Preparation method of fish chromosomes

Abstract

The invention discloses a preparation method of fish chromosomes, which comprises the following steps: selecting fish scales as a material, quickly culturing primary cells in an in-vitro culture manner, and directly preparing the chromosomes on a cell slide. According to the scale culture method, more cells can be quickly obtained, chromosome division phases prepared from adherent cells cultured by the scale culture method are more in number, large and attractive in shape and clear and complete in structure, and operation is performed on a cell slide, so that experimental steps are simplified, and chromosome preparation time is shortened. The method for preparing the chromosomes by using the cells cultured by the scales not only is efficient in process, but also does not need to dissect to take materials, is less harmful to fishes, is an optimal choice for fishes with small sizes and rare varieties, and provides an efficient and rapid way for rapid preparation of the chromosomes of the fishes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for preparing fish chromosomes. Background Art

[0002] The experimental process of the traditional method for preparing chromosomes by injecting PHA into fish kidney cells is complex and requires high operation requirements; the culture technology of peripheral blood cells needs to obtain lymphocytes, with a small number of cells and a long experimental period, making it difficult to quickly obtain good chromosome metaphases. This requires us to continuously develop new cell culture technologies, optimize culture conditions, and improve the experimental methods for chromosome preparation, so as to provide an excellent experimental method for fish cell culture technology and the technology for preparing chromosomes of cultured cells.

[0003] Therefore, it is necessary to develop a method for quickly preparing fish chromosomes. Summary of the Invention

[0004] The object of the present invention is to provide a method for preparing fish chromosomes, which has a short culture period (6 - 12 h), a high chromosome integrity rate (92.3%), and the number of repeated samplings per time ≥ 5 times.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for preparing fish chromosomes, and the method includes:

[0007] Select fish scales with good growth status, place them in cell slides and culture medium after aseptic cleaning for pre-treatment culture of fish scales for 6 - 12 hours, wherein the culture medium is DMEM medium containing 15% fetal bovine serum and 100 U / mL double antibiotics;

[0008] Induce cell synchronous division through colchicine treatment, adopt hypotonic combined with multiple Carnoy's fixative treatments, and finally obtain chromosome metaphases by Giemsa staining combined with the method of covering with nail polish in an inverted manner.

[0009] Further, the temperature for pre-treatment culture of fish scales is 26°C.

[0010] Further, the concentration of the double antibiotic gradient penetration system is:

[0011] Initial soaking stage: penicillin 10000 U / mL + streptomycin 10000 U / mL, mixed with 1×PBS in a ratio of 1:10;

[0012] Culture stage: working concentration penicillin 100 U / mL + streptomycin 100 U / mL to maintain a sterile environment throughout the cell culture process.

[0013] Further, the colchicine treatment step includes:

[0014] Add colchicine with a final concentration of 10 μg / mL to the culture medium, and precisely control the action time to be 4 ± 0.5 hours.

[0015] Further, the hypotonic fixation treatment includes:

[0016] Treat with 0.075 mol / L KCl solution at 20 - 22 °C for 2.5 hours, and gently pipette outside the cell smear every 30 minutes to make the hypotonic treatment complete; the fixative is methanol: glacial acetic acid = 3:1, and fix three times.

[0017] Further, the chromosome slide-making steps include:

[0018] When performing Giemsa staining, the pH value of the staining solution and the phosphate buffer is 7.0 - 7.2, and the staining time is strictly controlled at 30 ± 2 minutes;

[0019] The baking temperature is to heat at a position 2 cm away from the outer flame of the alcohol lamp for 5 seconds, and after natural cooling to room temperature, fix it on the glass slide.

[0020] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0021] 1. A method for preparing fish chromosomes provided by the present invention uses fish scales as raw materials for primary cell culture and chromosome preparation. After culturing with DMEM medium at 26°C for 6 - 12 hours, multiple layers of cells can be observed growing around the scales. Compared with other tissue culture methods, this method can culture cells more quickly, and there is no need to collect cells after the culture. During the culture process, the cells adhere to the cell culture slides and chromosome preparation experiments can be directly carried out on the slides, further simplifying the experimental steps. At the same time, by using the method of culturing cells in vitro, the experimental conditions can be better controlled, and the same experimental object can be repeatedly experimented multiple times to obtain ideal results, without being restricted by the diseases or deaths of the fish. The method of preparing chromosomes by culturing cells with scales is an ideal way. The primary cells cultured from scales have a strong metabolism rate and a strong division ability, which is conducive to obtaining clear and complete chromosome metaphases. Moreover, the types of cells after primary culture are generally relatively single, avoiding the interference of impurities. The prepared chromosome specimens have a clear background and are convenient for observation and analysis. In summary, fish scales not only have a large number, are easy to obtain, do not require dissection for sampling, cause less harm to experimental fish, and reduce the experimental cost, but also do not need to be digested and dispersed. After washing, they can be directly placed in the medium for culture, simplifying many steps compared with other traditional tissue culture methods and making cell culture more rapid. The primary cells cultured have a strong division ability, a short cell generation time, and a low mutation probability. When preparing chromosomes, there is no need to collect cells and perform high-altitude dropping, and it can be directly carried out on the cell culture slides, with a wide range of applications. It is an optimal choice especially for preparing chromosomes of small-sized and rare fish species.

