CN106434558B - Method for separating, culturing and verifying functions of fish mononuclear/phagocyte - Google Patents

Abstract

The application provides a separation, culture, transformation and function verification method for fish monocytes, which successfully obtains fish phagocytes by optimizing culture parameters and conditions; phagocytic function of phagocytic cells of fish obtained by phagocytic cell phagocytic exogenous chicken erythrocyte and in vitro respiratory burst detection. The method of the application realizes in vitro separation, transformation, continuous culture and the like of the fish phagocytes, has the advantages of less required samples, short operation time, simple flow and high accuracy, and can be widely applied to research of fish immune-related functions on the cell level.

Description

Method for separating, culturing and verifying functions of fish mononuclear/phagocyte

Technical Field

The application belongs to the technical field of biological cell culture, and particularly relates to a method for separating and culturing fish mononuclear/phagocyte and verifying functions.

Background

With the recent expansion of the cultivation scale, the development of the cultivation industry of fishes with high economic value (such as cynoglossus semilaevis) is seriously hampered by the disease problem caused by harmful bacteria such as vibrio anguillarum and the like. At present, no effective immune control technology and method exist, and the occurrence of diseases is still controlled by adopting traditional modes such as chemical medicines, antibiotics and the like. Peripheral blood mononuclear cells (precursors of phagocytes) are an important component of fish humoral immunity and play an important role in the processes of fish specific immunity and nonspecific immunity. At present, the immune function research of fish monocytes such as cynoglossus semilaevis still belongs to a starting stage, the action mechanism of the fish monocytes is not clear, and the cynoglossus semilaevis phagocytes which can be continuously cultured for cell function research are not obtained so far.

The precursor of phagocytes is mononuclear cells, which are spherical, pear-shaped or irregular, and have elongated pseudopodiform cytoprocess on their surface. The nuclei of monocytes are smaller, kidney-shaped or horseshoe-shaped, often on one side, with a nuclear to cytoplasmic ratio <1. In Giemsa staining, the nuclei of monocytes are in a grid. Under transmission electron microscopy, there are many mitochondria in the cytoplasm, and sometimes golgi apparatus, rough endoplasmic reticulum, ribosome, phagocytized senescent cells or nuclei thereof, and the like.

Monocytes have a strong phagocytic capacity and exert important nonspecific phagocytic functions in the fish organism. It can capture and fuse foreign substances and its own senescent cells through the pseudopodiform cell process and can produce active deformation movements. In addition, monocytes are also very sensitive to environmental changes, and in fish living in severely contaminated waters, the monocyte content can be approximately 50 times higher than normal. The fish may also cause a sudden increase in the number of monocytes after infection by parasites or artificial injection of substances such as colloidal carbon particles. Therefore, by measuring the quantity of the fish monocytes, the health condition of the fish and the environmental quality of the living water area can be primarily known.

Monocytes and phagocytes transformed with monocytes play an important role in the resistance and defense against pathogen infection, and in particular play a great role in intracellular pathogen defense. In addition, phagocytes are also present in fragments after phagocytosis by phagocytes, and they play a vital role in innate immunity and acquired immunity. They all exhibit different functions, morphologies, and metabolic effects, depending on the environment within the tissue and the different differentiation and activity stages they are in. Monocytes are derived from hematopoietic progenitor cells in the bone marrow, which migrate through the blood circulation of 2-3 d to individual tissues, differentiating into phagocytes in the tissues.

The cell which can survive in vitro for a long time and can be subcultured is the basis for performing researches such as cell immunology. Common monocyte isolation methods include: plasma gel method, standard Ficoll method and Percoll-paque method. But these methods alone utilize a mixed suspension that is prone to lymphocyte, granulocyte, platelet and various erythrocytes.

