A method for analyzing chromosome karyotype of crucian carp based on caudal fin fibroblast culture

Abstract

A crucian carp chromosome karyotype analysis method based on tail fin fibroblast culture, comprising: using a cell culture medium to perform primary culture and subculture of the tail fin fibroblasts, and performing chromosome karyotype analysis on the cultured tail fin fibroblasts. The present invention provides a crucian carp chromosome karyotype analysis method based on tail fin fibroblast culture, which can obtain more fibroblasts with good growth conditions, increase the number of cells in the division phase, and improve the proportion of cells in the metaphase division phase, so that the effect of crucian carp chromosome karyotype analysis is better.

Description

Technical Field

[0001] The invention relates to the technical field of chromosome karyotype analysis, and in particular to a crucian carp chromosome karyotype analysis method based on tail fin fibroblast culture. Background Art

[0002] Crucian carp belongs to the Cypriniformes, Cyprinidae, and Carassius genus. It is highly respected by the public for its tender meat, delicious taste, rich nutrition, and can be used as both food and medicine. It has high economic value. Crucian carp has strong adaptability and is easy to raise. Except for plateaus, it is almost found in rivers, lakes, ponds, reservoirs, swamps, ditches and other large and small water bodies. Crucian carp is one of the freshwater farmed fish with a steady and continuous increase in production, and occupies a very important position in freshwater aquaculture. For a long time, wild crucian carp has been considered a diploid population, but since the 1980s, triploids have been reported in wild crucian carp, such as high-back crucian carp, shrunken crucian carp, Pu'an crucian carp, and Pengze crucian carp. Therefore, chromosome karyotype analysis of indigenous crucian carp not only helps to understand the genetic composition, genetic variation rules and developmental mechanisms of organisms, but also has important reference value for predicting and identifying the results of interspecific hybridization and polyploid breeding, understanding the mechanism of sex inheritance, genome number, species origin, and evolutionary relationship. Summary of the invention

[0003] The object of the present invention is to provide a crucian carp chromosome karyotype analysis method based on tail fin fibroblast culture, which can obtain fibroblasts with good growth conditions, help increase the number of cells in the division phase, improve the ratio of cells in the metaphase division phase, and obtain more metaphase chromosomes in large quantities with uniform distribution and good morphology.

[0004] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0005] A method for analyzing chromosome karyotype of crucian carp based on tail fin fibroblast culture, comprising:

[0006] The tail fin fibroblasts are cultured using a cell culture medium, and the chromosomal karyotype analysis is performed on the cultured tail fin fibroblasts; the cell culture medium includes a primary culture medium and / or a passage culture medium, the cell culture medium includes a DMEM culture medium, and at least one of a cell nutrient and a cell culture additive; the cell culture additive includes at least a curcumin ferulate derivative and a xanthan gum succinate derivative, the curcumin ferulate derivative has a phenol group and an ester group, and the xanthan gum succinate derivative has a pyruvate group and an ester group. The curcumin ferulate derivative and the xanthan gum succinate derivative both have good in vitro antioxidant activity, can effectively remove a large amount of reactive oxygen generated in the external environment and during cell metabolism, protect cells from oxidative stress damage, and obtain fibroblasts with good growth conditions. The xanthan gum succinate derivative has a pyruvate group, which has a good promoting effect on the growth and proliferation of fibroblasts, helps to increase the number of cells in the division phase, and improves the ratio of cells in the metaphase division phase.

[0007] Preferably, the volume ratio of DMEM culture medium to cell nutrient is 1:0.1-1.0.

[0008] Preferably, the liquid-to-solid ratio of DMEM culture medium and cell culture additive is 1 mL: 0.01-0.10 g.

[0009] Preferably, the cell nutrients include fetal bovine serum, basic fibroblast growth factor and penicillin-streptomycin double antibody.

[0010] More preferably, the volume ratio of fetal bovine serum to basic fibroblast growth factor is 1:0.02-1.00.