[0022] 2. The method of this embodiment explores a low-cost and high-efficiency cell culture and chromosome preparation method. The obtained fish chromosome division index is high, the number is large, the structure is clear and complete, the morphology is large and beautiful, and the background is clean without impurities, providing reference opinions in the technology of rapidly preparing fish chromosomes, which is of great significance for studying the genetic composition, species evolution, and genetic relationship of fish, etc.

[0023] 3. Compared with the prior art, it has the following advantages:

[0024] (1) Efficiency breakthrough: The culture period is shortened from 72 hours to 6 - 12 hours (a shortening of 83%); the same sample can be repeatedly experimented ≥5 times per single sampling (only 1 time for the traditional method).

[0025] (2) Quality improvement: The chromosome division index reaches 42.3 ± 3.1% (a 126% increase compared with the traditional method), and the cell adhesion rate is 95.6 ± 1.8% (a 39.9% increase compared with the conventional method).

[0026] (3) Economic value: The cost is reduced by 66.7% (the cost of a single experiment is ￥50 vs ￥150), and the sampling of live rare species is reduced by 90%. Brief Description of the Drawings

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 Cell diagram (10×) cultured from fish scales;

[0029] Figure 2 Chromosome diagram (20×) prepared from cells cultured from fish scales;

[0030] Figure 3 Chromosome diagram (100×) prepared from cells cultured from fish scales;

[0031] Figure 4 Chromosome diagram (100×) prepared from cells cultured from fish scales. Detailed Embodiments

[0032] The following will specifically describe the present invention in combination with the detailed embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed embodiments and examples are used to illustrate the present invention, rather than limiting the present invention.

[0033] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of conflict, this specification prevails.

[0034] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or by existing methods.

[0035] The following will specifically describe the present application in combination with examples, comparative examples, and experimental data.

[0036] Example 1. A method for preparing fish chromosomes

[0037] The embodiment of the present invention provides a method for preparing fish chromosomes, including the following steps:

[0038] Step S1: Select fish scales with good growth status. After aseptic cleaning, place them on cell slides and in a culture medium for pre-treatment culture of fish scales for 6 - 12 hours. The culture medium is DMEM medium containing 15% fetal bovine serum and 100 U / mL of double antibiotics.

[0039] Step S2: Induce synchronous cell division by colchicine treatment. Use hypotonic combined with multiple Carnoy's fixative treatments, and finally prepare slides by Giemsa staining combined with the method of inverting nail polish to obtain chromosome metaphases.

[0040] In this embodiment, in step S1, the conditions for pre-treatment culture of fish scales are culturing at 26°C for 12 h.

[0041] In step S2, the concentration of colchicine is 10 μg / mL.

[0042] Specifically, the method specifically includes:

[0043] 1. Pre-treatment culture of fish scales

[0044] Turn on the ultraviolet table, place reagents such as DMEM medium, double antibiotics, 10×PBS, colchicine, sterile water, pipettes, pipette tips, 15-mm cell slides, sterilized forceps, 35-mm disposable cell culture dishes, 0.22-μm and 0.45-μm bacterial filters, 50-mL centrifuge tubes and other experimental equipment in the laminar flow hood, and perform ultraviolet sterilization for 30 min; perform aseptic operation in the laminar flow hood to prepare cell culture medium, 1×PBS and colchicine solution. Each 100 mL of the culture medium includes: 84 mL of DMEM medium, 15 mL of fetal bovine serum, 1 mL of double antibiotics (penicillin 10000 U / mL + streptomycin 10000 U / mL); prepare 1000 μg / mL colchicine, after preparation, filter it once with a 0.45-μm bacterial filter and then filter it once with a 0.22-μm bacterial filter; select a fish with healthy physique and no diseases or injuries on the body surface, gently pluck the scales with sterilized forceps, and place them in a 35-mm disposable culture dish containing 1×PBS, ensuring that the buffer covers the scales; wash the scales more than 3 times in the culture dish containing 1×PBS by the cross method, about 1 min each time. During the process of transferring the scales into the culture dish, only pick one scale with the forceps at a time to avoid incomplete cleaning caused by overlapping scales; add 3 mL of 1×PBS and 300 μL of double antibiotics solution (1×PBS: double antibiotics = 10:1) to the culture dish and mix well. Put the washed scales into the culture dish, ensure that the solution covers the scales, and soak for 30 min; prepare 35-mm culture dishes, place 3 15-mm cell slides in each dish, add 2 mL of cell culture medium, and make it evenly cover the cell slides. Pick up the soaked scales with new sterilized forceps and place them on the surface of the cell slides, with the concave side facing down, gently press the scales with the forceps to make them fit with the slides, and do not overlap the scales. Write the label on the culture dish and gently transfer it to a 26°C constant temperature CO2 incubator for 12 h;

[0045] 2. Chromosome preparation

[0046] On the second day, gently take out the culture dish from the constant temperature incubator and observe the cell growth status under an inverted microscope. When it is found that more than 6 layers of cells have grown at the edge of the scales, start the chromosome preparation experiment;

[0047] Add 20 μL of 1000 μg / mL colchicine to the area outside the cell slides in the culture dish, gently pipette and mix well to make its final working concentration 10 μg / mL, gently put it back into the 26°C constant temperature CO2 incubator, and act for 4 h;

[0048] Gently take out the culture dish, completely remove the solution in the dish, add 1×PBS and wash 3 times. Add 3 mL of 0.075 mol / mL KCL hypotonic solution outside the cell smear, and perform hypotonic treatment at 20 - 22 °C for 2.5 h. During this period, gently pipette outside the cell smear every 30 min to make the hypotonic treatment more complete;

[0049] Prepare Carnoy's fixative (methanol: glacial acetic acid = 3:1) in advance and pre-cool it in the refrigerator at 4 °C. After the hypotonic treatment is completed, remove the hypotonic solution, add 1 mL of freshly prepared pre-cooled Carnoy's fixative and fix for 1 min;

[0050] Remove the fixative, add 3 mL of freshly prepared pre-cooled fixative, and fix in the refrigerator at 4 °C for 1 h. During this period, gently pipette outside the cell smear every 20 min;

[0051] Remove the fixative, add 3 mL of freshly prepared pre-cooled fixative, and fix in the refrigerator at 4 °C for 2 h. During this period, gently pipette outside the cell smear every 30 min;

[0052] Remove the fixative, add 1 mL of freshly prepared pre-cooled fixative, immediately pick up the cell smear gently with forceps, quickly pass it through the outer flame of the alcohol lamp once, and dry and cool it at room temperature;

[0053] Prepare Giemsa stain solution according to the ratio of sodium dihydrogen phosphate: disodium hydrogen phosphate: Giemsa stock solution = 4:4:1. Use a pipette to suck the stain solution and drop it on the cell smear for staining for 30 min;

[0054] Pick up the cell smear with forceps, use a pipette to suck water and gently rinse the back of the smear until there is no stain residue on the smear. Place the smear face up on a clean filter paper and air dry it at room temperature;

[0055] Drop a small drop of nail polish on a clean glass slide, invert the cell smear onto the nail polish, and gently press it with forceps to fix it;

[0056] After the cell smear is completely fixed on the glass slide, observe and analyze it under a microscope, and take pictures for preservation.

[0057] Example 2

[0058] In this example, in step S1, the conditions for pre-treatment and culture of fish scales are 27 °C, and other methods are the same as those in Example 1.

[0059] Example 3

[0060] In this example, in step S1, the conditions for pre-treatment and culture of fish scales are 28 °C, and other methods are the same as those in Example 1.

[0061] Example 4

[0062] In this embodiment, in the step S1, the pretreatment culture time of fish scales is 6 h, and other methods are the same as those in Embodiment 1.

[0063] Embodiment 5

[0064] In this embodiment, in the step S1, the pretreatment culture time of fish scales is 9 h, and other methods are the same as those in Embodiment 1.

[0065] Embodiment 6

[0066] In this embodiment, in the step S2, the colchicine concentration is 5 μg / mL, and other methods are the same as those in Embodiment 1.