Disclosure of Invention

Aiming at overcoming the defects of the prior art, the application provides a separation, culture, transformation and function verification method for fish monocytes. The specific scheme is as follows:

a method for separating and culturing fish mononuclear/phagocyte, comprising the steps of:

step one: isolation and culture of monocytes;

step two: in vitro culture of monocytes induced as phagocytes;

further, in the first step, the specific steps are as follows:

s1, extracting peripheral blood of fish, and uniformly diluting and mixing whole blood with Hank' S buffer solution in a ratio of 1:2.5-1:3 to obtain diluted blood;

s2, taking lymphocyte separation liquid with the weight of 1.5 times of diluted blood and the specific gravity of 1.05-1.09 and release blood in a centrifuge tube;

s2, centrifuging for 18-21 minutes at 1800-2100rpm of a horizontal centrifuge, and collecting a mononuclear cell layer;

s4, adding 4-5 times of Hank' S buffer solution to wash for 2 times, centrifuging at 900-1100rpm for 5min, and collecting cell sediment;

s5, re-suspending in a DMEM complete medium, inoculating in a culture flask, and placing in a 24 ℃ incubator for culturing 3h;

s6, washing the supernatant with Hank' S buffer solution, and adding fresh culture medium to continue culturing the adherent cells;

s7, centrifuging to obtain a monocyte layer, namely lymphocyte and monocyte;

s8, re-suspending the separated mononuclear cells in a complete DMEM medium containing 10-15% of fetal calf serum and added with an adherence promoting growth factor bFGF;

s9, after culturing for 2-4 hours, washing away suspended lymphocytes to obtain adherent mononuclear cells.

Further, in the second step, the specific steps are as follows:

s1, adding 10-15% of lymphocytes in a complete DMEM culture medium containing mononuclear cells and 15% of fetal calf serum;

s2, changing fresh DMEM complete medium for half a day, and culturing for 3-12 days.

A method of functional verification of a mononuclear/phagocyte cell comprising the steps of:

s1, incubating 50ul of the separated mononuclear cell with phorbol ester (PMA) stimulant at 23-25 ℃ for 15min;

s2, adding 25ul of dihydrorhodamine, and incubating for 5min at 24 ℃ in a dark place;

s3, washing for 2 times by adopting PBS buffer solution;

s4, centrifuging at 1500rpm for 5min;

s5, removing supernatant, and adding 0.5ml of PBS buffer solution to resuspend cells;

s6, detecting by using an up-flow cytometer.

The beneficial effects of the application are as follows:

according to the application, the cell separation liquid is used for density gradient centrifugation, the fish mononuclear cells (phagocytes) are successfully separated and purified by correcting various parameters and experimental conditions in the experimental process, and then the cynoglossus semilaevis mononuclear cells are cultured for more than one week in an adherent growth mode by optimizing nutrition and culture conditions, so that the method can be suitable for subsequent experiments. The application successfully detects respiratory burst of phagocytes and provides materials for further researching biological characteristics and immunological functions of mononuclear/phagocytes.

Drawings

FIG. 1 shows the monocyte morphology (A: 12h for culture; B: 24h for culture; C: 32h for culture; D: 38h for culture) in the examples of the present application;

FIG. 2 is a graph showing the growth of monocytes in accordance with the present application;

FIG. 3 is a graph showing respiratory burst detection under PMA stimulation in an embodiment of the present application;

FIG. 4 shows respiratory burst detection after Vibrio anguillarum infection in an embodiment of the application;

FIG. 5 shows monocytes and phagocytes according to an embodiment of the present application;

FIG. 6 shows several forms of phagocytes according to embodiments of the application;

FIG. 7 is an illustration of an aged phagocyte according to an embodiment of the present application;

FIG. 8 shows giemsa stained phagocytes according to an embodiment of the application;

FIG. 9 shows phagocytes that phagocytose chicken erythrocytes in an embodiment of the application;

FIG. 10 shows the metaphase of chromosome division of phagocytes on day 5 of culture in examples of the present application;

FIG. 11 shows the distribution of the chromosome number of phagocytes in the examples of the present application.

Detailed Description

In order to make the technical solution of the present application better understood by those skilled in the art, the technical solution of the present application will be clearly and completely described in the following with reference to the accompanying drawings, and based on the embodiments of the present application, other similar embodiments obtained by those skilled in the art without making any inventive effort should be included in the scope of protection of the present application.

