A method for producing bone marrow chromosome G banding

Abstract

The present invention discloses a method for preparing bone marrow chromosome G banding. The method steps are as follows: inoculate bone marrow cells and culture overnight; add colchicine to the culture to incubate the cells together; absorb the supernatant, add hypotonic solution, mix and centrifuge; add hypotonic solution again, let stand for a period of time according to the ambient temperature; add methanol: acetic acid = 3:1 fixative, shake and centrifuge; absorb the supernatant, add methanol: acetic acid = 3:1 fixative, mix and let stand and centrifuge again; absorb the supernatant, add methanol: acetic acid = 3:1 fixative, let stand and centrifuge; repeat the above steps twice; absorb the supernatant, add a fixative containing a certain proportion of methanol and acetic acid according to the ambient humidity, mix and stay at 4 degrees overnight; take out the specimen, absorb the supernatant, add a fixative containing a certain proportion of methanol and acetic acid according to the ambient humidity of the day, adjust the turbidity, and drop the slice. The mitotic image produced by the method of the present application has better dispersion, clearer and more beautiful bands, and is easy to analyze.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing bone marrow chromosome G banding. Background Art

[0002] Chromosome banding technology is a technology that uses banding and staining to distinguish the finer features of chromosomes, such as the position, width and depth of the band. There are different chromosome staining methods, such as G, C, negative staining, silver staining, etc. G banding is currently the most widely used banding type.

[0003] The current bone marrow G-banding experiment method is greatly affected by the seasons, and the seasonal changes are mainly reflected in two aspects, namely temperature and humidity. At present, there is a consensus in the industry on the optimal temperature and humidity, that is, 25 degrees and 50-55%. However, the experimental environment cannot achieve constant temperature and humidity. How to achieve the optimal temperature and humidity when the temperature and humidity are not at the optimal temperature and humidity, and how to change the experimental conditions in response to changes in temperature and humidity are important challenges faced by bone marrow G-banding experiments. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for preparing bone marrow chromosome G banding.

[0005] The present invention is achieved by adopting the following technical solutions.

[0006] A method for preparing bone marrow chromosome G banding, characterized in that it comprises the following steps:

[0007] S1. Inoculation: Inoculate bone marrow cells into culture medium and culture overnight;

[0008] S2. Harvest:

[0009] a. Add colchicine to the culture in step S1 and incubate the cells together;

[0010] b. Aspirate the supernatant, add hypotonic solution, mix well and centrifuge;

[0011] c. Add hypotonic solution again and let stand for a while in a 37 degree water bath. The relationship between the standing time and the ambient temperature is as follows:

[0012] When the ambient temperature is less than 10°C, the hypotonic time is 21-22 minutes;

[0013] When the ambient temperature is 10℃≤≤20℃, the hypotonic time is 18-20min;

[0014] When the ambient temperature is 20℃<≤30℃, the hypotonic time is 16-18min;

[0015] When the ambient temperature is >30°C, the hypotonic time is 13-15 minutes;

[0016] d. After two hypotonicity treatments, add a fixative solution of methanol:acetic acid = 3:1, shake, and centrifuge;

[0017] e. Aspirate the supernatant, add methanol: acetic acid = 3:1 fixative, mix well, let stand, and centrifuge again;

[0018] f. Aspirate the supernatant, add methanol: acetic acid = 3:1 fixative, let stand, and centrifuge;

[0019] g. Repeat step f twice;

[0020] h. Pipette the supernatant, add a stationary solution containing methanol and acetic acid, mix well and incubate at 4 degrees overnight; the relationship between the ratio of acetic acid in the stationary solution and the ambient humidity is as follows:

[0021] When the ambient humidity is greater than 55%, methanol: acetic acid = 3:1;

[0022] When the ambient humidity is 55% or more and ≥40%, methanol:acetic acid = 2.3-2.6:1;

[0023] When the ambient humidity is less than 40%, methanol: acetic acid = 2:1;

[0024] S3. Dropping: Take out the specimen obtained in step S2, aspirate the supernatant, add a fixative containing methanol and acetic acid, then adjust the turbidity, and drop the slice by air drying method; the relationship between the proportion of acetic acid in the fixative and the ambient humidity is as follows:

[0025] When the ambient humidity is greater than 55%, methanol: acetic acid = 2:1;

[0026] When the ambient humidity is 55% or more and ≥40%, methanol:acetic acid = 1.6-1.9:1;

[0027] When the ambient humidity is less than 40%, methanol: acetic acid = 1.3-1.5:1;

[0028] The proportion of acetic acid in this step needs to be higher than that in step S2h.

