CN112680416A - Tilapia in vitro immunosuppression model and construction method thereof - Google Patents
Tilapia in vitro immunosuppression model and construction method thereof Download PDFInfo
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Abstract
The invention discloses a tilapia in-vitro immunosuppression model and a construction method thereof, wherein cyclophosphamide is adopted to induce tilapia in-vitro immunocytes, and the concentration of the cyclophosphamide is within 50 mM. Compared with the prior art, the invention has the following advantages: (1) the invention adopts cyclophosphamide to induce the tilapia in vitro immunosuppression model for the first time, and has very important significance for researching fish targeted drugs, specifically giving targeted therapy and adjusting fish immunologic function; (2) the method adopts cyclophosphamide to act on the tilapia in vitro immune cells, optimizes the use dosage and the acting time, avoids hyperfunction model, and ensures that 100 percent of immunosuppressive model is obtained.
Description
Technical Field
The invention belongs to the technical field of aquaculture, and relates to preparation of a cell model, in particular to a tilapia in-vitro immunosuppression model and a construction method thereof.
Background
The immune system is an important defense line for resisting bacterial and viral infection, and inhibition or reduction of the autoimmune function enables fish bodies to generate pathological cell signal transduction pathways to be changed, so that the immune response capability of the bodies is reduced, the sensitivity to diseases is enhanced, the resistance is weakened, other diseases can be induced, and the morbidity and the mortality are greatly increased. Therefore, the method for inducing the immune suppression of tilapia and constructing a model by using Cyclophosphamide (CTX) has very important significance for developing targeted drugs, specifically providing targeted therapy and regulating the immune function of fishes.
CTX is the strongest known alkylating agent immunosuppressant, has strong and lasting effect and is applied in clinic for many times, but the mechanism of CTX acting on the body is quite complex, usually different doses and different acting time can cause two different results of immunosuppression and hyperfunction of the body, and the mechanism of immunosuppression is not clear at present. CTX is widely applied to animals as an immunosuppressant, mainly focuses on pigs, chickens and mice, and aquatic animals such as tilapia and the like have no relevant report.
Disclosure of Invention
The technical problem to be solved is as follows: in order to overcome the defects of the prior art and obtain a method for inducing a tilapia in vitro immunosuppression model by cyclophosphamide, the invention provides the tilapia in vitro immunosuppression model and a construction method thereof.
The technical scheme is as follows: the method for constructing the tilapia in-vitro immunosuppression model adopts cyclophosphamide to induce tilapia in-vitro immunocytes, and the concentration of the cyclophosphamide is within 50 mM.
Preferably, the concentration of cyclophosphamide is 40 mM.
Preferably, the method specifically comprises the following steps: separating immune cells of tilapia, pre-culturing, removing culture supernatant, continuously culturing with L-15 culture medium containing cyclophosphamide, discarding supernatant to determine proliferation activity of immune cells, and simultaneously determining nitric oxide and superoxide anion content in part of immune cells.
Preferably, the immune cells are head and kidney macrophages, peripheral blood leukocytes and spleen cells; wherein the part of immune cells are head and kidney macrophages and spleen cells.
Preferably, the method for isolating head kidney macrophages is as follows: randomly drawing tilapia, anesthetizing by MS-222 (ethyl m-aminobenzoate mesylate), collecting blood, dissecting head and kidney of tilapia under aseptic condition, grinding, passing the grinding fluid through a 100-mesh nylon net, adding the filtered fluid onto the liquid surface of 34%/51% Percoll (multipurpose density gradient separation fluid), and centrifuging at 4 ℃ and 350g for 25min to obtain macrophage at the liquid surface junction.
Preferably, the method for separating peripheral blood leukocytes is as follows: randomly extracting tilapia, anesthetizing by MS-222, and using 0.1% KMnO4Sterilizing body surface, and scrubbing with 75% alcohol cotton ball; and (3) tail vein blood drawing is carried out under the aseptic condition, whole blood and 0.1% FCS-L-15 culture medium are diluted according to the proportion of 1/1 and then are added into a centrifuge tube filled with 60% Percol1 separating medium, and centrifugation is carried out for 15min at the temperature of 4 ℃ and 350g, so that white blood cells at the 60% liquid surface junction are obtained.
