CN106893691B - Shrimp lymphocytes, methods for purification thereof and methods for in vitro assessment of innate immune response - Google Patents
Shrimp lymphocytes, methods for purification thereof and methods for in vitro assessment of innate immune response Download PDFInfo
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Abstract
The invention relates to a purification method of shrimp lymphocytes, which comprises the steps of pretreating a shrimp body fluid sample containing glucosamine and low-concentration sodium ions as an anti-agglutinant, and culturing the sample by using a cell culture solution containing low-concentration sodium ions, so that the cell survival rate of the shrimp lymphocytes can be effectively improved, and the cell yield and the cell purity can be improved. The obtained shrimp lymphocytes can be used for evaluating the innate immune response in vitro.
Description
Technical Field
The invention relates to an invertebrate lymphocyte and a purification method thereof, in particular to a shrimp lymphocyte and a purification method thereof and a method for evaluating innate immune response in vitro by using the shrimp lymphocyte.
Background
Shrimps belong to invertebrates, and the immune system of invertebrates generally uses a humoral coagulation mechanism (coagulotion), i.e., a humoral coagulation mass forms a physical barrier as a first line of defense against pathogenic invasion, preventing microbial pathogens from entering the body.
Since shrimps have only innate immunity (nature) and do not have adaptive immunity (adaptive immunity) developed by vertebrates against pathogens, specific memory immune cells induced after infection with a specific pathogen cannot be generated, and thus vaccines for shrimps cannot be developed.
The most effective strategy for increasing the disease resistance of the shrimps is to develop an immunomodulator capable of promoting the innate immunity of the shrimps. However, a shrimp cell line capable of being maintained for a long period of time has not been established so far.
In view of the above, there is a need to develop a primary shrimp lymphocyte cell that provides a platform for in vitro (in vitro) screening.
Disclosure of Invention
The invention provides a method for purifying shrimp lymphocytes, which comprises the steps of pretreating a shrimp body fluid sample containing glucosamine and low-concentration sodium ions as an anti-agglutinant, and culturing the sample by using a cell culture solution containing the low-concentration sodium ions, so that the shrimp lymphocytes with high cell survival rate, cell yield and cell purity can be obtained.
Another aspect of the present invention provides an anti-agglutinating agent for shrimp lymphocytes, which comprises glucosamine and a low concentration of sodium ions, thereby inhibiting the cell agglutination rate of the shrimp lymphocytes and improving the in vitro cell survival rate thereof.
In still another aspect, the present invention provides a cell culture solution for shrimp lymphocytes, comprising an L-15 culture solution, fetal bovine serum, and low-concentration sodium ions, thereby increasing the cell survival rate of the shrimp lymphocytes and the time for in vitro culture.
In yet another aspect, the present invention provides a method for in vitro assessment of shrimp innate immune response, which comprises co-culturing shrimp lymphocytes obtained by the above method with a test sample to assess whether the test sample has activity of stimulating shrimp innate immune response.
According to the above aspect of the present invention, a method for purifying shrimp lymphocytes is provided. In one embodiment, a shrimp body fluid sample is first provided, wherein the shrimp body fluid sample is derived from white shrimp (Litopenaeus vannamei). Then, a pretreatment is carried out, and the shrimp body fluid sample is uniformly mixed with an anti-agglutinating agent to form a single-cell suspension, wherein the anti-agglutinating agent can comprise, for example, less than 10.0g/L of sodium chloride, 5.5g/L of citric acid, 8.0 to 10.0g/L of sodium citrate, 19.8 to 20.0g/L of glucose and 21.563g/L of glucosamine, and the anti-agglutinating agent has a pH value of 7.0. Then, a centrifugation step was performed to remove the supernatant of the single cell suspension and obtain a cell pellet. Thereafter, a culturing step is performed to uniformly suspend and culture the cell precipitate in a cell culture solution containing, for example, 2-fold concentration (2X) of L-15 culture solution, 5g/L of sodium chloride, 10g/L of glucose, 15% (v/v) of fetal bovine serum and having a pH of 7.0, to obtain shrimp lymphocytes.
