rate of 160 μg/mL Con a to RAW264.7 cells; (e) is the activity of RAW264.7 cells.
Fig. 4 is the promotion of Reactive Oxygen Species (ROS), nitric Oxide (NO), and Malondialdehyde (MDA) production in RAW264.7 macrophages by ConA. In the figure, (a) activates RAW264.7 macrophages for ConA, increases intracellular ROS, and measures ROS levels using the reactive oxygen species assay kit (DCFH-DA). Data are expressed as a percentage of increase in Median Fluorescence Intensity (MFI); (b) Cells were treated with ConA (0, 80 and 160. Mu.g/mL) at various concentrations for 12h and the NO content was determined by Griess and TBARS; (c) Cells were treated with ConA (0, 80 and 160. Mu.g/mL) at various concentrations for 12h and MDA content was determined by Griess and TBARS methods. Data are shown as mean ± SD (n=3, p <0.05, p < 0.01).
FIG. 5 is the effect of ConA on the expression of TNF- α by RAW264.7 macrophages. (a) Flow cytometry for expression of TNF- α by macrophages induced for different concentrations of ConA; (b) The amount of TNF- α expressed by RAW264.7 cells was induced for different concentrations of ConA.
Fig. 6 is the inhibition of binding of ConA to RAW264.7 macrophages by mannan. (a) Flow cytometry for inhibition of 80 μg/mL ConA binding to RAW264.7 macrophages for different concentrations of mannan; (b) Binding rates of 80 μg/mLConA to RAW264.7 cells were inhibited for different concentrations of mannan; (c) Activity of RAW264.7 cells at different time under the action of 100. Mu.g/mL of mannan; (d) Flow cytometry for inhibition of 160 μg/mL ConA binding to RAW264.7 macrophages for different concentrations of mannan; (e) Inhibiting the binding rate of 160 mug/mL Con A and RAW264.7 cells for different concentrations of mannans; (f) Activity of RAW264.7 cells at different concentrations of mannan.
FIG. 7 is the effect of mannan on ConA-induced macrophage TNF- α expression. (a) is a flow cytometer; (b) Effect of 100 μg/mL mannan on 160 μg/mLConA and 80 μg/mLConA induced RAW 264.7 cells to express TNF- α.
FIG. 8 is the effect of Con A on macrophage-derived TNF- α mediated hepatocyte damage. (a) is a flow cytometer; (b) is the rate of apoptosis of BRL3a hepatocytes.
FIG. 9 is a graph showing the effect of different co-culture methods on macrophage-derived TNF- α mediated hepatocyte damage. (a) And (b) shows the effect of 80. Mu.g/mL ConA on hepatocyte damage. (c) And (d) shows the effect of 160 μg/mLConA on hepatocyte damage. (e) And (f) shows the effect of direct co-culture and indirect co-culture on hepatocyte damage at 160 μg/mLConA incubated for 12 h.
Detailed Description
The following describes the technical scheme in the embodiment of the present invention in detail with reference to the embodiment of the present invention and the accompanying drawings. It should be noted that variations and modifications can be made by those skilled in the art without departing from the principles of the present invention, which are also considered to be within the scope of the present invention.
Experimental materials: mouse RAW 264.7 macrophage-like cell line, mouse BRL3a liver cell line, fluorescein isothiocyanate-labeled Canavalia ectropis A (ConA-FITC), mannan, TNF-alpha antibody, fetal bovine serum, high-sugar DMEM medium, penicillin/streptomycin mixed solution, dimethyl sulfoxide, LABSELECT Transwell cells and the like.
Example 1
ConA induces macrophage activation: optimal experimental conditions for ConA to induce macrophage activation were determined by concentration-dependent assay of ConA binding to macrophages and time-dependent assay of ConA binding to macrophages.
1. Concentration-dependent assay of ConA binding to macrophages: cell inoculation is carried out on 24-well plates according to 1X 10 6/RAW 264.7 macrophages per well, after 800 mu L of high-sugar DMEM culture medium is added into each well, conA-FITC is sequentially added into each well according to the concentration of 0, 5, 10, 20, 40, 80, 160 and 320 mu g/mL, and after 12 hours of macrophage induction, a flow cytometer is adopted to detect the number of FITC + RAW264.7 cells; meanwhile, CCK-8 experiments were performed to examine the effect of different ConA concentrations on RAW264.7 macrophage activity. Thus, the optimal concentration of ConA to induce macrophage activation was determined.
Fig. 2 is a graph showing the concentration-dependent results of binding of Con a to RAW264.7 macrophages, as can be seen from the graph: 160 μg/mL of Con A had the highest binding rate to RAW264.7 cells (a and b). CCK8 experiment results show that the activity of RAW264.7 macrophages gradually increases with the concentration of Con A, and the activity of RAW264.7 macrophages reaches a peak value when the concentration of Con A is 160 mug/mL. As ConA concentration increased to 320. Mu.g/mL, cell activity decreased (c).
