Method for improving toxicity resistance of insect cells

Abstract

A method for improving toxicity resistance of insect cells is characterized in that Sf9 cells may be poisoned by an exogenous toxic pollutant-perfluoro or polyfluoroalkyl compound (PFASs) in a culture process, and the Sf9 cells have cell apoptosis characteristics such as cytoplasm granulation, shrinkage, gap enlargement, fuzzy contour, cell nucleus migration and apoptotic body appearance due to the substance, so that the Sf9 cells can be degraded, and the toxicity resistance of the insect cells can be improved. The cells are inactivated and die. In order to solve the problem of poisoning of PFASs pollution to Sf9 cells, the invention provides a method capable of effectively relieving or preventing the poisoning, and the method is based on a cholesterol protective agent with the concentration of 0.07-5.4 [mu] g mL <-1 >. The specific implementation method is as follows: before culture of the Sf9 cells, cholesterol with the concentration is added into a culture medium in advance, so that cholesterol transformation disorder caused by interference of PFASs in the growth and proliferation process of the cells is avoided, and normal life activity of the Sf9 cells is maintained.

Description

Technical Field

[0001] The present invention relates to the technical field of insect cell culture, and in particular to a method for improving the toxicity resistance of fall armyworm (Sf9) cells to perfluoroalkyl and polyfluoroalkyl compounds (PFASs). Background Art

[0002] The fall armyworm (Spodoptera frugiperda) is a species of the genus Spodoptera in the family Noctuidae in the order Lepidoptera. Its cells are a type of insect cell commonly used in biological experiments, referred to as Sf9 cells. The cell is usually used as an insect platform to produce various proteins and viruses, or to conduct basic research.

[0003] PFASs are widely used as various hydrophobic materials, anti-stick coatings, firefighting foams, etc. They may enter the environment from various production materials, tools, coatings, etc. We found that during the culture of Sf9 cells, exogenous organic pollutants, per- or polyfluoroalkyl compounds (PFASs), can be toxic to them, causing the cells to show apoptotic features such as granulation, shrinkage, enlarged gaps, blurred outlines, nuclear displacement, and the appearance of apoptotic bodies, which eventually completely inactivate and kill the cells.

[0004] Hormone synthesis is crucial in the process of Sf9 cells maintaining normal life activities. Our research found that in the insect cell hormone synthesis pathway, the conversion of cholesterol is an important upstream link in the synthesis of a series of hormones. Experiments have confirmed that PFASs organic pollutants will seriously hinder the cholesterol conversion process inside Sf9 cells, thereby causing a lack of cholesterol in the intracellular environment, slow or blocked hormone synthesis, and ultimately causing Sf9 cells to enter a state of apoptosis and die. Summary of the invention

[0005] Technical problem to be solved: To solve the problem in the technical background, the present invention provides a method that can effectively alleviate or prevent the toxic effects of PFASs organic pollutants on the growth and proliferation of Sf9 cells during the culture process. Specifically, during the Sf9 cell culture stage, a certain concentration of cholesterol is added to the culture medium, and cholesterol is directly supplied artificially to avoid cholesterol conversion disorders in Sf9 cells due to the toxicity of PFASs pollutants, greatly improve the tolerance of Sf9 cells to PFASs, enable Sf9 cells to reproduce and grow normally, and ensure the quality of cell culture.

[0006] Technical solution: Application of sterols in the preparation of products that improve Sf9 cell tolerance to the toxicity of perfluoroalkyl and polyfluoroalkyl compounds.

[0007] The above-mentioned sterol is cholesterol or its derivatives.

[0008] The above-mentioned perfluoro and polyfluoroalkyl compounds are chlorinated polyfluoroalkyl ether sulfonic acids.

[0009] A protective agent for improving the tolerance of Sf9 cells to the toxicity of perfluorinated and polyfluorinated alkyl compounds, comprising cholesterol or a derivative thereof.

[0010] The content of the above cholesterol or its derivatives is 0.07~5.4μg mL -1 .