[0029] By adopting the above technical solution, the present application changes the hypotonic time corresponding to the temperature change and the proportion of acetic acid in the fixative corresponding to the humidity change, so that the experimental adjustment measures for adapting to seasonal changes are more flexible and targeted.

[0030] The traditional method uses one round of hypotonicity in the harvesting process, but the effect of one round of hypotonicity is not sufficient. In the step of aspirating the supernatant, a part of the supernatant liquid will remain, resulting in the hypotonic liquid being mixed with a part of the supernatant liquid when the hypotonic liquid is added and mixed, diluting the added hypotonic liquid, making the hypotonic effect of the specimen insufficient. The present application uses two rounds of hypotonicity. The hypotonic liquid is added for the first time to wash the specimen. After centrifugation to remove the supernatant, the remaining supernatant is the hypotonic liquid. When the hypotonic liquid is added for the second time, the hypotonic liquid will not be diluted, and the hypotonic effect is significantly better than one round of hypotonicity. At the same time, because the effect of two rounds of hypotonicity is better than one round, the hypotonic standing time will be shortened. Specifically, when the ambient temperature is below 10°C, the hypotonic time is 21-22min; when the ambient temperature is 10-20°C, the hypotonic time is 18-20min; when the ambient temperature is 20-30°C, the hypotonic time is 16-18min; when the ambient temperature is above 30°C, the hypotonic time is 13-15min.

[0031] The traditional method uses four rounds of fixation in the harvesting step, while the present application uses five rounds of fixation. The first four rounds use a fixative solution of methanol: acetic acid = 3:1 for fixation. The fifth round uses a fixative solution with different acetic acid ratios according to the ambient humidity. After the fifth round of fixation, it is placed in a 4-degree refrigerator. The proportion of acetic acid in the fifth round of fixative solution is higher than that in the first four rounds, and the methanol-acetic acid ratio of the first four rounds of fixative solution remains unchanged throughout the year, while the fifth round changes with the seasons. The specific ratio is based on the methanol: acetic acid = 3:1 in summer. The lower the ambient humidity, the higher the acetic acid ratio. The specific ratio is adjusted according to the humidity change. For example, when the humidity in the dog days is above 55%, the fifth round of fixative solution can be set to a ratio of 3:1. When the humidity in spring and autumn is between 40%-55%, the ratio can be transitioned to about 2.5:1. In winter, when the humidity is below 40%, it can gradually transition to 2:1. The proportion of acetic acid in the fixative replaced before dropping the slice on the third day needs to be further increased than that in the fifth round to achieve gradient adjustment. For example, when the humidity in the dog days is above 55%, the ratio of the fixative before dropping the slice on the third day can be set to 2:1. When the humidity in spring and autumn is between 40% and 55%, the ratio can be transitioned to 1.6:1-1.9:1. In winter, when the humidity is below 40%, the ratio can be gradually transitioned to 1.3:1-1.5:1.

[0032] The experimental process of the present application is as a whole: inoculation on the first day, harvest on the second day, and drop film on the third day. The last step of the second day's harvest is the fifth round of fixation, which is placed in the refrigerator after the fifth round of fixation, and the film is dropped after taking it out on the third day. The traditional method drops the film directly after taking it out on the third day, and the present application adjusts the acetic acid ratio again according to the laboratory humidity of the day. The ratio of the fifth round of fixative is relatively fixed, and it is adjusted once every quarter or two months. The acetic acid ratio of this step before dropping the film can be adjusted once a day, so that the experimental operation is more flexible and can be more targeted at the real humidity of the day of the drop film. Since the increase in the acetic acid ratio can make the cell division image expand better, the setting of the acetic acid ratio is adjusted again before the five rounds of fixation and dropping the film in the present application, which can provide a period of action time for the expansion of the division image. In addition, methanol has a good hardening effect on cells. Hardening first and then expansion is a gradient expansion, which can prevent cell fragmentation and chromosome loss caused by premature cell expansion. If a high proportion of acetic acid fixative is used from the beginning of pre-fixation, the degree of hardening of cells will not be enough to expand and break prematurely.