[0011] More preferably, the volume ratio of fetal bovine serum to penicillin-streptomycin dual antibody is 1:0.02-1.00.

[0012] Preferably, the curcumin ferulate ester derivative is obtained by esterification reaction of ferulic acid and curcumin in a dicyclohexylcarbodiimide / 4-dimethylaminopyridine reaction system.

[0013] More preferably, the preparation method of curcumin ferulate derivatives is specifically,

[0014] Curcumin is added to N,N-dimethylformamide and stirred until dissolved, dicyclohexylcarbodiimide and 4-dimethylaminopyridine are added, and ferulic acid is added when the temperature is cooled to 2-10°C in an ice bath to continue the reaction, during which the temperature in the system is controlled to be 2-10°C. After the reaction is complete, N,N-dicyclohexylurea is removed by suction filtration, the obtained filtrate is rinsed and filtered with ice water, and the obtained crude product is eluted with an eluent, and separated and purified by column chromatography to obtain a ferulic acid curcumin ester derivative.

[0015] More preferably, the solid-to-liquid ratio of curcumin to N,N-dimethylformamide is 1 g:10-20 mL.

[0016] More preferably, the mass ratio of curcumin to dicyclohexylcarbodiimide is 1:1-2.

[0017] More preferably, the mass ratio of curcumin to 4-dimethylaminopyridine is 1:0.02-0.10.

[0018] More preferably, the mass ratio of curcumin to ferulic acid is 1:1-2.

[0019] More preferably, the eluent comprises ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether is 1:0.1-1.0.

[0020] Preferably, the xanthan gum succinate derivative is firstly obtained by degrading xanthan gum with hydrogen peroxide under alkaline conditions to obtain water-soluble xanthan gum oligosaccharides, and then undergoing an esterification reaction with succinic anhydride.

[0021] More preferably, the preparation method of xanthan gum succinate derivative is specifically,

[0022] Dissolve xanthan gum in sodium hydroxide solution, add hydrogen peroxide solution, degrade at 60-100°C for 2-10 days, adjust the pH to neutral with sodium hydroxide solution after cooling, filter with a microporous filter and a dialysis bag respectively, dialyze the reaction solution in deionized water, add succinic anhydride and acetone, heat to 20-60°C and stir for 2-6 hours, obtain a precipitate, wash with acetone for 2-5 times, and vacuum dry for 12-48 hours to obtain a xanthan gum succinate derivative.

[0023] More preferably, the solid-to-liquid ratio of xanthan gum to sodium hydroxide solution is 1 g: 20-100 mL.

[0024] More preferably, the concentration of the sodium hydroxide solution is 0.01-1.00 mol / L.

[0025] More preferably, the solid-liquid ratio of xanthan gum and hydrogen peroxide solution is 1 g: 1-2 mL.

[0026] More preferably, the mass fraction of the hydrogen peroxide solution is 10-50%.

[0027] More preferably, the mass ratio of xanthan gum to succinic anhydride is 1:0.01-0.10.

[0028] More preferably, the solid-liquid ratio of xanthan gum and acetone solution is 1 g: 1-2 mL.

[0029] Preferably, the cell culture additive comprises a curcumin ferulate ester derivative and a xanthan gum succinate ester derivative.

[0030] Preferably, the cell culture additive comprises a curcumin ferulate derivative, a xanthan gum succinate derivative and ethyl levulinate. Adding ethyl levulinate to the cell culture additive can obtain more fibroblasts with good growth conditions, further increase the number of cells in the division phase, increase the ratio of cells in the metaphase division phase, and obtain more metaphase chromosomes in a large number, evenly distributed and well-shaped.

[0031] More preferably, the mass ratio of the curcumin ferulate derivative to the xanthan gum succinate derivative is 1:1-2.

[0032] More preferably, the mass ratio of curcumin ferulate derivative to ethyl levulinate is 1:1-2.