[0011] An anti-PFASs toxicity Sf9 cell culture medium for enhancing the toxicity resistance of Sf9 cells to PFASs and maintaining their normal growth and proliferation, comprising the following components according to volume concentration: Active ingredient concentration: cholesterol 0.07-5.4 μg mL -1 ; Culture medium: Sf9 insect cell serum-free and animal-free medium II (cholesterol-free); the active ingredient is cholesterol (Cholesterol), and the culture medium is " Sf9 insect cell serum-free, animal-free, cholesterol-free medium II (cholesterol-free) (hereinafter referred to as " Sf9 medium”).

[0012] The specific preparation of cell protection medium and cell culture steps are as follows:

[0013] (1) Preparation of culture medium. Take cholesterol (also known as cholesterol, English name; Cholesterol, chemical formula: C 27 H 46 O, CAS: 57-88-5) mother liquor (4000 mg L -1 ), according to the required medium volume and cholesterol concentration, transfer a certain amount of cholesterol stock solution to Sf9 medium and diluted to the target concentration (0.2, 0.6, 1.7, 5, 15 μg mL -1 ). Mix well and divide into portions according to the required volume of culture medium.

[0014] (2) Plate laying. First, subculture with cholesterol-free and serum-free medium. Take 9×10 7 Place Sf9 cells in a 50 mL centrifuge tube, add culture medium to 45 mL, and mix with a Pasteur pipette. Add 1000 μL of cholesterol-free culture medium to each well of a six-well plate, and then add 1000 μL of culture medium to each well, plate 54 wells, and mix. Culture overnight in a 27°C incubator.

[0015] (3) Change the medium. Discard 500 μL of culture medium from each well and pipette 600 μL of culture medium containing 0, 3, 9, 30, 90, 270 μg / mL F-53B and 600 μL of culture medium containing 0, 0.2, 0.6, 1.7, 5, 15 μg / mL cholesterol into a six-well plate. The corresponding culture medium concentrations are 0, 1, 3, 10, 30, 90 μg / mL F-53B and 0, 0.07, 0.2, 0.6, 1.8, 5.4 μg / mL cholesterol. Culture in a 27°C incubator for 24, 48, and 72 h. A total of 36×3 treatments, 3 replicates, and 324 samples.

[0016] (4) Take photos at 0, 24, 48, and 72 hours. Observe cell morphology using an inverted phase contrast microscope.

[0017] (5) Sampling the supernatant at 24, 48, and 72 h. Pipette the supernatant into a 1.5 mL EP tube, centrifuge at 3000 rpm for 10 min, remove the precipitate, dispense into 1000 μL and 500 μL portions, and freeze at -80°C for later use.

[0018] (6) Cell sampling at 24, 48, and 72 h. Add 200 mL of TRIzol to each well of the six-well plate after removing the culture medium. Quickly transfer the digestion solution to a 1.5 mL EP tube and freeze at -80°C for later use.

[0019] Application of an anti-PFASs toxicity Sf9 cell culture medium: This culture medium is suitable for preventing or alleviating the cell apoptosis phenomenon of Sf9 cells caused by PFASs toxicity, so that Sf9 cells maintain normal proliferation and growth in the presence of PFASs toxicity.

[0020] Beneficial effects: After using this culture medium, it can effectively prevent or alleviate the apoptosis characteristics of Sf9 cells caused by PFASs toxicity, such as cytoplasmic granulation, shrinkage, enlarged gaps, blurred outlines, nuclear displacement and the appearance of apoptotic bodies, significantly reduce the number of cell deaths, and restore cell viability to normal levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Sf9 cell culture test results under different cholesterol conditions (0 μg mL -1 F-53B);

[0022] Figure 2 Results of Sf9 cell culture tests under different cholesterol conditions (1 μg mL -1 F-53B); (Note: The blank part of the picture is the missing sample; the same below.)