According to another aspect of the present invention, there is provided a cell culture solution of shrimp lymphocytes, wherein the anti-agglutinating agent may comprise, for example, less than 10.0g/L of sodium chloride, 5.5g/L of citric acid, 8.0 to 10.0g/L of sodium citrate, 19.8 to 20.0g/L of glucose, and 21.563g/L of glucosamine, and the anti-agglutinating agent has an acid-base number of pH 7.0.
According to an embodiment of the present invention, the anti-clumping agent may further include 2.5g/L EDTA.
According to yet another aspect of the present invention, a method for in vitro assessment of shrimp innate immune responses is provided. In one embodiment, the co-culture step is performed first, and the shrimp lymphocytes obtained by the above method are cultured in the above cell culture solution for 30 minutes to 72 hours, wherein the cell culture solution contains or does not contain the sample to be tested. Then, the cell survival rate of the shrimp lymphocytes is detected, wherein the cell survival rate of the shrimp lymphocytes not cultured with the sample to be detected is a reference value, and the cell survival rate of the shrimp lymphocytes cultured with the sample to be detected is a measured value. And then, performing a judging step, and judging that the sample to be detected has the activity of stimulating the shrimp innate immune response if the measured value is higher than the reference value.
According to an embodiment of the present invention, before the co-culturing step, a pre-culturing step may be optionally performed to culture the shrimp lymphocytes in a cell culture solution without the test sample for at least 24 hours.
By applying the purification method of the shrimp lymphocytes, the body fluid sample of the shrimp, which contains glucosamine and the anti-agglutinant of low-concentration sodium ions, is pretreated, and then the cell culture solution containing the low-concentration sodium ions is used for culturing, so that the cell survival rate of the shrimp lymphocytes can be effectively improved, and the cell yield and the cell purity can be improved. The obtained shrimp lymphocytes can be used as an in vitro screening platform to evaluate the innate immune stimulation activity of a sample to be tested.
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In order to make the above and other objects, features, advantages and embodiments of the present invention more comprehensible, thereof, the accompanying drawings are provided, in which:
fig. 1 is a schematic diagram of a shrimp collection site according to an embodiment of the invention.
FIG. 2 is a schematic view of a shrimp body fluid being extracted at a specific angle according to one embodiment of the present invention.
FIG. 3 shows a two-dimensional scattergram of a flow cytometer for shrimp lymphocytes according to an embodiment of the invention.
FIG. 4 shows cell survival rates of shrimp lymphocytes treated with the anti-agglutinating agents according to preparation examples 1 to 5 of the present invention and preparation comparative example 1.
FIG. 5 shows the cell survival rates of the anti-agglutinating agent according to preparation examples 1 to 5 of the present invention and preparation comparative example 2 after treatment of shrimp lymphocytes.
FIGS. 6A and 6B show light microscope photographs of shrimp lymphocytes cultured in the cell culture solutions of production comparative example 3 (FIG. 6A) and production example 7 (FIG. 6B), respectively, for 120 minutes.
Detailed Description
In summary, the present invention provides a method for purifying shrimp lymphocytes, which comprises the steps of pre-treating a sample of prawn body fluid containing glucosamine and low-concentration sodium ions as an anti-agglutinant, and culturing the sample with a cell culture medium containing low-concentration sodium ions, so as to obtain shrimp lymphocytes with high cell survival rate, high cell yield and high cell purity.
In a broad sense, the term "shrimp lymphocytes" as used herein refers to primarily cultured lymphocytes purified in vivo from, for example, white shrimp (Litopenaeus vannamei), but the species of the family shrimp to which the present invention is applicable is not limited to those exemplified herein. Generally, the shrimp lymphocytes of the present invention may comprise three groups of cells with higher cell purity, namely, granulocytes (granulocytes), hemigranulocytes (hemigranulocytes), and hyaline cells, wherein the proportion of the granulocytes (granulocytes) is, for example, 36%, 9%, and 55%, respectively, but the proportion of each cell of the three groups of shrimp lymphocytes is not limited to the above, and the proportion of any one group of the three groups of liquid cells may be more or less, depending on the type of shrimp used.
