Slide preparation method for observing chromosome aberration of bone marrow cells of mammals

Abstract

The invention provides a slide preparation method for observing mammalian bone marrow cell chromosome aberration, and belongs to the technical field of biological detection. The slide specimen preparation method provided by the invention can meet the observation requirement of a standard operation procedure, the prepared slide specimen is superior in quality, the experimental operation period is shortened, and the slide specimen preparation method has wide application potential. The specimen prepared by the method has more metaphase division phase cells, the chromosome morphology is kept stable, and the slide preparation effect is remarkable. By using sodium citrate with an anticoagulation characteristic, the interference of peripheral blood mixing on the slide preparation process is effectively reduced, and the selective collection of metaphase division phase cells is realized, so that the concentration of target cells is improved. Besides, the method avoids the chromosome expansion phenomenon, so that the chromosome morphology is relatively thick in the observation process, and chromosome loss caused by serious rupture of a cell membrane is not easily caused.

Description

Technical Field

[0001] The invention relates to the field of biological detection technology, and in particular to a preparation method for observing chromosome aberration of mammalian bone marrow cells. Background Art

[0002] In biomedical research and clinical diagnosis, accurate observation and analysis of cells are crucial. Especially in the field of cytogenetics, the observation of chromosome changes during cell division is important for understanding the transmission of genetic information and identifying chromosomal abnormalities. Traditional slide specimen preparation methods play a key role in this process, but they have some technical and operational limitations.

[0003] Chromosomal aberration refers to abnormal changes in chromosome structure or number observed in the metaphase of cell mitosis. These changes can be structural, such as chromosome breakage, deletion, exchange or internal exchange, or numerical, involving chromosome aneuploidy or polyploidy. The study of chromosome aberration plays an important role in evaluating genetic toxicity, monitoring the effects of environmental pollutants, and diagnosing certain genetic diseases and cancers.

[0004] However, traditional slide preparation methods have obvious technical defects in ensuring the number of cells, the stability of chromosome morphology, and shortening the operation cycle. These defects limit the efficiency and accuracy of cytogenetic research and clinical diagnosis, and a new slide preparation method is urgently needed to overcome these problems in order to improve the quality of cytological research and diagnosis. When preparing metaphase cell specimens, traditional slide preparation methods often make it difficult to efficiently collect a sufficient number of target cells, resulting in insufficient representativeness of experimental results. In addition, unstable slide preparation quality and long operation cycle are also challenges faced by existing technologies. Therefore, the development of a slide specimen preparation method that can improve slide preparation quality, shorten the operation cycle, and effectively collect target cells is of great significance for promoting cytogenetic research and clinical applications. Summary of the invention

[0005] The object of the present invention is to provide a preparation method for observing chromosome aberrations in mammalian bone marrow cells, which can achieve better preparation effects.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a preparation method for observing chromosome aberration of mammalian bone marrow cells, comprising the following steps:

[0008] S1. flushing the femoral cavity with sodium citrate to obtain a bone marrow cell flushing solution;

[0009] S2, centrifuging the bone marrow cell washing fluid to obtain a cell precipitate;

[0010] S3, resuspending the cell pellet in complete culture medium, collecting the supernatant, and retaining the pellet after centrifugation to obtain non-adherent cells;

[0011] S4, mixing the non-adherent cells with the hypotonic solution, shaking in a water bath for 5 to 15 minutes, to obtain a cell suspension;

[0012] The cell suspension was fixed and stained.

[0013] Preferably, the concentration of the sodium citrate is 0.5-2%.

[0014] Preferably, in S2, the rotation speed of the centrifugal treatment is 1000-1200 rpm.

[0015] Preferably, in S2, the centrifugal treatment time is 3 to 8 minutes.

[0016] Preferably, in S3, the resuspension culture time is 2 to 3 hours.

[0017] Preferably, in S4, the hypotonic fluid is a KCl solution.

[0018] Preferably, the concentration of the KCl solution is 0.05-0.1 mol / L.

[0019] Preferably, the temperature of the water bath shaking treatment is 36-38°C.

[0020] Preferably, the rotation speed of the water bath oscillation treatment is 30 to 50 rpm.

[0021] Preferably, the fixing solution used for the fixation comprises methanol and glacial acetic acid;

[0022] The volume ratio of methanol to glacial acetic acid is 2 to 4:1;

[0023] The staining adopts Giemsa staining solution.