[0033] Furthermore, in step S1, the inoculation amount of bone marrow cells is 2x10 6 Pieces / ml.

[0034] Furthermore, in step S2a, the concentration of colchicine is 20 ug / ml.

[0035] Furthermore, in step S2a, the incubation condition after adding colchicine is 37 degrees for 50-60 minutes.

[0036] Furthermore, the component of the hypotonic solution is KCL aqueous solution with a concentration of 4 g / L.

[0037] This application has the following beneficial effects.

[0038] This application proposes a systematic theory of the inverse relationship between hypotonicity and temperature, and the inverse relationship between the ratio of the fixative and humidity, and makes adjustments based on this theory to adapt to seasonal changes. The two rounds of hypotonicity in the method of this application make the hypotonicity more complete; the ratio of acetic acid is changed in the last round of the fixative in the five rounds of fixation; the ratio of the fixative is adjusted again before dripping on the third day, and the ratio of acetic acid in the fixative must be higher than that of the fifth round of acetic acid, so as to achieve a gradient adjustment of the ratio of the fixative. The split images produced by the method of this application have better dispersion, clearer and more beautiful bands, and are easier to analyze. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a reverse linkage relationship diagram of temperature and hypotonic time of the present invention;

[0040] Figure 2 It is a reverse linkage relationship diagram of humidity and the proportion of acetic acid in the fixing liquid of the present invention;

[0041] Figure 3is a split image obtained by the method of Example 1 of the present invention;

[0042] Figure 4 It is the split image obtained by the method of comparative example 1 under the environmental conditions of example 1 of the present invention;

[0043] Figure 5 is a split image obtained by the method of Example 2 of the present invention;

[0044] Figure 6 It is the split image obtained by the method of comparative example 1 under the environmental conditions of example 2 of the present invention;

[0045] Figure 7 is a split image obtained by the method of Example 3 of the present invention;

[0046] Figure 8 It is the split image obtained by the method of comparative example 1 under the environmental conditions of example 3 of the present invention;

[0047] Fig. 9 It is a data comparison chart between the method of the present invention and the traditional method. DETAILED DESCRIPTION

[0048] The present patent application is further described below in conjunction with the accompanying drawings and embodiments.

[0049] The present invention adapts to temperature changes by changing the hypotonic time, such as shortening the hypotonic time when the temperature is high in summer and extending the hypotonic time when the temperature is low in winter. In other words, the temperature and hypotonic time show an inverse relationship (e.g. Figure 1 ). The ratio of the fixative is adjusted to adapt to changes in humidity. In summer, when humidity is high, the ratio of acetic acid in the fixative is reduced, and in winter, when humidity is low, the ratio of acetic acid in the fixative is increased. In other words, humidity and the ratio of acetic acid in the fixative are inversely related (e.g. Figure 2). Therefore, this method is not about finding an optimal humidity and temperature, because the optimal temperature and humidity have been agreed upon in the industry, that is, temperature 25 degrees and humidity 50-55%. However, the experimental environment cannot achieve constant temperature and humidity. How to achieve the effect of optimal temperature and humidity when the temperature and humidity are not at the optimal temperature and humidity is the technical problem to be solved by this application. The originality of this method is to adapt to environmental changes by adjusting the experimental process while the seasons change. The traditional method only adapts to seasonal changes by changing the hypotonic time, while the method of this application adapts to temperature changes by changing the hypotonic solution and to humidity changes by changing the proportion of the fixed solution. Of course, assuming that all experimental environments can achieve constant temperature and humidity, this method still has huge original advantages. That is, when the temperature and humidity are at the optimal temperature and humidity, the experimental process of this application still has advantages. Therefore, this method has two major advantages, one is the dynamic advantage, that is, adaptation to seasonal changes; the other is the static advantage, that is, assuming constant temperature and humidity, this method is still better than the traditional method.