In addition to the variety of shrimp species that affects the resulting shrimp lymphocytes, different collection points can also affect the number of cells obtained, the purity of the cells. The invention can obtain more cells and shrimp lymphocytes with higher cell purity from a specific collection point. Referring to fig. 1 and 2, fig. 1 is a schematic view of a shrimp body collecting point according to an embodiment of the present invention, and fig. 2 is a schematic view of a shrimp body fluid being extracted at a specific angle according to an embodiment of the present invention.
In fig. 1, from the collection point 103, the first pair of the first abdominal and thoracic portions of the shrimp body may be spread to expose the junction 101 between the first thoraco-concha and the shrimp body. Then, as shown in fig. 2, the head of the shrimp is facing downward, and the needle (not shown) is aligned with the collection point 103 of the joint 101, and the needle is dropped at an angle (e.g. θ) smaller than 45 degrees in fig. 2, wherein the angle θ is defined as the angle between the abdominal surface 203 of the shrimp body 201 and the needle body 205. After the tip of the needle is inserted to a depth of 0.3cm to about 0.5cm, shrimp body fluid (transparent or slightly blue) is slowly withdrawn. According to some embodiments of the present invention, about 0.4mL of body fluid may be obtained from the collection point 103. According to other embodiments of the present invention, only about 0.2mL of body fluid is obtained from the collection point 105.
Another influencing factor is that the constituents of shrimp body fluid have a clotting factor and a phenol oxidase activating system (prophenoxidase activating system) in shrimp circulating blood, and the degranulation of the constituents causes the agglutination activity of shrimp body fluid, which causes the aggregation of cells, thereby causing the rapid death of the cells in the test tube. In vivo (in vivo) studies have found that transaminase (TGase) activity of shrimp body fluid components induces coagulation of plasma Clotting Protein (CP). The transaminase enzyme requires the presence of cofactors (e.g.calcium ions) in its action. If the transaminase gene of shrimp is deleted, the number of bacteria in lymph is relatively high. In addition, some studies have shown that proteolytic enzymes (e.g., trypsin) used to isolate mammalian cells can increase the yield (yield rate) of cells isolated from shrimp tissue.
As used herein, the term "anti-clumping agent" refers to a composition that includes glucosamine and low levels of sodium ions. In one embodiment, the anti-clumping agent may comprise, for example, less than 10.0g/L sodium chloride, 5.5g/L citric acid, 8.0 to 10.0g/L sodium citrate, 19.8 to 20.0g/L glucose, and 21.563g/L glucosamine, and the anti-clumping agent has a pH of 7.0. In one example, the anti-clumping agent may include 8.2g/L NaCl, 5.5g/L citric acid, 8.8g/L Na citrate, 19.8g/L glucose, and 21.563g/L glucosamine, and the anti-clumping agent has a pH of 7.0. In another example, the anti-agglutinating agent may further optionally comprise ethylenediaminetetraacetic acid (EDTA) to further improve the cell survival rate of the shrimp lymphocytes, wherein the concentration of EDTA may be, for example, 2.5 g/L.
In some embodiments, the cell viability of shrimp lymphocytes treated with the above described anti-agglutinating agent after 30 minutes of culture can be, for example, at least 90%. In other embodiments, the cell viability of shrimp lymphocytes treated with the above described anti-agglutinating agents after 60 minutes of culture can be, for example, at least 85%. In other embodiments, the shrimp lymphocytes treated with the above described anti-agglutinating agent can have a cell viability of, for example, at least 73% after 120 minutes of culture.
The term "cell culture medium" as used herein refers to a medium containing L-15 medium, fetal calf serum, and low-concentration sodium ions. In one embodiment, the cell culture solution may comprise, for example, 2 times (2X) L-15, less than 10.0g/L NaCl, 10g/L glucose, and 15% (v/v) fetal bovine serum, and the cell culture solution has a pH of 7.0. In one example, the concentration of sodium chloride in the cell culture solution may be, for example, 5 g/L. In another example, the aforementioned cell culture fluid can further comprise an antibiotic, wherein the antibiotic can comprise, for example, streptomycin, penicillin (penicillin), and amphotericin. In the above examples, the cell culture solution may optionally comprise 100. mu.g/mL streptomycin, 100I.U./mL penicillin, and 0.25mg/mL amphotericin.