[0024] Beneficial effects of the present invention:

[0025] The slide specimen preparation method proposed in the present invention can meet the observation requirements of standard operating procedures, the prepared slide specimens are of high quality, and the experimental operation cycle is shortened, showing broad application potential. The number of metaphase cells in the specimens prepared by this method is large, the chromosome morphology remains stable, and the preparation effect is remarkable. By using sodium citrate with anticoagulant properties, the interference of peripheral blood mixing in the preparation process is effectively reduced, and the selective collection of metaphase cells is achieved, thereby increasing the concentration of target cells. In addition, this method avoids the phenomenon of chromosome expansion, so that the chromosome morphology is relatively coarse during the observation process, and it is not easy to cause chromosome loss due to severe rupture of the cell membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is an example photo of the film-making effect of Example 1;

[0027] Figure 2 This is a sample photo of the film production effect of Comparative Example 1;

[0028] Figure 3 This is a sample photo of the film production effect of Comparative Example 4;

[0029] Figure 4 This is an example photo of the production effect of comparative example 5. DETAILED DESCRIPTION

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1

[0032] With reference to the Technical Guidelines for Drug Genotoxicity Studies (2018) and ICH S2(R1) (Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, November 2011), this study selected SD rats as experimental animals, and the number of chromosomes of rats is 42.

[0033] Animal Grouping

[0034] Group design: vehicle control group, CPA group;

[0035] Number of animals: 10 per group;

[0036] Gender ratio: half male and half female;

[0037] Grouping method: First, 20 rats with uniform weight were selected from all rats that passed the adaptation period and included in the formal experiment; then, the rats were randomly grouped according to their weight and gender using the Pristima 7.2.0 version data system.

[0038] Specific grouping information is shown in Table 1:

[0039] Table 1 Rat grouping

[0040]

[0041] Note: The first digit of the animal number represents the group (1 and 2 represent the vehicle control group and CPA group, respectively); the second letter represents the gender: M for male and F for female; the last three digits represent the animal serial number.

[0042] Dosage design

[0043] The dose of cyclophosphamide group was 20 mg / kg, and a negative group (sterile water for injection) was also set up.

[0044] See Table 2 for detailed information:

[0045] Table 2 Rat bone marrow chromosome aberration test dosage design table

[0046]

[0047] Drug administration

[0048] Route of administration: oral gavage;

[0049] Dosing frequency: negative control group once a day for 3 consecutive days; CPA group once a day from the second day of the experiment for 2 consecutive days;

[0050] Dosing volume: 10mL / kg;

[0051] Dosage: Calculate the dosage based on the body weight measured before administration;

[0052] The day of first dosing was defined as trial day 1.

[0053] Observation and inspection

[0054] General status observation

[0055] Observed animals: All surviving animals in each group;

[0056] Observation content and frequency:

[0057] Near-death and death observation: at least once in the morning and afternoon every day;

[0058] Daily observation: at least once a day. If there are any toxic symptoms, the number of observations may be increased. This includes but is not limited to the animal's mental state, food intake, water intake, fur, excrement, death, and other abnormal symptoms.

[0059] Detailed observation: at least once a week, including but not limited to the animal's physical appearance, general behavior, mental state, glandular secretions, skin and mucous membrane color, respiratory status, genitals, death, and other abnormal symptoms.

[0060] weight

[0061] Measurement time: once a day before administration and before autopsy;

[0062] Animals tested: All surviving animals in each group.

[0063] Disposal of Dying Animals

[0064] The animal's condition and observation time were recorded. After the body weight was measured, appropriate amount of sodium pentobarbital was used for anesthesia and euthanasia was performed by cervical dislocation according to the animal's moribund condition.

[0065] Specimen preparation and observation

[0066] Preparation of specimens:

[0067] (1) 3.5 h before the animals were sacrificed, 4 mg / kg of colchicine was injected intraperitoneally;

[0068] (2) According to the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition (the American Veterinary Medical Association, 2020), animals were euthanized by blood sampling from the abdominal aorta under sodium pentobarbital anesthesia (approximately 60 mg / kg, intraperitoneal injection) based on the measured body weight.

[0069] (3) Place the animal on a dissecting board, cut open the thigh skin, and cut off the end of the femur connected to the ilium, being careful not to cut the femur. Separate the femur from the muscle, remove the muscle attached to both sides of the femur, and then gently wipe it with gauze or absorbent paper to remove any residue.