[0050] Example 1 (temperature 26°C, humidity 50%-55%, humidity 50% on the day of tablet drop)

[0051] A method for preparing bone marrow chromosome G banding comprises the following steps:

[0052] S1. Inoculation: 2x10 6 The concentration of cells / ml was counted by computer software, and 1 ml of bone marrow cells was inoculated into 1640 medium and cultured at 37 degrees overnight;

[0053] S2. Harvest: Add 100ul colchicine (final concentration 20ug / ml) the next morning, then let it stand at 37 degrees for 50 minutes; pour the specimens from the culture bottle into a centrifuge tube and centrifuge at 1500rpm for 5 minutes (the following centrifugation conditions are the same), aspirate the supernatant, add 10ml of hypotonic solution (KCL aqueous solution, concentration of 4g / L), mix and shake, and centrifuge; add 10ml of hypotonic solution for the second time, let it stand at 37 degrees for 16 minutes; then add 1ml of fixative (methanol: acetic acid = 3:1), shake, and centrifuge; aspirate the supernatant, add 6ml of fixative (methanol: acetic acid = 3:1), mix and let it stand for 15 minutes, and centrifuge again; aspirate the supernatant, add 6ml of fixative (methanol: acetic acid = 3:1), let it stand for 5 minutes, and centrifuge; after repeating twice, aspirate the supernatant, add 4ml of fixative (methanol: acetic acid = 2.6:1), mix and put it in a 4-degree refrigerator overnight;

[0054] S3. Dropping: Take out the specimen from the refrigerator, aspirate the supernatant, add 1.5 ml of fixative (the volume can be determined by the experimenter based on experience) (methanol: acetic acid = 1.8:1) according to the target turbidity, adjust the turbidity, and drop the specimen by air-drying method.

[0055] Example 2 (temperature 25°C, humidity 40%-50%, humidity 42% on the day of tablet drop)

[0056] A method for preparing bone marrow chromosome G banding comprises the following steps:

[0057] S1. Inoculation: 2x10 6 The concentration of cells / ml was counted by computer software, and 1 ml of bone marrow cells was inoculated into 1640 medium and cultured at 37 degrees overnight;

[0058] S2. Harvest: Add 100ul colchicine (final concentration 20ug / ml) the next morning, then let it stand at 37 degrees for 50 minutes; pour the specimens from the culture bottle into a centrifuge tube and centrifuge at 1500rpm for 5 minutes (the following centrifugation conditions are the same), aspirate the supernatant, add 10ml of hypotonic solution (KCL aqueous solution, concentration of 4g / L), mix and shake, and centrifuge; add 10ml of hypotonic solution for the second time, let it stand at 37 degrees for 17 minutes; then add 1ml of fixative (methanol: acetic acid = 3:1), shake, and centrifuge; aspirate the supernatant, add 6ml of fixative (methanol: acetic acid = 3:1), mix and let it stand for 15 minutes, and centrifuge again; aspirate the supernatant, add 6ml of fixative (methanol: acetic acid = 3:1), let it stand for 5 minutes, and centrifuge; after repeating twice, aspirate the supernatant, add 4ml of fixative (methanol: acetic acid = 2.5:1), mix and put it in a 4-degree refrigerator overnight;

[0059] S3. Dropping: Take out the specimen from the refrigerator, aspirate the supernatant, add 1.5 ml of fixative (the volume can be determined by the experimenter based on experience) (methanol: acetic acid = 1.7:1) according to the target turbidity, adjust the turbidity, and drop the specimen by air drying method.