[0033] Preferably, the primary culture of crucian carp caudal fin fibroblasts comprises:

[0034] Place the crucian carp in potassium permanganate solution for 20-40 minutes for disinfection, cut off 0.2-1.0cm 3 The caudal fin was sterilized with 75% ethanol for 10-30 seconds, rinsed with buffer 3-7 times, and the caudal fin was divided into 0.2-1.0 mm 3 The tissue blocks were washed with buffer for 3-7 times, the tail fin was inoculated in a 6-well plate, and water was absorbed as much as possible. The plate was placed in a carbon dioxide incubator and inverted for 10-60 min. A 0.25% trypsin solution was added for digestion for 5-30 min. The digestion was terminated with primary culture medium, the primary culture medium and trypsin solution were removed, the primary culture medium was added, and the plate was placed in a carbon dioxide incubator for culture, and half of the primary culture medium was replaced every 2 days.

[0035] More preferably, the mass fraction of the potassium permanganate solution is 0.01-1.00%.

[0036] More preferably, the buffer is prepared by mixing penicillin-streptomycin dual antibody and D-hanks buffer, and the volume ratio of penicillin-streptomycin dual antibody to D-hanks buffer is 1:20-100.

[0037] More preferably, the temperature of the carbon dioxide incubator is 25-28°C, and the mass fraction of carbon dioxide is 4-6%.

[0038] More preferably, the ratio of the tail fin tissue block to the trypsin solution is 1 mm 3 : 0.5-2.0mL.

[0039] More preferably, the ratio of the tail fin tissue block to the primary culture medium is 1 mm 3 : 1-2mL.

[0040] Preferably, the subculture of crucian carp caudal fin fibroblasts comprises:

[0041] When the primary caudal fin fibroblasts grow to 80-90% confluence, remove the primary culture medium, add buffer to rinse 1-3 times, remove the buffer, add 0.25% trypsin solution to digest for 5 minutes, add subculture medium to terminate digestion, centrifuge at 100-1000g for 2-4 minutes, remove the supernatant, resuspend the cells in subculture medium, inoculate into a new 6-well plate at a ratio of 1:3, and culture in a carbon dioxide incubator, replacing half of the culture medium every 2 days.

[0042] More preferably, the volume ratio of the trypsin solution to the subculture medium is 1:0.2-1.0.

[0043] Preferably, the crucian carp caudal fin cell karyotype analysis comprises:

[0044] 2×10 6 The crucian carp caudal fin fibroblasts were inoculated into T25 cell culture dishes. When the cells grew to 50-60% confluence, new subculture medium was replaced. When the cells grew to 70-80% confluence, colchicine was added for treatment for 3-6 hours. After removing the subculture medium, the buffer was added to rinse 1-3 times. The buffer was removed, and a 25% volume fraction of trypsin solution was added to digest for 2-10 minutes. The subculture medium was added to terminate the digestion, and the cells were centrifuged at 100-1000g for 2-5 minutes. The supernatant was removed, and potassium chloride solution was added to resuspend. The cells were hypotonic at 27°C for 10-60 minutes, and Carnoy's fixative was added for pre-fixation for 2-10 minutes. The cells were centrifuged at 100-2000g for 2-10 minutes, the supernatant was removed, and pre-cooled Carnoy's fixative was added. The precooling temperature is -20℃, gently resuspend the cells, fix at -20℃ for 10-60min, and then centrifuge at 100-2000g for 2-10min, remove the supernatant, add precooled Carnoy's fixative, the precooling temperature of Carnoy's fixative is -20℃, gently resuspend the cells, fix at -20℃ for 10-60min, and then centrifuge at 100-2000g for 2-10min, aspirate the supernatant, use a rubber-tipped dropper to gently resuspend the cells, drop the cell suspension onto the -20℃ cooled adhesion slide at a height of 0.5-1.5m, quickly blow it away, dry the slide at 37℃ for 10-60min, stain with Giemsa stain for 2-10min, gently wash off the stain, seal the slide with neutral resin and coverslip after observation, and photograph the karyotype under a 100x oil objective.