The present application has been completed based on the following findings by the inventors: in fish peripheral blood cells, various cells are caused to undergo density gradient centrifugation due to differences in morphology and weight: erythrocytes and granulocytes with the greatest specific gravity, secondary mononuclear cells (including lymphocytes and monocytes), and thrombus cells with the smallest specific gravity are at different levels when separated. Therefore, by correcting the centrifugal speed, the centrifugal time, and the density ratio of the separation liquid, the cells can be separated by the density gradient centrifugation method. After centrifugation, the plasma layer can be divided into a transparent plasma layer and a white membrane-shaped mononuclear cell layer from top to bottom, the transparent separation liquid layer has the greatest specific gravity of granulocytes and erythrocytes, and the granulocytes and erythrocytes are sunk at the lowest part, so that the purpose of coarse separation is achieved; secondly, the difference exists between the ability of the lymphocytes and the phagocytes to adhere when the lymphocytes and the phagocytes in the tunica albuginea are cultured in the culture medium, so that the phagocytes can be successfully separated by not distinguishing the adhesion force, and the serum in the cell culture medium can promote the cell growth and is beneficial to cell adhesion, so that the phagocytes and the lymphocytes can be effectively separated by correcting the addition amount and the addition timing of the serum and by correcting the concentration of pancreatin after a certain time of culture; in addition, in vitro monocytes are activated by phagocyte colony stimulating factors (M-CSF) to phagocytes, which are characterized by phagocytic activity, respiratory burst, etc. This is the largest difference between lymphocytes and phagocytes, which can be identified by this condition.

Examples: taking cynoglossus semilaevis as an example, the application discloses a method for culturing fish phagocytes, verifying functions and transforming the phagocytes.

1. Separation and culture of cynoglossus semilaevis peripheral blood mononuclear cells

The cynoglossus semilaevis is anesthetized in ice water, 70% alcohol cotton balls wipe back tail veins, heparin anticoagulation tubes extract 2ml of peripheral blood, whole blood and Hank's buffer solution are diluted and mixed uniformly in an ultra-clean workbench, 3ml of lymphocyte separation solution with the specific gravity of 1.05-1.09 is taken in a 15ml centrifuge tube, 2ml of diluted blood is added to centrifuge for 18-21 minutes at 1800-2100rpm of a horizontal centrifuge, a mononuclear cell layer is collected, hank's buffer solution with the volume of 4-5 times is added to wash for 2 times, centrifugation is carried out at 900-1100rpm for 5 minutes to collect cell sediment, the cell sediment is resuspended in DMEM complete medium, the cell sediment is inoculated in a culture flask and placed in a 24 ℃ incubator to be cultured for 3 hours, hank's buffer solution is added to wash out supernatant, adherent cells are continuously cultured, fresh complete DMEM medium is replaced by half of the specific gravity every other day, and cell morphology is observed under an inverted microscope.

According to the characteristic that lymphocytes are not adhered to the outside of a body and are in a suspension state, and the monocytes are very easy to adhere to the wall, the separated mononuclear cells are resuspended in a complete DMEM (medium) containing 10-15% of fetal calf serum and added with an adhesion promoting growth factor bFGF, and the suspension lymphocytes are washed after 2-4h of culture, so that the adhered monocytes are obtained, and the monocytes can be cultured for more than one week and are shown in a morphological figure 1 under an inverted microscope. The cell body diameter is 12-16 mu m, the cells are in irregular shapes such as a sector shape, a polygon shape and the like, the cell mass is high, and the adhesive property is strong.

MTT method for detecting monocyte in vitro growth curve

The isolated monocytes were inoculated into 96-well culture plates and incubated at 24℃in an incubator. 50ul of MTT solution is added at 0h, 6h, 12h, 18h, 24h, 36h and 48h respectively, and the incubator continues to incubate for 4h. The supernatant was washed, 150ul of DMSO was added to each well, the plate was shaken for 10min, and the optical density of each well was measured by an ELISA reader at 570nm and repeated three times. The in vitro growth curve of cynoglossus semilaevis mononuclear cells detected by MTT method is shown in figure 2. The cells can be well adhered after being inoculated for 3 hours, proliferate rapidly after 12 hours, the cell quantity is basically stable after 48 hours, and the cells show aggregation growth phenomenon and irregular morphology.