[0023] Figure 3 Results of Sf9 cell culture tests under different cholesterol conditions (3 μg mL -1 F-53B);

[0024] Figure 4 Results of Sf9 cell culture tests under different cholesterol conditions (10 μg mL -1 F-53B);

[0025] Figure 5 Results of Sf9 cell culture tests under different cholesterol conditions (30 μg mL -1 F-53B);

[0026] Figure 6 Results of Sf9 cell culture tests under different cholesterol conditions (90 μg mL -1 F-53B). DETAILED DESCRIPTION

[0027] Example 1

[0028] We tested the effect of the culture medium by using the PFASs pollutant F-53B (a type of PFASs pollutant with wide application) and cholesterol simultaneously for the culture of Sf9 cells.

[0029] By changing the concentration of F-53B and soluble cholesterol in the Sf9 cell culture medium, the effects of F-53B and cholesterol on the growth state of Sf9 cells were observed based on a series of indicators such as cell morphology and activity proliferation. The cholesterol pathway was verified to be the key pathway for F-53B cytotoxicity by dhcr-24qPCR and DHCR-24 enzyme level differences.

[0030] The detailed technical solution is as follows:

[0031] 1. Sf9 cell culture

[0032] (1) Sf9 cell recovery. First, the frozen Sf9 cells were quickly taken out from -80℃ and placed in a 37℃ water bath for 1 minute to thaw; then 1mL of frozen cells was added to 10mL of insect cell culture medium for cell counting (cell inoculation density ≥5×10 5 viable cells / mL); then, culture at 27°C for 12-24 hours, switch to fresh culture medium; finally, wait for the cells to develop well, and then continue to subculture. Sf9 cells were cultured using conventional methods. The cell growth medium was InsectProSf9 insect cell serum-free and animal-free culture medium II. The volume of the 10cm dish culture medium was 8mL / dish. Insect cells were cultured at 27°C, and the cell culture medium was changed every 2 days. Cell subculture and cryopreservation recovery were performed according to conventional methods.

[0033] (2) Plate laying. Use cholesterol-free and serum-free medium for subculture. Take 9×10 7Place Sf9 cells in a 50 mL centrifuge tube, add medium to 45 mL, and mix with a Pasteur pipette. Add 1000 μL of cholesterol-free medium to each well of a six-well plate, then add 1000 μL of medium to each well, plate 54 wells, and mix. Incubate overnight in a 27°C incubator.

[0034] (3) Change the medium. Discard 500 μL of culture medium from each well and pipette 600 μL of culture medium containing 0, 3, 9, 30, 90, 270 μg / mL F-53B and 600 μL of culture medium containing 0, 0.2, 0.6, 1.7, 5, 15 μg / mL cholesterol into a six-well plate. The corresponding culture medium concentrations are 0, 1, 3, 10, 30, 90 μg / mL F-53B and 0, 0.07, 0.2, 0.6, 1.8, 5.4 μg / mL cholesterol. Culture in a 27°C incubator for 24, 48, and 72 h. A total of 36×3 treatments, 3 replicates, and 324 samples.

[0035] (4) Take photos. Take photos at 0, 24, 48, and 72 hours. Observe cell morphology using an inverted phase contrast microscope.

[0036] (5) Supernatant sampling. Supernatant samples were collected at 24, 48, and 72 h. The supernatant was pipetted into a 1.5 mL EP tube and centrifuged at 3000 rpm for 10 min. The precipitate was removed and divided into 1000 μL and 500 μL aliquots and stored at -80°C for later use.

[0037] (6) Cell sampling at 24, 48, and 72 h. Add 200 mL of TRIzol to each well of the six-well plate after removing the culture medium. Quickly transfer the digestion solution to a 1.5 mL EP tube and freeze at -80°C for later use.

[0038] 2. Sf9 cell activity detection

[0039] (1) Plate laying. Use cholesterol-free and serum-free medium for subculture. Take logarithmic phase cells and add 100 μL to each well of a 96-well plate. Adjust the density of the cells to be tested to 5000 / well and culture at 27°C overnight.