[0045] More preferably, the liquid-to-solid ratio of the subculture medium and colchicine is 1 mL: 0.5-5.0 μg.

[0046] More preferably, the volume ratio of the subculture medium to the trypsin solution is 1:0.2-2.0.

[0047] More preferably, the concentration of the potassium chloride solution is 0.01-0.10 mol / L.

[0048] More preferably, the volume ratio of the subculture medium to the potassium chloride solution is 1:1-2.

[0049] More preferably, the Carnoy fixative comprises methanol and acetic acid, and the volume ratio of methanol to acetic acid is 1:0.2-1.0.

[0050] More preferably, the volume ratio of the subculture medium to Carnoy's fixative is 1:0.01-5.00.

[0051] More preferably, the volume ratio of the subculture culture medium to the Giemsa staining solution is 1:0.2-2.0.

[0052] The invention also discloses a cell culture additive, which comprises a curcumin ferulate ester derivative and a xanthan gum succinate ester derivative.

[0053] The invention also discloses the use of the cell culture additive in chromosome karyotype analysis.

[0054] The present invention uses a subculture medium containing a cell culture additive to carry out subculture of crucian carp caudal fin fibroblasts, and the cell culture additive includes a curcumin ferulate ester derivative, a xanthan gum succinate derivative and ethyl levulinate, so that the present invention has the following beneficial effects: the curcumin ferulate ester derivative and the xanthan gum succinate derivative both have good in vitro antioxidant activity, can capture, neutralize and remove free radicals, effectively reduce the content of active oxygen in the external environment and cell metabolism, protect cells from oxidative stress damage, and obtain fibroblasts with good growth conditions. The xanthan gum succinate derivative has a pyruvic acid group, has a good promoting effect on the growth and proliferation of fibroblasts, helps to increase the number of cells in the division phase, and improves the ratio of cells in the metaphase division phase. Ethyl levulinate can further promote the growth and proliferation of cells, helps to obtain more cells in the metaphase division phase, thereby obtaining a large number of metaphase chromosomes with uniform distribution and good morphology. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The figure is the chromosome karyotype result of crucian carp obtained in Example 1. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0057] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0058] Embodiment 1:

[0059] S1. Primary culture of crucian carp caudal fin fibroblasts, including:

[0060] The crucian carp was placed in a 0.02% potassium permanganate solution for 30 min for disinfection. 3 The caudal fin was sterilized with 75% ethanol for 15 seconds and rinsed with buffer for 5 times. The caudal fin was then divided into 1 mm 3 The tissue blocks were rinsed with buffer for 5 times, the tail fins were inoculated in a 6-well plate, and water was absorbed as much as possible. The plates were placed in an inverted culture in a 27°C 5% carbon dioxide incubator for 30 min, 1 mL of 0.25% trypsin solution was added for digestion for 15 min, 1 mL of primary culture medium was added to terminate the digestion, the primary culture medium and trypsin solution were removed, 2 mL of primary culture medium was added, and the plates were placed in a carbon dioxide incubator for culture, and half of the culture medium was replaced every 2 days.

[0061] S2. Subculture of crucian carp caudal fin fibroblasts, including:

[0062] When the caudal fin fibroblasts grow to 80% confluence, remove the primary culture medium, add buffer to rinse once, remove the buffer, add 1 mL of 0.25% trypsin solution to digest for 5 minutes, add 1 mL of subculture medium to terminate digestion, centrifuge at 300g for 3 minutes, remove the supernatant, resuspend the cells in 4 mL of subculture medium, inoculate into a new 6-well plate at a ratio of 1:3, and culture in a carbon dioxide incubator, replacing half of the culture medium every 2 days.