3. Determination of monocyte respiratory burst

Adding phorbol ester (PMA) stimulant 50ul to the separated mononuclear cells, incubating for 15min at 23-25 ℃, adding dihydrorhodamine 25ul, incubating for 5min at 24 ℃ in a dark place, washing for 2 times by using PBS buffer, centrifuging at 1500rpm for 5min, removing supernatant, adding 0.5ml of PBS buffer to resuspend cells, detecting by using an upflow cytometer, and setting a control group, wherein the mononuclear cells are not added in the control group; meanwhile, the cynoglossus semilaevis is directly infected by vibrio anguillarum: an experimental group and a control group were set, and the experimental group was 3.18X10 ⁵ The cynoglossus semilaevis is intraperitoneally injected with CFU/g (semi-lethal dose, LD 50), and the control group is injected with PBS at a dose corresponding to the body weight in the reference experimental group. After 24h, the peripheral blood of the experimental group fish and the control group fish are respectively aseptically taken, mononuclear cells are obtained by separation according to the method, 50ul of the phorbol ester (PMA) stimulant is added, incubation is carried out for 15min at 24 ℃, 25ul of the dihydrorhodamine is added, incubation is carried out for 5min at 24 ℃ in a dark place, PBS buffer is used for washing for 2 times, centrifugation is carried out at 1500rpm for 5min, supernatant is removed, 0.5ml of PBS buffer is added for resuspension of cells, and the cells are respectively detected by a flow cytometer.

The flow cytometer detection results are shown in fig. 3, wherein the proportion of cells which fluoresce in the normal cell control group is 0.4%, and the proportion of cells which fluoresce in the experimental group under the stimulation of PMA is 80.67%, which indicates that the separated monocytes have respiratory burst function. The respiratory burst of mononuclear cells isolated after Vibrio anguillarum infection is shown in FIG. 4, the proportion of fluorescing cells in the control group is 4.18%, and the proportion of fluorescing cells in the experimental group is 83.81%, which indicates that the pathogenic stimulus significantly enhances the respiratory burst intensity of mononuclear cells.

4. In vitro culture of monocytes induced to phagocytes

Monocytes and lymphocytes are co-cultured in vitro to stimulate activation of monocytes to become typical phagocytes. Thus, 5-10% by number of lymphocytes were added to a complete DMEM medium containing 15% fetal bovine serum, and half a day apart was exchanged for fresh DMEM complete medium. After 3d of cell culture, the cells were observed under an inverted phase contrast microscope (see FIG. 5). Phagocytes adhere to the wall, the cells stretch and firmly adhere, the volume is obviously increased, the irregular shape is gradually changed into an oval shape, the shape is a omelette shape, and a few of phagocytes are long fusiform shapes. There may be partial fusion between cells, visible platelike pseudopodia and protrusion around or at both poles, no apparent proliferation of cells (FIG. 6), culturing to 13 days, and aging of phagocytes (FIG. 7).

5. Phagocyte staining observations

Culturing phagocytes until the phagocytes are cultured for 7 days, washing off lymphocytes suspended in a culture flask by using PBS buffer solution, adding methanol to fix the adherent phagocytes, dripping diluted giemsa staining solution to cover the bottom of the flask, staining at room temperature for 20min, sucking the staining solution, flushing the adherent cells by using distilled water, and observing by using an inverted microscope, wherein the phagocytes have larger volume and irregular morphology. The cytoplasm is rich, the color is light pink, and the cytoplasm is rich in particles and a few vacuoles. The nucleus blue-purple-red, oval, kidney-shaped or irregular, has typical phagocytic morphological characteristics (fig. 8).