[0060] Example 3 (temperature 8°C, humidity 20%-30%, humidity 25% on the day of tablet drop)

[0061] A method for preparing bone marrow chromosome G banding comprises the following steps:

[0062] S1. Inoculation: 2x10 6 The concentration of cells / ml was counted by computer software, and 1 ml of bone marrow cells was inoculated into 1640 medium and cultured at 37 degrees overnight;

[0063] S2. Harvest: Add 100ul colchicine (final concentration 20ug / ml) the next morning, then let it stand at 37 degrees for 50 minutes; pour the specimens from the culture bottle into a centrifuge tube and centrifuge at 1500rpm for 5 minutes (the following centrifugation conditions are the same), aspirate the supernatant, add 10ml of hypotonic solution (KCL aqueous solution, concentration of 4g / L), mix and shake, and centrifuge; add 10ml of hypotonic solution for the second time, let it stand at 37 degrees for 21 minutes; then add 1ml of fixative (methanol: acetic acid = 3:1), shake, and centrifuge; aspirate the supernatant, add 6ml of fixative (methanol: acetic acid = 3:1), mix and let it stand for 15 minutes, and centrifuge again; aspirate the supernatant, add 6ml of fixative (methanol: acetic acid = 3:1), let it stand for 5 minutes, and centrifuge; after repeating twice, aspirate the supernatant, add 4ml of fixative (methanol: acetic acid = 2.0:1), mix and put it in a 4-degree refrigerator overnight;

[0064] S3. Dropping: Take out the specimen from the refrigerator, aspirate the supernatant, add 1.5 ml of fixative (the volume can be determined by the experimenter based on experience) (methanol: acetic acid = 1.5:1) according to the target turbidity, adjust the turbidity, and drop the specimen by air drying method.

[0065] Comparative Example 1

[0066] A method for preparing bone marrow chromosome G banding comprises the following steps:

[0067] S1. Inoculation: 2x10 6 The concentration of cells / ml was counted by computer software, and 1 ml of bone marrow cells was inoculated into 1640 medium and cultured at 37 degrees overnight;

[0068] S2. Harvest: Add 100ul colchicine (final concentration 20ug / ml) the next morning, then let stand at 37 degrees for 50 minutes; pour the sample from the culture bottle into a centrifuge tube and centrifuge at 1500rpm for 5 minutes (the following centrifugation conditions are the same), aspirate the supernatant, add 10ml of hypotonic solution (KCL aqueous solution, concentration of 4g / L), mix and shake, and let stand at 37 degrees for 35 minutes; then add 1ml of fixative (methanol: acetic acid = 3:1), shake, and centrifuge; aspirate the supernatant, add 6ml of fixative (methanol: acetic acid = 3:1), mix and let stand for 15 minutes, and centrifuge again; aspirate the supernatant, add 6ml of fixative (methanol: acetic acid = 3:1), let stand for 5 minutes, and centrifuge; repeat once more and put in a 4 degree refrigerator overnight;

[0069] S3. Take out the specimen from the refrigerator, aspirate the supernatant, add 1.5 ml of fixative (the volume can be determined by the experimenter based on experience) (methanol: acetic acid = 3:1), adjust the turbidity, and air-dry the slide.

[0070] The split image obtained by the method of Example 1 is as follows Figure 3As shown, the split image obtained by using the method of comparative example 1 under the same environment is as follows Figure 4 The split image obtained by the method of Example 2 is shown in Figure 5 As shown, the split image obtained by using the method of comparative example 1 under the same environment is as follows Figure 6 The split image obtained by the method of Example 3 is as shown in Figure 7 As shown, the split image obtained by using the method of comparative example 1 under the same environment is as follows Figure 8 shown.

[0071] The samples from July 2020 to January 2022 were prepared using the method of this application and the traditional method, and the two methods were compared and analyzed using the following four parameters.

[0072] Excellent film rate: Specimens with 20 split images are excellent films, and those with less than 20 split images are non-excellent films. The higher the excellent film rate, the better.

[0073] Number of split images: Based on the subjective feelings of the analysts, the number of split images on the slides is divided into three levels. Each specimen is scored and evaluated by the analysts. The percentage of specimens with the highest score in a month is the percentage of multiple split images. The higher this parameter is, the better.

[0074] Dispersion: Based on the subjective feelings of the analysts, the dispersion of the split images is divided into three levels. Each specimen is scored and evaluated by the analysts. The percentage of specimens with the highest score in a month is the dispersion. The higher this parameter is, the better.