[0040] (2) Change the medium. Replace the medium with medium containing 0, 1, 3, 10, 30, and 90 μg mL -1 F-53B, and 0, 0.07, 0.2, 0.6, 1.8, 5.4 μg mL -1 100 μL of cholesterol-containing culture medium was added and cultured at 27°C.

[0041] (3) CCK-8 staining: Every 24 hours, add 10 μL of CCK-8 solution to each well of plates (I and II), protect from light, and continue incubation for 2 hours.

[0042] (4) Absorbance measurement: The absorbance at 450 nm was measured for 4 consecutive days (corresponding to 24, 48, 72 and 96 h, respectively).

[0043] Case 1~324:

[0044] An anti-PFASs toxicity Sf9 cell culture medium for enhancing the tolerance of Sf9 cells to PFASs toxicity and maintaining their normal growth and proliferation, comprising the following components according to volume concentration:

[0045] Active ingredient concentration: Cholesterol 0-5.4 μg mL -1

[0046] PFASs pollutant concentration: F-53B 0~90μg mL -1

[0047] Culture medium: Sf9 Insect Cell Serum-Free and Animal Origin-Free Medium Ⅱ (Cholesterol-Free)

[0048] Among them, the active ingredient is cholesterol, the PFASs pollutant is F-53B, and the culture medium is " Sf9 insect cell serum-free, animal-free, and cholesterol-free medium II (cholesterol-free).

[0049] For the results of cell tests in cases 1 to 324, please refer to the attached drawings in the specification. Figure 1 to Figure 6 (Some test results in the examples were damaged due to a malfunction of the shooting instrument, and the missing pictures have been left blank at the corresponding positions in the accompanying drawings. The missing data does not affect the overall test results).

[0050] The test results are summarized as follows:

[0051] 1) Effects of F-53B on Sf9 cell morphology

[0052] To verify the changes in Sf9 cell viability under different F-53B and cholesterol concentrations, light microscopy observation and photography were performed in the experiment ( Figures 1 to 6 ). As can be seen from the light microscopy photos, Sf9 cells survived normally in the cholesterol-free control treatment, with a basically round appearance and clear cell outlines. As the concentration of F-53B increased, cell apoptosis gradually intensified, and at 30 and 90 μg mL - 1 The cells treated with F-53B underwent dramatic morphological changes, showing typical morphological characteristics of apoptosis, including cell granulation, cell shrinkage, enlarged gaps, blurred outlines, nuclear displacement, and the appearance of apoptotic bodies. Over time, all cells died ( Figure 5 , 6In addition to changing the cell morphology, F-53B also affected the proliferation of cells, and the cell density was significantly reduced compared with the control group.

[0053] (2) The alleviating effect of cholesterol on F-53B cytotoxicity

[0054] As cholesterol concentration increased, the cell morphology in each test condition gradually recovered, and the degree of recovery increased with the increase of cholesterol concentration ( Figures 2 to 6 ). F-53B was at 1.8 μg mL -1 Cholesterol added, 30 μg mL -1 The cell viability of the F-53B-treated group returned to normal, 90 μg mL -1 The F-53B treated group showed no significant change compared to the cholesterol-free group, with very few cells surviving and a cell death rate of approximately 100% ( Figure 6 ). At 5.4 μg mL -1 Cholesterol was added ( Figure 5 , 6 ), cells treated with two concentrations of F-53B could survive normally, and the death phenomenon was significantly reduced.

Claims

1. Sterols in the preparation of Sf9 Application of products in cell tolerance to toxicity of per- and polyfluoroalkyl compounds.

2. The use according to claim 1, characterized in that: The sterol is cholesterol or a derivative thereof.

3. The use according to claim 1, characterized in that: The perfluoro and polyfluoroalkyl compounds are chlorinated polyfluoroalkyl ether sulfonic acids.

4. An improvement Sf9 A protective agent for cells to resist the toxicity of perfluoroalkyl and polyfluoroalkyl compounds, characterized in that Contains cholesterol or its derivatives.

5. The culture medium according to claim 3, characterized in that The content of cholesterol or its derivatives is 0.07-5.4 μg mL -1 .

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