2. Time-dependent assay of ConA binding to macrophages: cell inoculation is carried out on 24 pore plates according to 1X 10 6/RAW 264.7 macrophages per pore, after 800 mu L of high-sugar DMEM culture medium is added into each pore, conA-FITC is respectively added into each pore according to the concentration of 80 mu g/mL and 160 mu g/mL, the macrophages are respectively stimulated for 0h, 3h, 6h, 12h and 24h at 37 ℃, and the cell number of the FITC + RAW 264.7 is detected by adopting a flow cytometer; meanwhile, CCK-8 experiments were performed to examine the effect of ConA stimulation time on RAW 264.7 macrophage activity. Thus, the optimal time for ConA to induce macrophage activation was determined.
FIG. 3 is a graph showing the time-dependent results of binding of Con A to RAW264.7 macrophages. As can be seen from fig. 3: at 24h, 80 μg/mLConA and 160 μg/mLCon A both bind to RAW264.7 cells at the highest rate, indicating that Con A binding to macrophage surface mannose receptor is time dependent (a-d). CCK8 experiment results show that the activity of RAW264.7 macrophages gradually increases with the prolongation of the action time of Con A, and the activity of RAW264.7 macrophages reaches a peak value when Con A acts for 12 hours. When ConA was applied for 24 hours, the cell activity was decreased (e). Thus, the duration of action of Con A was limited to within 12h in the subsequent experiments.
Fig. 4 is a graph of the promotion of Reactive Oxygen Species (ROS), nitric Oxide (NO), and Malondialdehyde (MDA) production by ConA in RAW264.7 cells. (a) (b) and (c) are results of treating cells with ConA (0, 80 and 160. Mu.g/mL) at various concentrations for 12h, measuring NO, MDA content by Griess method and TBARS method, and measuring ROS by flow cytometry, as follows: the content of oxidative stress reaction products (e.g., ROS, MDA, NO) was highest at a concentration of ConA of 160. Mu.g/mL.
FIG. 5 is the effect of ConA concentration on macrophage TNF- α expression. As can be seen from the figures: compared with the normal group, the TNF-alpha expression of the RAW264.7 cells of the ConA-FITC 80 mug/mL group and the ConA-FITC 160 mug/mL group is obviously increased, and the ConA-FITC 160 mug/mL group is higher than the ConA-FITC 80 mug/mL group.
The comprehensive preparation method comprises the following steps: finally, determining the binding rate of Con A and RAW 264.7 cells when the RAW 264.7 macrophages are stimulated for 12 hours by 160 mug/mLConA-FITC, wherein the activity of the RAW 264.7 cells reaches the optimal state; meanwhile, the RAW 264.7 macrophage expresses the highest level of cytokines (TNF-alpha) and oxidative stress reaction products (e.g., ROS, MDA, NO).
Example 2
Detection of inhibition of ConA by mannan induced macrophage activation:
Mannan inhibition of ConA induces macrophage activation: conA acts as a recognizable lectin capable of binding to alpha-d-mannitol and alpha-d-glucosyl groups. ConA is presumed to transmit specific signals into cells through mannose receptor binding on the surface of macrophages, causing a series of signaling cascades that regulate the expression of related genes and the secretion of related cytokines. The experiment is to investigate the mechanism and target of binding of ConA and macrophage by adding mannose as a specific blocker of mannose receptor.
The experimental method comprises the following steps: cell inoculation is carried out on the 24-well plate according to 1X 10 6/RAW 264.7 macrophage per well, after 800 mu L of high-sugar DMEM culture medium is added into each well, conA-FITC (80, 160 mu g/mL) DMEM solution is added into the experimental group to induce for 12 hours; the inhibitor groups were pretreated with mannan (0, 25, 50, 100, 200, 400. Mu.g/mL) for 12h and then induced with ConA-FITC DMEM solution for 12h. FITC + RAW 264.7 cell numbers were detected using a flow cytometer.
FIG. 6 shows inhibition of binding of ConA to RAW264.7 macrophages by mannan. It can be derived that: 25 μg/mL of mannan can obviously inhibit the combination of 80 μg/mL ConA and RAW264.7 cells, and 200 μg/mL of mannan can reach saturation inhibition; whereas 50. Mu.g/mL of mannan significantly inhibited the binding of 160. Mu.g/mL ConA to RAW264.7 cells.
FIG. 7 is the effect of 100. Mu.g/mL mannan on ConA-induced macrophage TNF- α expression. The result was a significant decrease in the levels of ConA-induced macrophage TNF- α expression following the addition of 100 μg/mL mannan.
The comprehensive experimental result shows that: conA-induced macrophage activation can be significantly inhibited by 100 μg/ML of Mannan (MMR), indicating that the mechanism of Con A-induced RAW 264.7 macrophage activation is associated with mannose receptor (MMR is an inhibitor of mannose receptor). It was shown that the mechanism of mediating hepatocyte damage following ConA-induced macrophage activation is associated with the induction of macrophage-expressed cytokines and the induction of oxidative stress.