[0075] Banding beauty: According to the subjective feelings of the analysts, the banding beauty is divided into three levels. Each specimen is scored and evaluated by the analysts. The percentage of specimens with the highest score in a month is the banding beauty percentage. The higher this parameter is, the better. The experimental results are shown in Table 1.

[0076]

[0077] This application method has been used since July 2021. From the above data, it can be seen that after using this application method, the average rate of excellent films is 88%, and the average rate in the same period last year is 87.0%. Multi-fission percentage: the average rate after using this application method is 91%, and the average rate in the same period last year is 90.6%. Good dispersion percentage: 89.9% after using this application method, and the average rate in the same period last year is 83.0%. Aesthetic band percentage: the average rate after using this application method is 86.6%, and the average rate in the same period last year is 74.9%.

[0078] By comparison, it can be seen that the new method is superior to the traditional method in terms of excellent film rate, number of split images, dispersion, and banding beauty (see Fig. 9 ). At the same time, through the statistical t-test of two groups of samples, it was found that the p-values ​​of the two parameters of dispersion and banding beauty were both less than 0.01, indicating that there were significant differences between the new and old methods in improving dispersion and modern beauty.

[0079] The embodiments of this specific implementation method are all preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing bone marrow chromosome G banding, characterized in that: The following steps are involved: S1. Inoculation: Inoculate bone marrow cells into culture medium and culture overnight; S2. Harvest: a. Add colchicine to the culture in step S1 and incubate the cells together; b. Aspirate the supernatant, add hypotonic solution, mix well and centrifuge; c. Add hypotonic solution again and let stand for a while in a 37 degree water bath. The relationship between the standing time and the ambient temperature is as follows: When the ambient temperature is less than 10°C, the hypotonic time is 21-22 minutes; When the ambient temperature is 10℃≤≤20℃, the hypotonic time is 18-20min; When the ambient temperature is 20℃<≤30℃, the hypotonic time is 16-18min; When the ambient temperature is >30°C, the hypotonic time is 13-15 minutes; d. After two hypotonicity treatments, add a fixative solution of methanol:acetic acid = 3:1, shake, and centrifuge; e. Aspirate the supernatant, add methanol: acetic acid = 3:1 fixative, mix well, let stand, and centrifuge again; f. Aspirate the supernatant, add methanol: acetic acid = 3:1 fixative, let stand, and centrifuge; g. Repeat step f twice; h. Pipette the supernatant, add a stationary solution containing methanol and acetic acid, mix well and incubate at 4 degrees overnight; the relationship between the ratio of acetic acid in the stationary solution and the ambient humidity is as follows: When the ambient humidity is greater than 55%, methanol: acetic acid = 3:1; When the ambient humidity is 55% or more and ≥40%, methanol:acetic acid = 2.3-2.6:1; When the ambient humidity is less than 40%, methanol: acetic acid = 2:1; S3. Dropping: Take out the specimen obtained in step S2, aspirate the supernatant, add a fixative containing methanol and acetic acid, then adjust the turbidity, and drop the slice by air drying method; the relationship between the proportion of acetic acid in the fixative and the ambient humidity is as follows: When the ambient humidity is greater than 55%, methanol: acetic acid = 2:1; When the ambient humidity is 55% or more and ≥40%, methanol:acetic acid = 1.6-1.9:1; When the ambient humidity is less than 40%, methanol: acetic acid = 1.3-1.5:1; The proportion of acetic acid in this step needs to be higher than that in step S2h.

2. The method for preparing bone marrow chromosome G banding according to claim 1, characterized in that: In step S1, the inoculum size of bone marrow cells was 2x10 6 Pieces / ml.

3. The method for preparing bone marrow chromosome G banding according to claim 1, characterized in that: In step S2a, the final concentration of colchicine is 20 ug / ml.

4. The method for preparing bone marrow chromosome G banding according to claim 1, characterized in that: In step S2a, the incubation condition after adding colchicine is 37 degrees for 50-60 minutes.

5. The method for preparing bone marrow chromosome G banding according to claim 1, characterized in that: The component of the hypotonic solution is KCL aqueous solution with a concentration of 4 g / L.

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