[0067] Embodiment 7

[0068] In this embodiment, in the step S2, the colchicine concentration is 15 μg / mL, and other methods are the same as those in Embodiment 1.

[0069] Comparative Example 1

[0070] In this comparative example, in the step S1, the pretreatment culture condition of fish scales is 25 °C, and other methods are the same as those in Embodiment 1.

[0071] Comparative Example 2

[0072] In this comparative example, in the step S1, the pretreatment culture time of fish scales is 24 h, and other methods are the same as those in Embodiment 1.

[0073] Comparative Example 3

[0074] In this comparative example, in the step S2, the colchicine concentration is 0, and other methods are the same as those in Embodiment 1.

[0075] Experimental Example 1

[0076] 1. Single-factor experiment on temperature

[0077] Table 1

[0078]

[0079] Conclusion: For every 1 °C deviation from 26 °C ± 1 °C in temperature, the cell attachment rate decreases by ≥ 6.1%, and the mitotic index decreases by ≥ 5.5% (p < 0.01).

[0080] 2. Single-factor experiment on culture time

[0081] Table 2

[0082]

[0083] Conclusion: The mitotic index reaches the peak at 12 hours of culture time, and both exceeding or falling short of this time significantly reduces it (p < 0.05).

[0084] 3. Single-factor experiment on colchicine concentration

[0085] Table 3

[0086]

[0087] Conclusion: When the colchicine concentration exceeds 10 μg / mL, the cell viability decreases significantly (p < 0.01).

[0088] In summary:

[0089] (1) When the temperature rises from 25°C to 26°C, the LDH activity (a marker of cell damage) decreases by 37%, and this phenomenon is not seen in the prior art.

[0090] (2) The 12-hour culture time is exactly at the peak of DNA replication (BrdU labeling rate of 89.2%), breaking through the misunderstanding of the traditional view that "the longer the better".

[0091] Experimental Example 2. Comparison between the present application and traditional methods

[0092] The method of the present application and the traditional method are listed and shown as follows.

[0093] Table 4

[0094] parameter method of the present invention traditional renal cell method significant difference pretreatment time 6 - 12 hours 72 hours p<0.001 chromosome integrity rate 92.3％ 67.8％ p<0.01 number of repetitions for single sampling ≥5 times 1 time p<0.001

[0095] As can be seen from Table 4, compared with the traditional method, the method of the present application has a significant improvement in the culture period, chromosome integrity rate, and the number of repeated samplings per time.

[0096] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0097] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0098] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing fish chromosomes, characterized in that, The method includes: Select fish scales in good growth condition, place them on cell slides and in culture medium after aseptic cleaning for pre-treatment culture of fish scales for 6 - 12 hours. The culture medium is DMEM medium containing 15% fetal bovine serum and 100 U / mL of double antibiotics. Induce cell synchronous division by colchicine treatment, use hypotonic combined with multiple Carnoy's fixative treatments, and finally obtain chromosome metaphases by Giemsa staining combined with the method of covering with nail polish in an inverted manner.

2. The method according to claim 1, characterized in that, The temperature for pre-treatment culture of the fish scales is 26°C.

3. The method according to claim 1, characterized in that, The concentration of the double-antibiotic gradient penetration system is: Initial soaking stage: penicillin 10000 U / mL + streptomycin 10000 U / mL, mixed with 1×PBS at a ratio of 1:10; culture stage: working concentration penicillin 100 U / mL + streptomycin 100 U / mL to maintain a sterile environment throughout the cell culture process.

4. The method according to claim 1, characterized in that, The steps of colchicine treatment include: Add colchicine with a final concentration of 10 μg / mL to the culture medium, and the action time is precisely controlled at 4 ± 0.5 hours.

5. The method according to claim 1, characterized in that, The hypotonic fixation treatment includes: Treat with 0.075 mol / L KCl solution at 20 - 22°C for 2.5 hours, and shake and mix evenly every 30 minutes. The fixative is methanol: glacial acetic acid = 3:1, and fix three times.

6. The method according to claim 1, wherein The steps of chromosome slide preparation include: When performing Giemsa staining, the pH value of the staining solution and phosphate buffer is 7.0 - 7.2, and the staining time is strictly controlled at 30 ± 2 minutes. The baking temperature is to heat at 2 cm away from the outer flame of the alcohol lamp for 5 seconds, and after natural cooling to room temperature, fix it on the glass slide.

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