In some embodiments, the shrimp lymphocytes obtained as described above may have a cell viability of, for example, at least 90% after being cultured in the above cell culture solution for 24 hours.
The method can obtain shrimp lymphocytes with higher cell survival rate, cell yield and cell purity, and can be further applied to in vitro evaluation of shrimp innate immune response. In one embodiment, the method for "assessing shrimp innate immune response in vitro" includes performing a co-culture step of culturing the obtained shrimp lymphocytes in the cell culture medium for 30 minutes to 72 hours, wherein the cell culture medium may or may not contain the sample to be tested. Then, the cell survival rate of the shrimp lymphocytes is detected, wherein the cell survival rate of the shrimp lymphocytes not cultured with the sample to be detected is a reference value, and the cell survival rate of the shrimp lymphocytes cultured with the sample to be detected is a measured value. And then, a judging step is carried out, and if the measured value is higher than the reference value, the sample to be detected is judged to have the activity of stimulating the innate shrimp immune response.
In other embodiments, the co-culturing step may be optionally preceded by a pre-culturing step, in which the shrimp lymphocytes are cultured in a cell culture medium without the test sample for at least 24 hours.
The present invention is described in more detail with reference to the following examples, which are not intended to limit the scope of the invention.
Example 1: purification of shrimp lymphocytes
1. Preparation examples 1 to 6 and preparation comparative examples 1 to 2
Preparation examples 1 to 6 and preparation comparative examples 1 to 2 anti-agglomerants were formulated according to table 1, wherein preparation comparative examples 1 to 2 are anti-agglomerants of known formulations and preparation examples 1 to 6 are anti-agglomerants according to several examples of the present invention. The above components are dissolved in sterile water, and the pH is adjusted to 7.0 with 1N sodium hydroxide aqueous solution, then the bacteria are removed by filtration with a filter membrane having a pore size of 0.22 μm, and the solution is left at 4 ℃ for use.
TABLE 1
2. Preparation example 7 and preparation comparative example 3
Preparation example 7 and preparation example 3 cell culture solutions were prepared according to table 2, wherein preparation example 3 was a culture solution of a known formulation, and preparation example 7 was a cell culture solution according to an embodiment of the present invention. The above components are dissolved in sterile water, and the pH is adjusted to 7.0 with 1N sodium hydroxide aqueous solution, then the bacteria are removed by filtration with a filter membrane having a pore size of 0.22 μm, and the solution is left at 4 ℃ for use.
TABLE 2
The formulations of the cell culture solutions of preparation example 7 and preparation comparative example 3 are shown in Table 2. In the preparation, after glucose is added into 800mL of sterile water and completely dissolved, sodium chloride and 2 packets of Leibovitz's L-15 powder (corresponding to L-15 with 2 times concentration (2X)) are added. After the above components are completely dissolved, the total volume is adjusted to 840mL by sterile water, the pH value is adjusted to 7.0 by 1N HCl, 10mL (1%, v/v) of antibiotic is added, and finally 150mL (15%, v/v) of Fetal Bovine Serum (FBS) is added, and the total volume is adjusted to 1000mL by sterile water. The prepared cell culture solution is filtered by a filter membrane with the pore size of 0.22 mu m to remove bacteria, and is placed at 4 ℃ for later use.
The cell culture solutions of the above preparation examples 7 and preparation comparative example 3 contained 100. mu.g/mL of streptomycin, 100I.U./mL of penicillin (penicillin), and 0.25mg/mL of amphotericin (CORNING).
3. Isolation of shrimp lymphocytes
10 white shrimps (Litopenaeus vannamei) are taken, and the body fluid cells of the shrimps are obtained according to different collection points.
When the shrimp lymphocytes are separated, at least two inflating pumps and fresh seawater are prepared to ensure the survival of the shrimps at any time. Meanwhile, 0.4mL of the anti-aggregation agents of preparation examples 1 to 6 and comparative examples 1 to 2 were drawn up into respective syringes (total syringe volume is 1mL) with different syringes, and placed on ice.