Preferably, the splenocytes are isolated as follows: randomly extracting tilapia, anesthetizing by MS-222, and using 0.1% KMnO4Sterilizing body surface, and scrubbing with 75% alcohol cotton ball; under the aseptic condition, dissecting spleen of tilapia, adding PBS (phosphate buffer solution) for grinding, then adding PBS with 5 times volume, centrifuging at 1500g for 10min, and discarding supernatant; adding erythrocyte lysate with 5-10 times of cell volume to resuspend cells, standing at room temperature for 5min, centrifuging at 1500g for 5min, discarding supernatant, washing with PBS 3 times, and resuspending cells with 5% FCS-L-15.
Preferably, the weight of the tilapia is 150 +/-5 g; the tilapia is fed at 27 + -2 deg.C, pH6.8-7.6, and DO>5mg/L，NH3<0.05mg/L，H2S<0.01 mg/L; the daily feeding amount is 2 percent of the fish body mass.
Preferably, the immune cell pre-culture method comprises the following steps: detecting and counting cell activity by trypan blue method, adjusting cell concentration to 107One cell/mL was inoculated into a 96-well plate at 100. mu.L/well, cultured at 27 ℃ for 2 hours, then non-adherent cells were washed off, and cultured at 27 ℃ in 5% FCS-L-15 medium at 100. mu.L/well.
The tilapia in vitro immunosuppression model is constructed by any one of the methods.
Has the advantages that: (1) the invention adopts cyclophosphamide to induce the tilapia in vitro immunosuppression model for the first time, and has very important significance for researching fish targeted drugs, specifically giving targeted therapy and adjusting fish immunologic function; (2) the method adopts cyclophosphamide to act on the tilapia in vitro immune cells, optimizes the use dosage and the acting time, avoids hyperfunction model, and ensures that 100 percent of immunosuppressive model is obtained.
Drawings
FIG. 1 is a graph of the effect of different concentrations of CTX on the proliferation activity rate of tilapia mossambica head and kidney macrophages;
FIG. 2 is the effect of different CTX concentrations on the proliferation activity rate of tilapia mossambica peripheral blood leukocytes;
FIG. 3 is the effect of different concentrations of CTX on the proliferative activity rate of spleen cells of tilapia;
FIG. 4 is a graph of the concentration of CTX on superoxide anion (O) in tilapia mossambica head and kidney macrophages2-) The effect of the content;
FIG. 5 is the comparison of different concentrations of CTX on superoxide anion (O) in spleen cells of tilapia mossambica2-) The effect of the content;
FIG. 6 is a graph of the effect of different concentrations of CTX on Nitric Oxide (NO) content in tilapia mossambica head and kidney macrophages;
FIG. 7 is a graph of the effect of different concentrations of CTX on Nitric Oxide (NO) content in spleen cells of tilapia.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
1 Material
Experimental fish
Healthy tilapia with the weight of 150 +/-5 g is provided by a fishery of the research center of freshwater fishery of the Chinese academy of aquatic sciences. Feeding in circulating water system (240L for each tank), wherein the feeding condition is (27 + -2) ° C, pH is 6.8-7.6, DO (dissolved oxygen)>5mg/L，NH3<0.05mg/L，H2S<0.01 mg/L. The daily feeding amount is 2 percent of the fish body mass.
2 method
2.1 preparation of CTX-induced tilapia in vitro immunosuppression model
After 2h of immune cell pre-culture, removing culture supernatant, and continuously culturing by using L-15 culture medium containing CTX (0, 10, 20, 30, 40 and 50mM) with different concentrations of 100 mu L/hole, wherein each experimental concentration is set to be 6 times; culturing at 27 deg.C for 6 hr, discarding supernatant to determine proliferation activity of head and kidney macrophage, peripheral blood leukocyte and spleen cell, and simultaneously determining Nitric Oxide (NO) and superoxide anion (O) in head and kidney macrophage and spleen cell2 -) And (4) content.
2.2 isolation of Tilapia mossambica head and kidney macrophages
Randomly drawing tilapia, and collecting blood after MS-222 anesthesia. Under the aseptic condition, the head and the kidney of the tilapia are dissected and taken, and are ground. Grinding the solution through a 100 mesh nylon net, adding the filtrate onto the liquid surface of 34%/51% Percol1 separating medium, and centrifuging at 4 deg.C and 350g for 25min to obtain macrophage at the liquid surface boundary. The cells were washed 2 times at 4 ℃ with centrifugation at 2000r/min for 10 min. Detecting and counting cell activity by trypan blue method, adjusting cell concentration to 107One cell/mL was inoculated into a 96-well plate at 100. mu.L/well, cultured at 27 ℃ for 2 hours, then nonadherent cells were washed off, and 100. mu.L/well of 5% FCS-L-15 (1% S/P, 0.2% heparin and 5% FCS) was added and cultured at 27 ℃ until use.