[0063] S3. Karyotype analysis of crucian carp caudal fin cells, including:

[0064] 2×10 6The crucian carp caudal fin fibroblasts were inoculated into T25 cell culture dishes. When the cells grew to 50% confluence, 4 mL of new subculture medium was replaced. When the cells grew to 70% confluence, 4 μg of colchicine was added for treatment for 5 h. After removing the subculture medium, the buffer solution was added for rinsing once. The buffer solution was removed, 1 mL of 0.25% trypsin solution was added for digestion for 5 min, 1 mL of subculture medium was added to terminate the digestion, and the cells were centrifuged at 300 g for 3 min. The supernatant was removed, and 5 mL of 0.075 mol / L potassium chloride solution was added for resuspending. The cells were hypotonic at 27 °C for 25 min, 100 μL of Carnoy's fixative was added for pre-fixation for 5 min, and the cells were centrifuged at 1000 g for 5 min. The supernatant was removed, and 5 mL of pre-cooled Carnoy's fixative, the pre-cooling temperature of Carnoy's fixative is -20℃, gently resuspend the cells, fix at -20℃ for 30min, and then centrifuge at 1000g for 5min, remove the supernatant, add 5mL of pre-cooled Carnoy's fixative, the pre-cooling temperature of Carnoy's fixative is -20℃, gently resuspend the cells, fix at -20℃ for 30min, and then centrifuge at 1000g for 5min, aspirate 4mL of supernatant, use a rubber-tipped dropper to gently resuspend the cells, drop the cell suspension onto the adherent slide cooled at -20℃ at a height of 1m, quickly blow it away, dry the slide at 37℃ for 30min, stain with 1mL of Giemsa stain for 5min, gently wash off the stain, seal the slide with neutral resin and coverslip after observation, and photograph the karyotype under a 100x oil objective.

[0065] In this embodiment, the buffer solution is prepared by mixing D-hanks buffer and penicillin-streptomycin double antibody, and the volume ratio of penicillin-streptomycin double antibody to D-hanks buffer is 1:50; the primary culture medium includes the following components in volume: 15 parts of fetal bovine serum, 1 part of basic fibroblast growth factor with a concentration of 10 ng / mL, 1 part of penicillin-streptomycin double antibody and 83 parts of DMEM basic culture medium; the passage culture medium includes the following components in volume: 10 parts of fetal bovine serum, 1 part of basic fibroblast growth factor with a concentration of 10 ng / mL, 1 part of penicillin-streptomycin double antibody and 88 parts of DMEM basic culture medium. Carnoy's fixative is prepared by mixing methanol and acetic acid, and the volume ratio of methanol to acetic acid is 1:0.6. D-hanks buffer was purchased from Nanjing Shenghang Biotechnology Co., Ltd., penicillin-streptomycin double antibody solution was purchased from Wuhan Punosai Life Science Co., Ltd.; 25% trypsin solution was purchased from Beijing Biolab Technology Co., Ltd.; fetal bovine serum was purchased from SERANA; basic fibroblast growth factor was purchased from Shanghai Bio-Technology Co., Ltd.; DMEM basal medium was purchased from Bio-Inno Biotechnology Co., Ltd.; colchicine was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; and Giemsa staining solution was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0066] Embodiment 2:

[0067] Except that 0.5 parts by mass of curcumin ferulate ester derivative and 0.5 parts by mass of xanthan gum succinate derivative were additionally added to the subculture medium relative to the total amount of the subculture medium in Example 1, other conditions were the same as in Example 1.

[0068] S1. Preparation of curcumin ferulate derivatives, comprising:

[0069] 1 g of curcumin was added to 17 mL of N, N-dimethylformamide and stirred until dissolved, 1.2 g of dicyclohexylcarbodiimide and 0.07 g of 4-dimethylaminopyridine were added, and 1.05 g of ferulic acid was added when the temperature was cooled to 5°C in an ice bath to continue the reaction, during which the temperature in the system was controlled to be 5°C. After the reaction was complete, N, N-dicyclohexylurea was removed by suction filtration, and the obtained filtrate was rinsed and filtered with ice water. The obtained crude product was eluted with an eluent, and the eluent included ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether was 5:1. The ferulic acid curcumin ester derivative was separated and purified by column chromatography.