After the white shrimps are moved to an operation table, the white shrimps are kept stand for 1 hour to stabilize the shrimps, the air inflation condition and the shrimp state are noticed at any time, and fresh seawater is added at proper time. Subsequently, the shrimp is appropriately immobilized, and then the abdomen of the shrimp is exposed, and as shown in FIG. 1, the body fluid of the shrimp is collected from two collection points, respectively shown as 103 and 105.
Refer to fig. 1 and 2. In fig. 1, from the collection point 103, the first pair of the first abdominal and thoracic portions of the shrimp body may be spread to expose the junction 101 between the first thoraco-concha and the shrimp body. Then, as shown in fig. 2, the shrimp is placed with its head facing downward and the needle (e.g., 26G x 1/2 "(0.45 x 13mm) (not shown)) is aligned with the collection point 103 of the junction 101 at an angle less than 45 degrees (e.g., angle θ) of fig. 2, where angle θ is defined as the angle between the shrimp body surface extension 203 and the needle body 205. After the tip of the needle is inserted to a depth of 0.3cm to about 0.5cm, shrimp body fluid (transparent or slightly blue) is slowly withdrawn. About 0.4mL of body fluid may be obtained from the collection point 103.
The collection from collection point 105 is the same as collection from collection point 103 described above, except that the needle is pointed at collection point 105 of junction 101, and the needle is pointed at an angle of less than 45 degrees (e.g., reference number θ) as in fig. 2. Only about 0.2mL of body fluid can be accessed by the collection point 105.
The obtained shrimp body fluid is uniformly mixed with the anti-agglomeration agent in the syringe in a volume ratio of 1:1 to form a mixed solution. Then, 10. mu.L of the mixture was added with 90. mu.L of a 0.5% Trypan blue solution (Biological Industries) to calculate the cell number and the cell viability. The obtained lymphocytes, the mean cells thereofNumber 1X 107Cells/shrimp. Thereafter, the cell subsets were analyzed by a commercially available flow cytometer, and the results are shown in FIG. 3.
Referring to FIG. 3, there is shown a two-dimensional scatter diagram of (FSC/SSC) of a flow cytometer (FACScan flow cytometer) for shrimp lymphocytes according to an embodiment of the present invention, in which the horizontal axis represents the side scattered light (FSC) signal intensity (x1,000) and the vertical axis represents the side scattered light (SSC) signal intensity (x1,000).
From the results shown in FIG. 3, it is understood that the shrimp somatic cells obtained in example 1 contain three groups of high-purity lymphocytes, namely 36% granular cells (G), 9% semi-granular cells (SG) and 55% hyaline cells (H).
Example 2: evaluation of anticoagulation Effect of anticoagulation agent prawn lymphocytes
1. Evaluation of anti-aggregating Effect of anti-aggregating agent glucosamine and/or Trypsin prawn lymphocytes
This example uses 18 white shrimps, the anti-agglutinant formulations of preparation examples 1 to 5 and preparation comparative example 1, to evaluate the effect of containing or not containing glucosamine or/and trypsin on shrimp lymphocyte anti-agglutination and cell viability.
The shrimp body fluid cells obtained from the collection site B (shown as 103 in fig. 1) were quickly mixed with the anti-agglutinating agent of preparation examples 1 to 5 and comparative preparation example 1 in a syringe at a volume ratio of 1:1 to prepare a mixed solution, and then 10 μ L of the mixed solution was added with 90 μ L of 0.5% trypan blue solution to calculate the number of cells and the survival rate of cells every 10 minutes, and the results are shown in table 3 and fig. 4 below.
TABLE 3
Referring to Table 3 and FIG. 4, the cell number (Table 3) and the cell survival rate (FIG. 4) of shrimp lymphocytes treated with the anti-agglutinating agents according to preparation examples 1 to 5 of the present invention and preparation comparative example 1, respectively, are shown. Curves 403, 405, 407, 409 and 411 in fig. 4 represent the numbers of lymphocytes of shrimps treated with the anti-agglutinating agents of preparation examples 1 to 5, respectively, and curve 401 represents the number of lymphocytes of shrimps treated with the anti-agglutinating agent of preparation comparative example 1.
As can be seen from the results of table 3 and fig. 4, the cell viability was higher after the anti-agglutinating agents of preparation examples 2 to 5, compared to the known anti-agglutinating agent of preparation example 1 (curve 401) and the anti-agglutinating agent of preparation example 1 (curve 403), as shown by curves 405, 407, 409 and 411 in fig. 4.
