Test method for chromosome aberration

Abstract

The invention relates to the technical field of cell biology, and discloses a chromosome aberration test method which comprises the following steps: inoculating CHL cells into a plate with the diameter of 100mm, and culturing in a carbon dioxide incubator at 37 DEG C for 24 hours; 4 hours before the cells are harvested, adding a colchicine solution to block the cells in a mitosis metaphase; adding 0.075 mol / L KCL solution for resuspending the cells, and putting the cells into a water bath of about 37 DEG C for hypotonic for 30 minutes to expand the cells; 5 min before the hypotonic treatment is finished, 1 mL of fixing liquid is added for pre-fixing, after uniform mixing, 200 G centrifugation is conducted for 5-7 min, and supernate is discarded; slowly adding 5mL of fresh stationary liquid, uniformly mixing, fixing in a water bath at about 37 DEG C for 30 minutes, reversing uniformly mixed cells every 10 minutes in the process to fully fix, centrifuging at 300G for 10 minutes, discarding supernate, and fixing twice by the same method; adding a set amount of stationary liquid to resuspend the cells, and fixing overnight at 4 DEG C; the chromosome aberration test method provided by the invention solves the technical problem that the chromosome aberration condition in the genetic toxicology test is not easy to observe in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, in particular to a method for testing chromosome aberration. Background Art

[0002] Chromosome aberration is a common genotoxicity test used to evaluate the potential carcinogenicity and teratogenicity of compounds or environmental factors that come into contact with the human body. This method can observe specific structural and quantitative changes in cellular chromosomes, making it invaluable for understanding the mechanisms and characteristics of the effects of agents on chromosomes. With its short testing cycle and intuitive results, it is widely used in genotoxicity testing for foods, drugs, medical devices, cosmetics, and pesticides.

[0003] In vitro mammalian cell chromosome aberration testing involves cell inoculation, toxin treatment, colchicine blocking, and slide preparation. The slide preparation process includes hypotonicity, fixation, slide placement, and staining. Each step of the test is crucial. Current testing procedures can affect the degree of chromosome dispersion and the number of metaphases, thereby affecting the interpretation of the final test results and reducing the usefulness of the research results. Therefore, a method is urgently needed to optimize the chromosome aberration testing process to more standardize the testing and improve the test quality. Summary of the Invention

[0004] The present invention provides a method for chromosome aberration testing, which solves the technical problem in the prior art that chromosome aberration is difficult to observe in genetic toxicology tests.

[0005] The present invention provides a method for testing chromosome aberrations, comprising:

[0006] Cell seeding: CHL cells were seeded in 100 mm diameter dishes, with 1×10 cells per dish. 6 Place in a 37°C carbon dioxide incubator for 24 hours;

[0007] Colchicine treatment: 4 h before harvesting cells, add colchicine solution to block the cells in metaphase; wherein the final concentration of the colchicine solution is 1 μg / mL;

[0008] Hypotonicity: Add 5 mL of 0.075 mol / L KCl solution to resuspend the cells and place in a 37°C water bath for 30 minutes to allow the cells to swell. Invert and mix the cells every 10 minutes to achieve full hypotonicity.

[0009] Fixation: 5 minutes before the end of hypotonic treatment, add 1 mL of fixative for pre-fixation, mix, centrifuge at 200G for 5-7 minutes, and discard the supernatant; slowly add 5 mL of fresh fixative, mix, and place in a 37°C water bath for 30 minutes. During the process, invert and mix the cells every 10 minutes to fully fix them. Centrifuge at 300G for 10 minutes, discard the supernatant, and fix twice in the same way; resuspend the cells in the prescribed amount of fixative and fix them at 4°C overnight.

[0010] Furthermore, before the step of colchicine treatment, the method further comprises:

[0011] Toxic treatment: Aspirate the culture medium from the cell culture dish, add different concentrations of the test substance or positive control substance and S9 mixture, and incubate in a carbon dioxide incubator for 3 hours, with 2 replicates per group; after completion, aspirate the complete culture medium, wash the cells three times with Hanks solution, add serum-containing culture medium, and culture in a carbon dioxide incubator again, and harvest the cells after 24 hours; wherein the test substance or positive control substance is mitomycin C or cyclophosphamide.

[0012] Furthermore, in the step of fall poisoning treatment, when the S9 mixture was not added, RPMI1640 culture medium was used to supplement it.

[0013] Furthermore, before the step of performing the hypotonic treatment, the method further comprises:

[0014] Digestion: Digest the cells with trypsin. After the cells are detached, add cell culture medium containing serum to terminate the trypsin action. Mix well, place in a 15 mL centrifuge tube and centrifuge at 200G for 5-7 minutes, and discard the supernatant.

[0015] Furthermore, after the fixing step, the method further includes:

[0016] Dropping slides: Add 0.5-1 mL of fresh fixative solution to resuspend the cells according to the amount of cell pellet. Slightly tilt the slide and drop the slide at arm height, dropping 50 μL on one end. For each treatment group, drop at least 4 slides. Place the slide at room temperature to dry naturally.