2.3 isolation of peripheral blood leukocytes from Tilapia mossambica
Randomly extracting tilapia, anesthetizing by MS-222, and using 0.1% KMnO4Sterilizing body surface, and scrubbing with 75% alcohol cotton ball; tail vein blood was drawn under aseptic conditions, whole blood was diluted with 0.1% FCS-L-15 (1% S/P, 0.2% heparin and 0.1% FCS) medium at a ratio of 1/1, an appropriate amount was added slowly to a centrifuge tube containing 60% Percol1 isolate, centrifuged at 4 ℃ and 350g for 15min to obtain white blood cells at the 60% interface of the liquid surface, and the procedure was as above.
2.4 separation of spleen cells of Tilapia mossambica
Separating from peripheral blood leukocyte, dissecting spleen of tilapia under aseptic condition, adding appropriate amount of PBS, and grinding. 5 volumes of PBS were added, 1500g was centrifuged for 10min and the supernatant was discarded. Adding erythrocyte lysate with 5-10 times of cell volume to resuspend cells, standing at room temperature for 5min, centrifuging at 1500g for 5min, and discarding supernatant. PBS wash 3 times, 5% FCS-L-15 resuspend cells, trypan blue count. Culturing at 27 deg.C for use.
2.5 detection of cell growth Activity by MTT method
After collecting the supernatant, the 96-well cell plates were transferred to a centrifuge at 350g for 10min, the supernatant was decanted, 100. mu.L of 15% FCS-L-15 and 20. mu.L of MTT were added to each well, and incubation was continued at 27 ℃ for 4 h. And (3) decanting the supernatant, adding 150 mu L DMSO into each well, oscillating for 10min to ensure that the crystals are fully dissolved and the wavelength of 570nm is selected, measuring the light absorption value of each well on an enzyme-linked immunosorbent assay instrument, and recording the result.
2.6 determination of superoxide anion
The procedure was performed according to the superoxide anion content detection kit (Solebao, BC 1290). Adding 1mL of the extractive solution into each group of cells, grinding, centrifuging at 4 deg.C and 12000 rpm for 20min, collecting 20 μ L of supernatant, determining protein content, and taking the rest supernatant as sample to be tested to determine O2 -Content, absorbance at 530 nm.
2.7 determination of nitric oxide
Adding 100 μ L NO detection lysis solution (Biyunyan, S0021) into each well of immunocyte, mixing, fully lysing, 10000-. The supernatant (75. mu.L) was transferred to another cell culture plate, and Griss reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% phosphoric acid) was added to determine absorbance at 540 nm.
3 results
Data analysis was performed using the SPSS20.0 software package, and all values are expressed as mean ± standard error. All data were analyzed by One-way ANOVA with Tukey multiple comparisons of data between different groups, with P <0.05 indicating significant differences.
As can be seen from FIGS. 1 to 3, after 0-50mM CTX acts on tilapia immune cells for 6h, the cell proliferation activity of head and kidney macrophages, peripheral blood leukocytes and splenocytes is remarkably reduced along with the increase of the acting concentration of CTX, and the survival rates of the head and kidney macrophages, peripheral blood leukocytes and splenocytes of the 50mM CTX acting group are respectively reduced to 76%, 88% and 67% of that of the blank group, which indicates that the activity of tilapia immune cells is inhibited.
As can be seen from FIG. 2, after 0-50mM CTX acted on tilapia immune cells for 6h, with the increase of the action concentration of CTX, O in head and kidney macrophages and splenocytes2-The content is obviously reduced, which indicates that the oxygen respiratory activity of immune cells of tilapia is inhibited.
As can be seen from FIG. 3, after 0-50mM CTX acts on tilapia immune cells for 6h, the NO content in head and kidney macrophages and splenocytes is remarkably reduced along with the increase of the acting concentration of CTX, which indicates that the nitrogen respiratory activity of tilapia immune cells is inhibited.
And (4) conclusion: and (3) selecting 40mM of CTX with damage of 6h as a condition for constructing an in-vitro immunosuppression model of the tilapia by combining the influence of CTX with different concentrations on the proliferation activity and oxygen nitrogen burst activity of immune cells of the tilapia.
Claims (10)
1. The method for constructing the tilapia external immunosuppression model is characterized in that cyclophosphamide is adopted to induce tilapia external immunocytes, and the concentration of the cyclophosphamide is within 50 mM.
2. The method for constructing an in vitro immunosuppression model of tilapia according to claim 1, wherein the concentration of cyclophosphamide is 40 mM.