Next, 9.38X10 was obtained after incubation for 0 to 60 minutes after treatment with the anti-clumping agent of preparation example 26The average number of lymphocytes per shrimp was more superior to those of preparation examples 1, 3 to 5, as shown in Table 3. Furthermore, after the anti-agglutinating agent of preparation example 2 was treated, the cell survival rate was maintained for a longer period of time, and the cell survival rate reached 90% after 20 minutes of culture and 70% after 60 minutes of culture, which are better than the effects of preparation examples 1, 3 to 5, as shown by curve 405 in FIG. 4.
2. Evaluation of anticoagulant Effect of glucosamine and/or ethylenediaminetetraacetic acid prawn lymphocytes
This example uses 9 white shrimp, the anticoagulant formulations of preparation examples 2, 6 and preparation comparative example 2 to evaluate the effect of the inclusion or exclusion of glucosamine or/and ethylenediaminetetraacetic acid (EDTA) on the anticoagulation and cell viability of shrimp lymphocytes.
The shrimp body fluid cells obtained from the collection site B (shown as 103 in FIG. 1) were mixed with the anti-agglutinating agents of preparation examples 2 and 6 and comparative preparation example 2 in a syringe at a volume ratio of 1:1, and then 10. mu.L of the mixture was added with 90. mu.L of a 0.5% Trypan blue solution, and the number of cells and the survival rate of cells were calculated every 10 minutes, as shown in FIG. 5.
Referring to FIG. 5, there is shown cell viability of shrimp lymphocytes treated with the anti-agglutinating agents according to preparation examples 2 and 6 of the present invention and preparation comparative example 2. Curve 501 and curve 503 of fig. 5 represent the number of shrimp lymphocytes after treatment with the anti-agglutinating agent of preparation examples 6 and 2, respectively, while curve 505 represents the number of shrimp lymphocytes after treatment with the anti-agglutinating agent of preparation comparative example 2. In the anti-agglomerating agent of preparation example 6, EDTA was further added to the anti-agglomerating agent of preparation example 2.
From the results of FIG. 5, it can be seen that the cell viability was higher after treatment with the anti-agglutinating agent of preparation example 6, as shown by curve 501, compared to the known anti-agglutinating agent of preparation comparative example 2 and the anti-agglutinating agent of preparation example 2 (curve 503). The cell survival rate after treatment with the anti-agglutinating agent of preparation 6 (curve 501) was better than that of the anti-agglutinating agent of preparation 2 (curve 503), wherein the cell survival rate after 30 minutes of culture was 90%, the cell survival rate after 60 minutes of culture was 85%, and the cell survival rate after 120 minutes of culture was 73%. Therefore, the anti-agglutinating agent of preparation example 6 is more effective, and can maintain cells for a longer time and with a higher survival rate.
Example 3: evaluation of the Effect of the cell culture solution on the culture of Primary cultured shrimp lymphocytes
In this example, 20 white shrimps were used, and shrimp lymphocytes were obtained according to the method of example 1 and placed in two tubes containing 20mL of the cell culture solutions of preparation example 7 and preparation comparative example 3, respectively, immediately after leaving the living body within 10 minutes, and the cells were uniformly dispersed.
After counting the cells according to the above method, 1X 106Cell concentration per mL, 10mL were seeded into a 10cm dish, slowly shaken until uniform, and then left at room temperature at 25 ℃ until they were attached. After the incubation for 24 hours, 36 hours, and 72 hours, the results are shown in fig. 6A and 6B.
Referring to FIGS. 6A and 6B, there are shown optical micrographs of shrimp lymphocytes cultured in the cell culture solutions of production comparative example 3 (FIG. 6A) and production example 7 (FIG. 6B) for 120 minutes, respectively. As is clear from the results shown in FIGS. 6A and 6B, the shrimp lymphocytes cultured in the cell culture medium of preparation example 7 still showed a cell survival rate of 90% at 24 hours, as shown in FIG. 6B. In contrast, the shrimp lymphocytes cultured in the cell culture medium of comparative example 3 had a less satisfactory culture effect and died all over 24 hours, as shown in FIG. 6A.