[0017] Staining: Stain with Giemsa stain for 15 min, rinse gently with deionized water, and dry the slides at 60°C.

[0018] Sealing: Soak the dry slide in xylene for 5 minutes, add 1-2 drops of neutral resin glue while it is still wet, seal the slide with a cover glass, and let it dry naturally.

[0019] Furthermore, after the step of sealing, the method further comprises:

[0020] Data processing: 100 metaphase cells with well-dispersed chromosomes were selected from each group for chromosome aberration analysis. Structural and quantitative abnormalities of chromosomes were observed and recorded. The obtained experimental data were statistically processed using the χ2 test.

[0021] The beneficial effects of the present invention are:

[0022] The processing method for a chromosome aberration test of the present invention optimizes cell inoculation conditions, thereby increasing the probability that cells in the test are infected during the more sensitive S phase; optimizes colchicine treatment conditions, thereby increasing the number of cells in the metaphase; optimizes hypotonic conditions, thereby increasing hypotonic efficiency; and optimizes fixation conditions, thereby increasing fixation efficiency. The slide stained by this processing method is uniformly stained with an appropriate depth, and can clearly identify the specific structure and quantity changes of cell chromosomes, and can easily observe a large number of metaphases for data analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the process of the chromosome aberration test method of the present invention.

[0024] Figure 2 This is the metaphase chromosome map of the negative control group (NC) in the present invention.

[0025] Figure 3 This is the metaphase map of the chromosomes of the positive control group (PC) in the present invention.

[0026] Figure 4 This is the metaphase map of chromosomes in the test group of the present invention.

[0027] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0028] It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0029] The present invention is a method for evaluating a genetic toxicity chromosome aberration test of a hair growth solution. The method optimizes cell inoculation conditions to increase the probability that cells are infected in the more sensitive S phase during the test; optimizes colchicine treatment conditions to increase the number of cells in the metaphase; optimizes hypotonic conditions to improve hypotonic efficiency; and optimizes fixation conditions to improve fixation efficiency. Glass slides stained by this treatment method are uniformly stained with appropriate depths, and can clearly identify specific structures and quantitative changes of cell chromosomes, and can easily observe a large number of metaphases for data analysis.

[0030] like Figure 1 As shown, the present invention provides a method for chromosome aberration testing, comprising:

[0031] S1. Cell inoculation: One day before the experiment, CHL cells (Chinese hamster lung cells) were inoculated into 100 mm diameter dishes, with 1×10 cells per dish. 6 The cells were cultured in a 37°C carbon dioxide incubator until use.

[0032] S2. Exposure: Aspirate the culture medium from the cell culture dish and add varying concentrations of the test substance or positive control (mitomycin C or cyclophosphamide) along with a mixture of S9 (if S9 is not added, use RPMI1640 culture medium to make up the difference). Incubate in a CO2 incubator for 3 hours, with two replicates per group. After completion, aspirate the complete culture medium, wash the cells three times with Hanks' solution, add serum-containing culture medium, and return to a CO2 incubator. Harvest the cells after 24 hours.

[0033] S3. Colchicine treatment: 4 h before harvesting cells, colchicine solution (final concentration 1 μg / mL) was added to block the cells in metaphase of mitosis.

[0034] S4. Chromosome preparation

[0035] 1) Digestion: Digest the cells with trypsin. After the cells are detached, add cell culture medium containing serum to terminate the trypsinization. Mix thoroughly, place in a 15 mL centrifuge tube, centrifuge at 200G for 5-7 minutes, and discard the supernatant.

[0036] 2) Hypotonicity: Add 5 mL of 0.075 mol / L KCl solution to each tube, gently pipette to evenly distribute the cells, and place in a 37°C water bath for 30 minutes to allow the cells to swell.

[0037] 3) Fixation: 5 minutes before the end of hypotonic treatment, add 1 mL of fixative (prepared immediately prior to use) for pre-fixation. Mix thoroughly, centrifuge at 200G for 5-7 minutes, and discard the supernatant. Slowly add 5 mL of fresh fixative, mix thoroughly, and place in a 37°C water bath for 30 minutes. Invert and mix the cells every 10 minutes to ensure full fixation. Centrifuge at 300G for 10 minutes, discard the supernatant, and repeat the fixation procedure twice. Resuspend the cells in the prescribed amount of fixative and incubate at 4°C overnight.

[0038] 4) Dropping slides: Add 0.5-1 mL of fresh fixative solution according to the amount of cell pellet to resuspend the cells. Slightly tilt the slide and drop the slide at arm height (the slide must be pre-cooled before use). Drop 50 μL on one end. For each treatment group, drop at least 4 slides. Place the slide at room temperature to dry naturally.

[0039] 5) Staining: Stain with Giemsa stain (prepared immediately before use) for 15 min, rinse gently with deionized water, and dry the slides at 60°C.