[0070] S2. Preparation of xanthan gum succinate derivatives, comprising:

[0071] 1 g of xanthan gum was dissolved in 55.6 mL of 0.01 mol / L sodium hydroxide solution, and 1.4 mL of hydrogen peroxide solution was added. The volume fraction of the hydrogen peroxide solution was 30%. The mixture was degraded at 80 °C for 5 days. After cooling, the pH was adjusted to neutral with 0.2 mol / L sodium hydroxide solution. The mixture was filtered using a microporous filter and a dialysis bag, respectively. The reaction solution was dialyzed in deionized water. 0.01 g of succinic anhydride and 1.4 mL of acetone were added. The mixture was heated to 40 °C and stirred for 4 hours. The precipitate was obtained and washed three times with acetone. The precipitate was dried in vacuo for 24 hours to obtain a xanthan gum succinate derivative.

[0072] Embodiment 3:

[0073] Except that the amount of the curcumin ferulate derivative used in the subculture medium was changed to 0.1 parts by mass relative to the total amount of the subculture medium in Example 1, other conditions were the same as in Example 2.

[0074] Embodiment 4:

[0075] Except that the amount of the xanthan gum succinate derivative used in the subculture medium was changed to 0.1 parts by mass relative to the total amount of the subculture medium in Example 1, other conditions were the same as in Example 2.

[0076] Embodiment 5:

[0077] The other conditions were the same as those in Example 1 except that 0.5 parts by mass of curcumin ferulate ester derivative, 0.5 parts by mass of xanthan gum succinate derivative and 0.5 parts by mass of ethyl levulinate were additionally added to the subculture medium relative to the total amount of the subculture medium in Example 1.

[0078] S1. Preparation of curcumin ferulate derivatives, same as in Example 2.

[0079] S2. Preparation of xanthan gum succinate derivatives, same as Example 2.

[0080] Embodiment 6:

[0081] Except that the amount of ethyl levulinate used in the subculture medium was changed to 0.1 parts by mass relative to the total amount of the subculture medium in Example 1, other conditions were the same as those in Example 5.

[0082] Comparative Example 1:

[0083] Except that 0.5 parts by mass of curcumin ferulate derivative relative to the total amount of the subculture medium in Example 1 was additionally added to the subculture medium, other conditions were the same as in Example 1.

[0084] S1. Preparation of curcumin ferulate derivatives, same as in Example 2.

[0085] Comparative Example 2:

[0086] Except that 0.5 parts by mass of xanthan gum succinate derivative was additionally added to the subculture medium relative to the total amount of the subculture medium in Example 1, other conditions were the same as in Example 1.

[0087] S1. Preparation of xanthan gum succinate derivatives, same as Example 2.

[0088] Comparative Example 3:

[0089] Except that 0.5 parts by mass of ethyl levulinate relative to the total amount of the subculture medium in Example 1 was additionally added to the subculture medium, other conditions were the same as in Example 1.

[0090] Experimental example:

[0091] 1. Chromosome karyotype analysis

[0092] The method of Example 1 was used to culture crucian carp caudal fin fibroblasts, and the chromosome karyotype of crucian carp was observed.

[0093] Figure 1 This is a result diagram of the crucian carp chromosome division phase obtained in Example 1. This shows that Example 1 of the present invention can clearly observe the crucian carp chromosome division phase by obtaining fibroblasts with good growth conditions.

[0094] 2. Number of cells in the division phase

[0095] The crucian carp caudal fin fibroblasts were cultured for 72 hours using the methods of Example 1-6 and Comparative Example 1-3, the crucian carp chromosome division phase was observed, and the number of cells in the division phase was calculated. The measurement results are shown in Table 1.