In summary, the above embodiments demonstrate that the method for purifying shrimp lymphocytes of the present invention successfully performs a pre-treatment on a shrimp body fluid sample with an anti-agglutinating agent containing glucosamine and low-concentration sodium ions at a specific collection point, and then performs a culture step with a cell culture solution containing low-concentration sodium ions, so as to effectively improve the cell survival rate of shrimp lymphocytes, and increase the cell yield and cell purity. The obtained shrimp lymphocytes can be further used as an in vitro screening platform to evaluate the innate immune stimulation activity of a sample to be tested.
For example, when the shrimp lymphocytes are used as an in vitro screening platform, a co-culture step may be performed to culture the obtained shrimp lymphocytes in the cell culture medium for 30 minutes to 72 hours, wherein the cell culture medium may or may not contain a sample to be tested. Then, the cell survival rate of the shrimp lymphocytes is detected, wherein the cell survival rate of the shrimp lymphocytes not cultured with the sample to be detected is a reference value, and the cell survival rate of the shrimp lymphocytes cultured with the sample to be detected is a measured value. And then, performing a judging step, and judging that the sample to be detected has the activity of stimulating the shrimp innate immune response if the measured value is higher than the reference value. In other embodiments, optionally, a pre-incubation step may be performed prior to the co-incubation step, such that the shrimp lymphocytes are incubated in a cell culture medium without the test sample for at least 24 hours.
It should be noted that although the present invention is illustrated by using a specific process, a reagent with a specific formula, a specific analysis method or a specific apparatus, the shrimp lymphocytes and the purification method thereof and the method for in vitro assessment of innate immune response of the present invention are described, but those skilled in the art will recognize that the present invention is not limited thereto, and that the shrimp lymphocytes and the purification method thereof and the method for in vitro assessment of innate immune response of the present invention can be performed by using other processes, reagents with other formulas, other analysis methods or other apparatuses without departing from the spirit and scope of the present invention.
The present invention is directed to a method for in vitro assessment of innate immune responses, which comprises pre-treating a sample of prawn body fluid with an anti-agglutinating agent comprising glucosamine and low-concentration sodium ions, and culturing the sample in a cell culture medium comprising low-concentration sodium ions, thereby effectively increasing the survival rate of the prawn lymphocytes and increasing the cell yield and purity. The obtained shrimp lymphocytes can be used as an in vitro screening platform to evaluate the innate immune stimulation activity of a sample to be tested.
Although the present invention has been described with respect to the above embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (4)
1. A method for purifying shrimp lymphocytes, comprising:
providing a shrimp body fluid sample, wherein the shrimp body fluid sample is derived from white shrimp (Litopenaeus vannamei);
performing pretreatment, and uniformly mixing the shrimp body fluid sample with an anti-agglutinating agent to form a single-cell suspension, wherein the anti-agglutinating agent consists of 8.2g/L of sodium chloride, 5.5g/L of citric acid, 8.8g/L of sodium citrate, 19.8g/L of glucose, 21.563g/L of glucosamine and 2.5g/L of ethylenediamine tetraacetic acid, and the anti-agglutinating agent has an acid-base value of pH 7.0;
performing a centrifugation step to remove supernatant of the single cell suspension and obtain a cell pellet; and
performing a culturing step of uniformly suspending and culturing the cell precipitate in a cell culture solution to obtain the shrimp lymphocytes, wherein the cell culture solution consists of 2-fold concentration of L-15 culture solution, 5g/L of sodium chloride, 10g/L of glucose and 15% (v/v) of fetal bovine serum, and the cell culture solution has a pH value of 7.0.
2. Use of an anti-agglutinating agent for shrimp lymphocytes in the anticoagulation of white shrimp lymphocytes, wherein the anti-agglutinating agent consists of 8.2g/L sodium chloride, 5.5g/L citric acid, 8.8g/L sodium citrate, 19.8g/L glucose, 21.563g/L glucosamine and 2.5g/L ethylenediaminetetraacetic acid, and the anti-agglutinating agent has an acid-base value of pH7.0 to obtain white shrimp lymphocytes.