Example 3
Establishment of macrophage/liver cell co-culture model
1. Establishment of an indirect co-culture model: RAW264.7 macrophages were seeded into the upper chamber of LABSELECT Transwell cells at a density of 2×10 5 and BRL3a hepatocytes were seeded into the lower chamber of Transwell cells at a density of 2×10 5. The Transwell chamber 24-well plates were then incubated overnight at 37 ℃ with 5% co 2 incubator. ConA-FITC at a concentration of 160. Mu.g/mL was added to the upper chamber of the Transwell chamber and induced for 12 hours, and the medium in the chamber was collected and used to detect the levels of transaminase, oxidative stress products, and Tumor Necrosis Factor (TNF) - α. RAW264.7 macrophages were washed twice with PBS to eliminate residual media for apoptosis detection.
2. Establishment of direct co-culture model: BRL3a hepatocytes were first seeded into 24-well plates at a density of 2.5×10 5 and incubated for 6h at 37 ℃ with a 5% co 2 incubator; RAW264.7 macrophages were then seeded into the same culture well at a density of 2.5X10 5, incubated with BRL3a hepatocytes, treated with 160. Mu.g/mL of ConA-FITC for 12h, and the supernatant was collected for detection of transaminase, oxidative stress products and Tumor Necrosis Factor (TNF) - α content.
FIG. 8 is the effect of Con A on macrophage-derived TNF- α mediated hepatocyte damage. Incubation with RAW264.7 cells for 12h with 0, 160. Mu.g/mLConA-FITC showed that: the number of apoptotic cells (%) of hepatocytes in ConA 160. Mu.g/mL group was significantly higher than that in the control group (flow cytometry detection).
FIG. 9 is a graph showing the effect of different co-culture methods on macrophage-derived TNF- α mediated hepatocyte damage. The results show that: a-b: compared with the blank group, 80 mug/mLConA causes less damage to liver cells, and only the ALT activity is increased. c-d: compared with a blank group, 160 mug/mLConA causes heavier hepatic cell injury, and AST and ALT activities are increased; e-f: under the same conditions (160. Mu.g/mLConA incubation for 12 h), the direct co-culture resulted in a greater damage to hepatocytes than the indirect co-culture (transwell).
The comprehensive results show that: when RAW264.7 macrophages and BRL3a hepatocytes are co-cultured indirectly, the damage degree of the hepatocytes is light; when RAW264.7 macrophages and BRL3a hepatocytes were co-cultured directly, the extent of hepatocytes damage was relatively high. Therefore, in the future, an indirect co-culture method may be considered for the study of the mechanism of hepatic cell damage in the early stage of autoimmune hepatitis; for example, when the mechanism of advanced or severe liver injury in autoimmune hepatitis is to be studied, direct co-culture may be considered.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (5)
1. A method of establishing a cellular model for studying autoimmune hepatitis, comprising the steps of:
Step 1, the canavalin A realizes the induction and activation of macrophages through the mannose receptor binding on the surfaces of the macrophages, and the specific process is as follows: cell inoculation is carried out on the 24-well plate according to 1X 10 6/RAW 264.7 macrophages in each well, after 800 mu L of high-sugar DMEM culture medium is added into each well, 0-320 mu g/mL of fluorescein isothiocyanate-marked canavalin A is respectively added into each well, and the macrophages are induced for 0-24 hours at 37 ℃;
Step 2, establishing a macrophage and liver cell co-culture model, wherein the macrophage and liver cell co-culture model comprises a macrophage and liver cell indirect co-culture cell model, and the specific method for establishing the macrophage and liver cell indirect co-culture cell model comprises the following steps: RAW264.7 macrophages were seeded into the upper chamber of LABSELECT Transwell cells at a density of 2X 10 5, BRL3a hepatocytes were seeded into the lower chamber of the Transwell cells at a density of 2X 10 5, then the Transwell cells were incubated overnight or 12h at 37℃and 5% CO 2 incubator, 160. Mu.g/mL of fluorescein isothiocyanate-labeled Canavanin A was added to the upper chamber of the Transwell cells, and RAW264.7 macrophages were induced for 12h.
2. The method for establishing a cell model for studying autoimmune hepatitis according to claim 1, characterized in that: the macrophage is a mouse RAW 264.7 macrophage, and the liver cell is a mouse BRL3a liver cell.
3. The method for establishing a cell model for studying autoimmune hepatitis according to claim 1, characterized in that: the macrophage and liver cell co-culture cell model also comprises a macrophage and liver cell direct co-culture cell model.
4. The method for establishing a cell model for studying autoimmune hepatitis according to claim 1, characterized in that: the optimal concentration of the fluorescein isothiocyanate-labeled canavalin A induced macrophages is 160 mug/mL, and the optimal time is 12 hours.
5. The method for establishing a cell model for studying autoimmune hepatitis according to claim 3, wherein the specific method for directly co-culturing the macrophage and the liver cells is as follows: BRL3a hepatocytes were first seeded into 24-well plates at a density of 2.5×10 5 and incubated for 6h at 37 ℃ with a 5% co 2 incubator; RAW264.7 macrophages were then seeded into the same culture well at a density of 2.5X10 5, incubated with BRL3a hepatocytes, and induced by the addition of 160. Mu.g/mL fluorescein isothiocyanate labeled Canavalia protein A for 12h.
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