6. Verification of phagocytic function of phagocytes

Chicken erythrocyte phagocytosis assay: culturing 5d 100ul/ml of 10-15% chicken erythrocyte suspension is added into the culture system, the culture is performed in a 24 ℃ incubator for 30 min to 1h, and after PBS flushing, the microscopic examination is performed. Phagocytes phagocyting chicken erythrocytes, a plurality of chicken erythrocytes are visible in the cytoplasm, and the nuclei are squeezed (see fig. 9). The external bacteria invade the phagocytes, the microscopic examination and observation of the turbid culture medium is carried out, the culture medium is continuously placed in a 24 ℃ incubator for culture, and the phagocytes are found to finally phagocytize the bacteria, so that the culture medium becomes clear.

7. Phagocyte chromosome karyotyping

Karyotyping was performed at day 5 of phagocyte culture and chromosome preparation was as follows: colchicine with the concentration of 1ug/ml is added into a culture flask, the culture medium is sucked and removed, PBS is used for flushing, 0.25% pancreatin digestion mixed solution is used for digestion, then the culture medium is added for blowing, phagocytes are tightly attached to the walls, cells which are difficult to digest are scraped by using the cells, then the cell suspension is transferred into a 15ml centrifuge tube, centrifugation is carried out at 1500rpm for 5min, the supernatant is sucked and removed, 5ml of 0.0375M KCl is added, then hypotonic treatment is carried out for 25min in a water bath at 37 ℃, 1ml of newly prepared precooled carnot fixing solution is slowly added, centrifugation is carried out at 1000rpm for min, the supernatant is sucked and removed, 2ml carnot fixing solution is added, ice bath fixing treatment is carried out for 15min, centrifugation at 1000rpm for 5min is repeated twice, 0.2-0.5ml carnot fixing solution is reserved according to the quantity of cells collected by centrifugation after the second fixing, and the glass slide is dripped by a cold dripping method, after drying, dyeing is carried out by 5% Gimsa for 25min, and after drying, oily microscopy is carried out.

Chromosome karyotyping was performed on phagocytes cultured for 5 days, and the number of chromosomes was varied from 28 to 56 among 100 divided phases counted, with 61% of the divided phase chromosomes being 42 (fig. 10), and all of the 42 chromosomes being terminal mitotic chromosomes and containing W-type chromosomes. The number of chromosomes was normally distributed, the frequency of occurrence of diploid chromosome numbers was highest, and the proportion of other aneuploidy was small (FIG. 11).

The foregoing detailed description of the application has been presented for purposes of illustration and description, but is not intended to limit the scope of the application, i.e., the application is not limited to the details shown and described.

Claims (1)

1. A method for culturing phagocytes of fish, characterized in that it comprises the steps of isolation and culture of monocytes, identification of the function of the monocyte body and induction of monocytes as phagocytes,

wherein the isolation and culture of monocytes comprises the steps of:

s1, extracting peripheral blood of fish, and uniformly diluting and mixing whole blood with Hank' S buffer solution in a ratio of 1:2.5-1:3 to obtain diluted blood;

s2, taking lymphocyte separating liquid with the weight 1.5 times of that of diluted blood and the specific gravity 1.05-1.09, and mixing the lymphocyte separating liquid with the blood in a centrifuge tube;

s3, centrifuging for 18-21min at 1800-2100rpm of a horizontal centrifuge, and collecting a mononuclear cell layer;

s4, adding 4-5 times of Hank' S buffer solution to wash for 2 times, centrifuging at 900-1100rpm for 5min, and collecting cell sediment;

s5, re-suspending in a DMEM complete culture medium, inoculating in a culture flask, and placing in a 24 ℃ incubator for culturing for 3 hours; s6, washing the supernatant with Hank' S buffer solution, and adding fresh culture medium to continue culturing the adherent cells;

s7, centrifuging to obtain a monocyte layer, namely lymphocyte and monocyte;

s8, re-suspending the separated mononuclear cells in a complete DMEM medium containing 10-15% of fetal calf serum and added with an adherence promoting growth factor bFGF;

s9, culturing for 2-4 hours, and washing away suspended lymphocytes to obtain adherent mononuclear cells;

the steps of inducing monocytes to phagocytes are as follows:

s1, adding 5-10% of lymphocytes in a complete DMEM culture medium containing mononuclear cells and 15% of fetal calf serum;

s2, changing fresh DMEM complete medium for half a day, and culturing for 3-12 days.