3. The application of a cell culture solution of shrimp lymphocytes in culturing the white shrimp lymphocytes, wherein the cell culture solution consists of 2-fold concentration of L-15 culture solution, 5g/L of sodium chloride, 10g/L of glucose and 15% (v/v) of fetal bovine serum, and the cell culture solution has an acid-base value of pH7.0 to culture the white shrimp lymphocytes.
4. A method for assessing shrimp innate immune response in vitro comprising:
providing shrimp lymphocytes, wherein the shrimp lymphocyte cell line is obtained by treating a body fluid sample of white shrimps with an anti-agglutinating agent for shrimp lymphocytes, wherein the anti-agglutinating agent for shrimp lymphocytes is composed of 8.2g/L sodium chloride, 5.5g/L citric acid, 8.8g/L sodium citrate, 19.8g/L glucose, 21.563g/L glucosamine and 2.5g/L ethylenediaminetetraacetic acid, and the anti-agglutinating agent has an acid-base value of pH 7.0;
performing a pre-culture step to allow the shrimp lymphocytes to have a cell survival rate of at least 90% after being cultured for 24 hours in a cell culture solution, wherein the cell culture solution consists of 2-fold concentration of L-15 culture solution, 5g/L of sodium chloride, 10g/L of glucose, 15% (v/v) of fetal bovine serum and has a pH value of 7.0;
performing a co-culture step, culturing the shrimp lymphocytes in the cell culture solution for 30 minutes to 72 hours, wherein the cell culture solution contains or does not contain a sample to be detected; and
detecting the cell survival rate of the shrimp lymphocytes, wherein the cell survival rate of the shrimp lymphocytes not cultured with the test sample is a reference value, and the cell survival rate of the shrimp lymphocytes cultured with the test sample is a measured value; and
and performing a judging step, and judging that the sample to be detected has the activity of stimulating the innate shrimp immune response if the measured value is higher than the reference value.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104278077A (en) * | 2014-08-14 | 2015-01-14 | 浙江省淡水水产研究所 | High-flux quick screening method of Macrobrachium rosenbergii immunopotentiators |
| CN104450706A (en) * | 2014-12-10 | 2015-03-25 | 中山大学 | Method for inducing blood lymphocyte of litopenaeus vannamei to generate UPR (unfolded protein response) by means of RNA interference technology |
-
2015
- 2015-12-18 CN CN201510960822.9A patent/CN106893691B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104278077A (en) * | 2014-08-14 | 2015-01-14 | 浙江省淡水水产研究所 | High-flux quick screening method of Macrobrachium rosenbergii immunopotentiators |
| CN104450706A (en) * | 2014-12-10 | 2015-03-25 | 中山大学 | Method for inducing blood lymphocyte of litopenaeus vannamei to generate UPR (unfolded protein response) by means of RNA interference technology |
Non-Patent Citations (6)
| Title |
|---|
| Morphologic, physiological and immunological changes of haemocytes from Litopenaeus vannamei treated by lipopolysaccharide;Jian-An Xian 等;《Aquaculture》;20091005;第139–145页 * |
| 中国对虾血淋巴凝集素的血凝活性与促噬活性;彭其胜 等;《水产学报》;20010630;第23卷(第3期);第197-202页 * |
| 中国对虾血淋巴液中的凝集素;彭其胜 等;《中国水产科学》;20001012;第7卷(第4期);第14-18页 * |
| 克氏原螯虾的血淋巴细胞原代培养;魏 静等;《畜牧与兽医》;19990930;第31卷(第5期);第2页右栏2.2血清凝集素对单糖和红细胞的亲和性，表1，第11页左栏1.2鳌虾血淋巴细胞的培养，2结果，第12页最后一段 * |
| 日本对虾血清凝集素的基本物理化学性质;廖绍安等;《中国水产科学》;20011215;第8卷(第4期);1.材料与方法 * |
| 魏 静等.克氏原螯虾的血淋巴细胞原代培养.《畜牧与兽医》.1999,第31卷(第5期),第2页右栏2.2血清凝集素对单糖和红细胞的亲和性，表1，第11页左栏1.2鳌虾血淋巴细胞的培养，2结果，第12页最后一段. * |
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