3. The method for constructing the tilapia in vitro immunosuppression model according to claim 1, wherein the method specifically comprises the following steps: separating immune cells of tilapia, pre-culturing, removing culture supernatant, continuously culturing with L-15 culture medium containing cyclophosphamide, discarding supernatant to determine proliferation activity of immune cells, and simultaneously determining nitric oxide and superoxide anion content in part of immune cells.
4. The method for constructing the tilapia in vitro immunosuppression model according to claim 3, wherein the immune cells are head and kidney macrophages, peripheral blood leukocytes and spleen cells; wherein the part of immune cells are head and kidney macrophages and spleen cells.
5. The method for constructing the tilapia in vitro immunosuppression model according to claim 4, wherein the method for separating head and kidney macrophages is as follows: randomly drawing tilapia, anesthetizing by MS-222, collecting blood, dissecting and taking tilapia head and kidney under aseptic condition, grinding, passing the grinding liquid through a 100-mesh nylon net, adding the filtrate on the liquid surface of 34%/51% Percol1 separating liquid, and centrifuging at 4 ℃ for 25min at 350g to obtain macrophage at the liquid surface boundary.
6. The method for constructing the tilapia mossambica in vitro immunosuppression model according to claim 4, wherein the method for separating peripheral blood leukocytes comprises the following steps: randomly extracting tilapia, anesthetizing by MS-222, and using 0.1% KMnO4Sterilizing body surface, and scrubbing with 75% alcohol cotton ball; and (3) tail vein blood drawing is carried out under the aseptic condition, whole blood and 0.1% FCS-L-15 culture medium are diluted according to the proportion of 1/1 and then are added into a centrifuge tube filled with 60% Percol1 separating medium, and centrifugation is carried out for 15min at the temperature of 4 ℃ and 350g, so that white blood cells at the 60% liquid surface junction are obtained.
7. The method for constructing the tilapia in vitro immunosuppression model according to claim 4, wherein the spleen cell isolation method comprises the following steps: randomly extracting tilapia, anesthetizing by MS-222, and using 0.1% KMnO4Sterilizing body surface, and scrubbing with 75% alcohol cotton ball; under the aseptic condition, dissecting spleen of tilapia, adding PBS (phosphate buffer solution) for grinding, then adding PBS with 5 times volume, centrifuging at 1500g for 10min, and discarding supernatant; adding erythrocyte lysate with 5-10 times of cell volume to resuspend cells, standing at room temperature for 5min, centrifuging at 1500g for 5min, discarding supernatant, washing with PBS 3 times, and resuspending cells with 5% FCS-L-15.
8. The method for constructing the tilapia in vitro immunosuppression model according to claim 3, wherein the weight of the tilapia is 150 +/-5 g; the tilapia is fed at 27 + -2 deg.C, pH6.8-7.6, and DO>5mg/L，NH3<0.05mg/L，H2S<0.01 mg/L; the daily feeding amount is 2 percent of the fish body mass.
9. The method for constructing the tilapia in vitro immunosuppression model according to claim 3, wherein the immune cell pre-culture method comprises the following steps: detecting and counting cell activity by trypan blue method, adjusting cell concentration to 107One cell/mL was inoculated into a 96-well plate at 100. mu.L/well, cultured at 27 ℃ for 2 hours, then non-adherent cells were washed off, and cultured at 27 ℃ in 5% FCS-L-15 medium at 100. mu.L/well.
10. An in vitro immunosuppression model of tilapia mossambica constructed by the method of any one of claims 1-9.
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| RU2008141628A (en) * | 2008-10-22 | 2010-04-27 | Леонид Юрьевич Телегин (RU) | METHOD FOR SUPPRESSING IMMUNE REACTIONS |
| CN102526716A (en) * | 2011-12-07 | 2012-07-04 | 蔡颖 | Preparation of specific tumor killing cell |
| CN105030794A (en) * | 2015-06-14 | 2015-11-11 | 四川农业大学 | Establishment of rhesus monkey immunosuppression model |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2008141628A (en) * | 2008-10-22 | 2010-04-27 | Леонид Юрьевич Телегин (RU) | METHOD FOR SUPPRESSING IMMUNE REACTIONS |
| CN102526716A (en) * | 2011-12-07 | 2012-07-04 | 蔡颖 | Preparation of specific tumor killing cell |
| CN105030794A (en) * | 2015-06-14 | 2015-11-11 | 四川农业大学 | Establishment of rhesus monkey immunosuppression model |
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