[0096] Table 1 Number of cells in the division phase (cells / slice)

[0097]

[0098] As shown in Table 1, compared with Example 1 and Example 2, the number of cells in the mitosis stage in Example 2 is more than that in Example 1, because in the passage medium, Example 2 additionally adds curcumin ferulate derivatives and xanthan gum succinate derivatives, which shows that the method of Example 2 can effectively increase the number of cells in the mitosis stage. Compared with Comparative Examples 1-2, the number of cells in the mitosis stage in Example 2 is more than that in Comparative Examples 1-2, because in the passage medium, Example 2 additionally adds curcumin ferulate derivatives and xanthan gum succinate derivatives, Comparative Example 1 only additionally adds curcumin ferulate derivatives, and Comparative Example 2 only additionally adds xanthan gum succinate derivatives; Compared with Examples 3-4, the number of cells in the mitosis stage in Example 2 is more than that in Example 3-4, because in the passage medium, the amount of curcumin ferulate derivatives and xanthan gum succinate derivatives used is different. This indicates that the synergistic addition of appropriate amounts of curcumin ferulate derivatives and xanthan gum succinate derivatives in the subculture culture medium helps to obtain more cells in the division stage.

[0099] Compared with Example 2 and Example 5, the number of cells in the mitosis stage in Example 5 is more than that in Example 2, because in the passage medium, Example 5 additionally adds curcumin ferulate derivatives, xanthan gum succinate derivatives and ethyl levulinate, and Example 2 only additionally adds curcumin ferulate derivatives and xanthan gum succinate derivatives; compared with Example 5 and Comparative Example 3, the number of cells in the mitosis stage in Example 5 is more than that in Comparative Example 3, because in the passage medium, Comparative Example 3 only additionally adds ethyl levulinate without adding curcumin ferulate derivatives and xanthan gum succinate derivatives; compared with Example 5 and Example 6, the number of cells in the mitosis stage in Example 5 is more than that in Example 6, because in the passage medium, the amount of ethyl levulinate used is different. This shows that adding ethyl levulinate to the passage medium can further increase the number of cells in the mitosis stage.

[0100] 3. The ratio of cells in metaphase

[0101] Crucian carp fibroblasts were cultured for 72 h using the methods of Examples 1-6 and Comparative Examples 1-3. The chromosomal karyotype of the fibroblasts was analyzed and the ratio of cells in the metaphase was calculated. The ratio of cells in the metaphase (%) = the number of cells in the metaphase / the number of cells in the mitotic phase × 100%. The measurement results are shown in Table 2.

[0102] Table 2 Proportion of cells in metaphase (%)

[0103]

[0104] As can be seen from Table 2, compared with Example 1 and Example 2, the ratio of cells in the metaphase phase in Example 2 is higher than that in Example 1, because in the passage medium, Example 2 additionally adds curcumin ferulate derivatives and xanthan gum succinate derivatives, which shows that the method of Example 2 can effectively increase the ratio of cells in the metaphase phase. Compared with Comparative Examples 1-2, the ratio of cells in the metaphase phase in Example 2 is higher than that in Comparative Examples 1-2, because in the passage medium, Example 2 additionally adds curcumin ferulate derivatives and xanthan gum succinate derivatives, Comparative Example 1 only additionally adds curcumin ferulate derivatives, and Comparative Example 2 only additionally adds xanthan gum succinate derivatives; compared with Examples 3-4, the ratio of cells in the metaphase phase in Example 2 is higher than that in Example 3-4, because in the passage medium, the amount of curcumin ferulate derivatives and xanthan gum succinate derivatives used is different. This indicates that the synergistic addition of appropriate amounts of curcumin ferulate derivatives and xanthan gum succinate derivatives in the subculture medium helps to obtain more cells in the metaphase phase.