[0070] (4) Cut off both ends of the femur to form a cavity, and rinse the femur from one end to the other with 5 mL of 1% sodium citrate. Collect the fluid flushed from the two femurs, centrifuge at 1100 rpm for 5 minutes, discard the supernatant, and obtain the precipitate.

[0071] (5) The precipitate was resuspended in complete medium and cultured for 2.5 hours. The non-metaphase cells gradually adhered to the wall. The culture flask was shaken slowly (35 rpm, 2 min), and the supernatant was collected (the non-adherent metaphase cells were obtained by collecting the supernatant). The culture was centrifuged at 1100 rpm for 5 minutes and the supernatant was discarded.

[0072] (6) Add 0.075 mol / L KCl hypotonic solution to the centrifuge tube to disperse the cells into a suspended state. Place the centrifuge tube in a water bath (constant temperature oscillating water bath, 37°C, 40 rpm) for 10 min and then take it out.

[0073] (7) Add cell fixative (methanol: glacial acetic acid = 3:1), mix well, centrifuge at 1000 rpm for 10 min, discard the supernatant, and repeat twice; drop the slides on frozen glass slides; stain with Gimsa at room temperature for 15 min, rinse off the staining solution on the slides with pure water, and examine under a microscope after natural drying.

[0074] Cell number observation

[0075] Observation of the number of metaphase cells: 1000 cells were observed for each animal in each group (if the number was less than 1000 due to the high toxicity of the test product, it was recorded and reflected truthfully), and the number of metaphase cells was recorded:

[0076] Cell division index (%) = number of cell division phases / total number of cells (1000) × 100%.

[0077] And calculate its mitotic index inhibition rate: mitotic index inhibition rate (%) = (mitotic index of negative control group - mitotic index of each dose group) / mitotic index of negative control group × 100%.

[0078] Observation of chromosome structural aberration: 200 metaphase cells were observed for each animal in each experimental group (if the number of metaphase cells could not be observed due to excessive toxicity of the test substance or the count was less than 200, it was observed as it was) and the chromosome structural aberration rate of each group was counted.

[0079] Chromosome structural aberration rate (%) = number of aberrant cells in each group / total number of metaphase cells in each group × 100%.

[0080] Distortion type observation

[0081] The number of cells with structurally aberrant chromosomes and the types of aberrations (including fragments, gaps, exchanges and projections, ring chromosomes, polyploidy, dicentric chromosomes, endoreduplication, chromosome fragmentation, deletions, adhesions, and microbodies) were recorded in each group. The gaps were recorded separately but not included in the aberration rate. The number of polyploidy and endoreduplication aberrations were recorded separately and not included in the aberration rate.

[0082] Fissure rate (%) = number of fissure cells in each group / total number of metaphase cells in each group × 100%;

[0083] Polyploidy rate (%) = number of polyploid cells in each group / total number of metaphase cells in each group × 100%;

[0084] Endoreplication rate (%) = number of endoreplication cells in each group / total number of metaphase cells in each group × 100%.

[0085] Statistical analysis

[0086] The chromosome aberration rate, gap rate, polyploidy rate and internal duplication rate of each group of animals are expressed as mean ± standard deviation. describe.

[0087] The differences between the positive group and the negative control group were compared using group t test or Mann-Whitney U rank sum test (LEVENE variance homogeneity test P ≤ 0.05). All tests were two-sided tests with α = 0.05, and all statistical analyses were performed by gender using Stata / IC 15.0 for Windows software.

[0088] The statistical results are shown in Table 3

[0089] Table 3 Results of the mammalian bone marrow cell chromosome aberration test

[0090]

[0091]

[0092] Note: * Compared with the negative control group, the difference in mean is statistically significant, proving that the positive results meet the requirements of the test system.

[0093] From the above results, it can be seen that the slide specimens prepared by the slide preparation method provided by the present invention meet the SOP observation requirements, have better preparation quality, and a shorter test operation cycle, and have good application prospects.

[0094] Comparative Example 1

[0095] The only difference from Example 1 is that the specimen preparation step (1) is replaced by: 2.5 hours before the animal is killed, 4 mg / kg of colchicine is injected intraperitoneally.

[0096] Example 1 Tablet making effect Figure 1 As shown, the tableting effect of Comparative Example 1 is as follows Figure 2 shown.