[0040] 6) Sealing: In order to preserve the slides for a long time, place the dry slides in xylene for 5 minutes, add 1-2 drops of neutral resin glue while they are still wet, cover them with a glass slide, and let them dry naturally.

[0041] 7) Data processing: 100 metaphase cells with well-dispersed chromosomes were selected from each group for chromosome aberration analysis. Structural and quantitative abnormalities of chromosomes were observed and recorded. The obtained experimental data were statistically analyzed using the χ2 test.

[0042] Result analysis:

[0043] Under the experimental conditions of the present invention, as shown in the table below, the chromosome aberration rates observed in the three treatment groups (positive control (+S9, 3h), (-S9, 3h), and (-S9, 24h)) were 47%, 38%, and 54%, respectively. The cell aberration rate in the positive control group was significantly higher than that in the negative control group (P < 0.05). Furthermore, no significant differences were observed between any dose group and the negative control group (P > 0.05), indicating that the test substance did not induce chromosome aberrations in cultured mammalian somatic cells. Therefore, the results of the in vitro mammalian chromosome aberration test of this hair growth liquid, with or without the activation system, were negative, indicating no mutagenicity.

[0044]

[0045] Note: Use χ 2 Statistical analysis was performed using the test, and *P<0.05 was compared with the negative control group.

[0046] like Figure 2-4 As shown, under the oil microscope, there are more chromosomes in the metaphase phase, with better color and good dispersion, which is conducive to reading the film and distinguishing the types of chromosome and chromatid aberrations.

[0047] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, apparatus, article, or method comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, apparatus, article, or method. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, apparatus, article, or method comprising the element.

[0048] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for chromosome aberration testing, characterized in that: include: Cell seeding: CHL cells were seeded in 100 mm diameter dishes, with 1×10 cells per dish. 6 Place in a 37°C carbon dioxide incubator for 24 hours; Colchicine treatment: 4 h before harvesting cells, add colchicine solution to block the cells in metaphase; wherein the final concentration of the colchicine solution is 1 μg / mL; Hypotonicity: Add 5 mL of 0.075 mol / L KCl solution to resuspend the cells and place in a 37°C water bath for 30 minutes to allow the cells to swell. Invert and mix the cells every 10 minutes to achieve full hypotonicity. Fixation: 5 minutes before the end of hypotonic treatment, add 1 mL of fixative for pre-fixation, mix, centrifuge at 200G for 5-7 minutes, and discard the supernatant; slowly add 5 mL of fresh fixative, mix, and place in a 37°C water bath for 30 minutes. During the process, invert and mix the cells every 10 minutes to fully fix them. Centrifuge at 300G for 10 minutes, discard the supernatant, and fix twice in the same way; resuspend the cells in the prescribed amount of fixative and fix them at 4°C overnight.

2. The method for chromosome aberration testing according to claim 1, wherein Before the colchicine treatment step, also include: Toxic treatment: Aspirate the culture medium from the cell culture dish, add different concentrations of the test substance or positive control substance and S9 mixture, and incubate in a carbon dioxide incubator for 3 hours, with 2 replicates per group; after completion, aspirate the complete culture medium, wash the cells three times with Hanks solution, add serum-containing culture medium, and culture in a carbon dioxide incubator again, and harvest the cells after 24 hours; wherein the test substance or positive control substance is mitomycin C or cyclophosphamide.

3. The method for chromosome aberration testing according to claim 2, wherein: In the fall poisoning treatment step, when the S9 mixture was not added, RPMI1640 culture medium was used to supplement it.

4. The method for chromosome aberration testing according to claim 1, wherein Before the hypotonic treatment step, it also includes: Digestion: Digest the cells with trypsin. After the cells are detached, add cell culture medium containing serum to terminate the trypsin action. Mix well, place in a 15 mL centrifuge tube and centrifuge at 200G for 5-7 minutes, and discard the supernatant.

5. The method for chromosome aberration testing according to claim 1, wherein: After the fixing step, the following steps are also included: Dropping slides: Add 0.5-1 mL of fresh fixative solution to resuspend the cells according to the amount of cell pellet. Slightly tilt the slide and drop the slide at arm height, dropping 50 μL on one end. For each treatment group, drop at least 4 slides. Place the slide at room temperature to dry naturally. Staining: Stain with Giemsa stain for 15 min, rinse gently with deionized water, and dry the slides at 60°C. Sealing: Soak the dry slide in xylene for 5 minutes, add 1-2 drops of neutral resin glue while it is still wet, seal the slide with a cover glass, and let it dry naturally.

6. The method for chromosome aberration testing according to claim 5, characterized in that: After the sealing step, the following steps are also included: Data processing: 100 metaphase cells with well-dispersed chromosomes were selected from each group for chromosome aberration analysis. Structural and quantitative abnormalities of chromosomes were observed and recorded. The obtained experimental data were statistically processed using the χ2 test.

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