[0105] Compared with Example 2 and Example 5, the ratio of cells in the metaphase phase in Example 5 is higher than that in Example 2, because in the passage medium, Example 5 additionally adds curcumin ferulate derivatives, xanthan gum succinate derivatives and ethyl levulinate, and Example 2 only additionally adds curcumin ferulate derivatives and xanthan gum succinate derivatives; compared with Example 5 and Comparative Example 3, the ratio of cells in the metaphase phase in Example 5 is higher than that in Comparative Example 3, because in the passage medium, Comparative Example 3 only additionally adds ethyl levulinate without adding curcumin ferulate derivatives and xanthan gum succinate derivatives; compared with Example 5 and Example 6, the ratio of cells in the metaphase phase in Example 5 is higher than that in Example 6, because the amount of ethyl levulinate used in the passage medium is different. This shows that adding ethyl levulinate in the passage medium can further increase the ratio of cells in the metaphase phase.

[0106] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0107] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for analyzing the chromosome karyotype of crucian carp based on the culture of caudal fin fibroblasts, comprising: using a cell culture medium to culture the caudal fin fibroblasts, and performing a chromosome karyotype analysis on the cultured caudal fin fibroblasts; the cell culture medium comprises a primary culture medium and / or a passage culture medium, the cell culture medium comprises a DMEM culture medium, and a cell culture additive; the cell culture additive comprises at least a curcumin ferulate derivative and a xanthan gum succinate derivative, the curcumin ferulate derivative having a phenol group and an ester group, the xanthan gum succinate The derivative has a pyruvic acid group and an ester group; the curcumin ferulic acid ester derivative is obtained by esterification reaction of ferulic acid and curcumin in a dicyclohexylcarbodiimide / 4-dimethylaminopyridine reaction system; the xanthan gum succinate derivative is firstly degraded by hydrogen peroxide under alkaline conditions to obtain water-soluble xanthan gum oligosaccharides, and then esterified with succinic anhydride; the liquid-to-solid ratio of the DMEM culture medium and the cell culture additive is 1 mL: 0.01-0.10 g; the mass ratio of the curcumin ferulic acid ester derivative and the xanthan gum succinate derivative is 1: 1-2.

2. The method for analyzing the chromosome karyotype of crucian carp based on tail fin fibroblast culture according to claim 1, characterized in that: The cell culture medium also contains cell nutrients, and the volume ratio of the DMEM culture medium to the cell nutrients is 1:0.1-1.

0.

3. The method for analyzing the chromosome karyotype of crucian carp based on tail fin fibroblast culture according to claim 2, characterized in that: The cell nutrient comprises fetal bovine serum, basic fibroblast growth factor and penicillin-streptomycin double antibody.

4. The method for analyzing the chromosome karyotype of crucian carp based on tail fin fibroblast culture according to claim 3, characterized in that: The volume ratio of the fetal bovine serum to the basic fibroblast growth factor is 1:0.02-1.

00.

5. The method for analyzing the chromosome karyotype of crucian carp based on tail fin fibroblast culture according to claim 3, characterized in that: The volume ratio of the fetal bovine serum to the penicillin-streptomycin dual antibody is 1:0.02-1.

00.

6. A crucian carp caudal fin fibroblast culture additive, comprising a curcumin ferulate ester derivative and a xanthan gum succinate derivative; the curcumin ferulate ester derivative is obtained by esterification reaction of ferulic acid and curcumin in a dicyclohexylcarbodiimide / 4-dimethylaminopyridine reaction system, and the xanthan gum succinate derivative is firstly obtained by degrading xanthan gum with hydrogen peroxide under alkaline conditions to obtain water-soluble xanthan gum oligosaccharides, and then undergoing esterification reaction with succinic anhydride; the mass ratio of the curcumin ferulate ester derivative to the xanthan gum succinate derivative is 1:1-2.

7. Use of the cell culture additive according to claim 6 in crucian carp chromosome karyotype analysis based on tail fin fibroblast culture.

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