[0097] Colchicine can inhibit mitosis to obtain a large number of metaphase cells. The present invention confirms that if colchicine is injected 2.5 hours before dissection, there are fewer metaphase cells, the chromosomes are slender, and it is difficult to observe the aberration; if colchicine is injected 3.5 hours before dissection, there are more metaphase cells, the chromosome morphology is stable, and the preparation effect is good.

[0098] Comparative Example 2

[0099] The only difference from Example 1 is that the specimen preparation step (4) is replaced by:

[0100] Cut off both ends of the femur to form a cavity, flush the femur from one end to the other with 5 mL of normal saline, collect the fluid flushed from the two femurs, centrifuge at 1100 rpm for 5 minutes, and discard the supernatant.

[0101] Observation of the tableting effect shows that the physiological saline background has more impurity fragments, while the 1% sodium citrate used in the present invention has a cleaner background. Therefore, the present invention confirms that sodium citrate has an anticoagulant effect and can effectively reduce the impact of peripheral blood incorporation during the tableting process.

[0102] Comparative Example 3

[0103] The only difference from Example 1 is that the specimen preparation step (5) is deleted, and 0.075 mol / L KCl hypotonic solution is directly added to the precipitate obtained in step (4) for subsequent treatment.

[0104] In the final preparation and counting process, the number of cells required for counting 100 metaphase cells in Example 1 was about 300, and that in Comparative Example 3 was about 1000, indicating that the solution provided by the present invention can selectively collect metaphase cells and increase the concentration of target cells.

[0105] Comparative Example 4

[0106] The only difference from Example 1 is that the specimen preparation step (6) is replaced by:

[0107] Add 0.075 mol / L KCl hypotonic solution into the centrifuge tube to disperse the cells into a suspended state. Manually blow the centrifuge tube and let it stand for 20 minutes.

[0108] The results of the comparative example are as follows Figure 3 As shown, Figure 1 Comparison of the preparation effect of Example 1 shows that the blowing is likely to be insufficient, the chromosomes are likely to clump, and are difficult to observe.

[0109] Comparative Example 5

[0110] The only difference from Example 1 is that the specimen preparation step (6) is replaced by:

[0111] Add 0.075 mol / L KCl hypotonic solution to the centrifuge tube to disperse the cells into a suspended state, and put the centrifuge tube into a water bath (constant temperature oscillating water bath, 37°C, 40 rpm) for 30 minutes and then take it out.

[0112] The results of the comparative example are as follows Figure 4 As shown, Figure 1 Compared with the preparation effect of Example 1 shown, it can be seen that the hypotonic time affects the preparation effect. If the time is longer, the chromosomes will swell, the chromosomes will be coarse during observation, and the chromosomes will be easily lost due to severe rupture of the cell membrane. The solution provided by the present invention can obtain better results.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A preparation method for observing chromosome aberrations in mammalian bone marrow cells, characterized in that: The following steps are involved: S1. flushing the femoral cavity with sodium citrate to obtain a bone marrow cell flushing solution; S2, centrifuging the bone marrow cell washing fluid to obtain a cell precipitate; S3, resuspending the cell pellet in complete culture medium, collecting the supernatant, and retaining the pellet after centrifugation to obtain non-adherent cells; S4, mixing the non-adherent cells with the hypotonic solution, shaking in a water bath for 5 to 15 minutes, to obtain a cell suspension; The cell suspension was fixed and stained.

2. The tableting method according to claim 1, characterized in that: The concentration of the sodium citrate is 0.5-2%.

3. The tableting method according to claim 1, characterized in that: In S2, the rotation speed of the centrifugal treatment is 1000-1200 rpm.

4. The tableting method according to claim 3, characterized in that: In S2, the centrifugal treatment time is 3 to 8 minutes.

5. The tableting method according to claim 1, characterized in that: In S3, the resuspension culture time is 2 to 3 hours.

6. The tableting method according to claim 1, characterized in that: In S4, the hypotonic solution is a KCl solution.

7. The film-making method according to claim 6, characterized in that: The concentration of the KCl solution is 0.05-0.1 mol / L.

8. The tableting method according to claim 1, characterized in that: The temperature of the water bath shaking treatment is 36-38°C.

9. The tableting method according to claim 8, characterized in that: The rotation speed of the water bath shaking treatment is 30 to 50 rpm.

10. The tableting method according to claim 1, characterized in that: The fixing solution used in the fixing comprises methanol and glacial acetic acid; The volume ratio of methanol to glacial acetic acid is 2 to 4:1; The staining adopts Giemsa staining solution.

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