CN102845335A - Dyeing method of yolk sacs and/or adipose cells and applications thereof - Google Patents
Dyeing method of yolk sacs and/or adipose cells and applications thereof Download PDFInfo
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Abstract
The invention relates to a dyeing method of yolk sacs and/or adipose cells, and particularly relates to a dyeing method of the yolk sacs and/or adipose cells of zebra fish, and a weight-reducing medicine screening model constructed by utilizing the method. Specially, resin dye is added in a culture solution for culturing juvenile fish of the zebra fish, the juvenile fish is incubated, the dyeing condition of the adipose cells of the zebra fish can be observed by a microscope, and the effect of the weight-reducing medicine can be judged according to the fluorescence intensity. The invention also relates to applications of the zebra fish used for screening the weight-reducing medicine. According to the method provided by the invention, the rapidness, intuition, flexibility and high efficiency can be realized, the high throughput screening can be conducted, and a new approach is provided for the construction of an animal model for evaluating the weight-reducing medicine.
Description
Technical field
The present invention relates to the colouring method of a kind of yolk sac and/or adipocyte, the particularly colouring method of a kind of zebra fish yolk sac and/or adipocyte, and the zebra fish slimming medicine screening model that utilizes the method to make up.
Background technology
Obesity has become one of hazards of human health, and along with the raising of material life condition, the adiposis incidence of disease is soaring year by year.Not only affect figure and activity, and cause easily the diseases such as hyperlipidemia, atherosclerotic, hypertension, coronary heart disease, diabetes.Suffer from obesity more than 10% in the whole world population according to incompletely statistics, developing country is particularly serious.The obesity crowd surpasses 30% in China's urban population, and Beijing surpasses 40%, and the trend of expansion is arranged.So prevention and obesity controlling are very urgent, the exploitation slimming medicine is imperative.Fat-reducing has become novel medical science problem.Slimming medicine, slim tea, diet food continue to bring out in recent years, and various fat-reducing researchs are like a raging fire.But the animal model of estimating the fat-reducing drug effect is very deficient, although there is high fat diet to make fat mouse model and trangenic mice model, it is under the undernatured state, and individual difference is very large, poor repeatability, and experimental data infers that by a large amount of error is very large.Develop a kind of can be intuitively as seen, experimental result need not infer in a large number that quick, directly perceived, sensitive, efficient, the high-throughout evaluation model that just can judge is very necessary.
Zebra fish is the vertebra model organism that development in recent years is got up.Zebra fish receives much attention with its unique advantage.The zebra fish oophyte is transparent, even in the advanced stage of growing, still can observe whole cells.Fluorescent staining or use other label can make cell-line observe clearlyer.Growth destiny that can not only each cell of tracing observation also can be observed the embryonic development situations such as the formation in cell movement, brain district of gastrul stage and heartbeat, and dysplastic mutant also is easy to be identified simultaneously.Embryonic development period, pattern of body form change is little, and full embryo tissue specimen manufacturing technology, and full embryo hybridization in situ technique can be implemented the embryo.Because the embryo is in vitro fertilization, ectogenesis, so can carry out independent operation to the embryo outside parent.And the mouse of normal operation belongs to placentalia, can not leave parent during embryo operation, just needs a large amount of inferences during experimental analysis, experimental result often with the physical presence different.In addition, the embryonic development of zebra fish very fast (hatching about 3d from being fertilized to), and also the embryo in same parent who comes grows synchronously, is easy to collect in a large number embryo's material same period of moment.
Therefore zebra fish has other biological unrivaled advantage, mainly comprise the following aspects: 1. the zebra fish nucleotide sequence analysis is finished substantially, and central nervous system, internal organs, blood and vision system have very high autoploidy with the people on molecular level; 2. the related gene of disease expression is identical with many vertebrates; 3. lay eggs the whole year, volume is little, egg laying amount is large, as the embryonic development model abundant research background is arranged; 4. use the zebra fish embryo that parent is had no effect, early stage health is transparent, can directly observe growth course; 5. zebrafish embryo and juvenile fish are very responsive to medicine, and denier will respond, and minimum detectable activity is in the ng/L level; 6. simple and easy to do, only need to be detected water or the fast injection that material is put into the cultivation embryo; Consumption seldom; Test period is short; The test specimens given figure can be very large, can be used as the high flux screening of drugs and toxicants, to guarantee the significance on the statistical significance.For this reason, OECD (OECD) with zebra fish classified as the standard fish that healthy toxicity and environmental toxicity detect (
Http:// www.oecd.org/).International Standards Organization (ISO) has been recommended as zebra fish river toxicity test fingerling and has formulated corresponding standard (ISO07346).
Because zebra fish has above-mentioned characteristic, if use it for drug screening, has a lot of advantages, but at present relevant report is arranged not yet.
Summary of the invention
The nutrition of zebra fish juvenile fish is that the yolk sac by fertilized egg provides, and has a large amount of fat in the yolk sac.The inventor is surprised to find that, just it can be dyeed by fatty dyestuff.Dyestuff is dissolved in the solution of raising the zebra fish juvenile fish jointly hatches, can observe its fat stains situation.Because above-mentioned discovery becomes possibility so that utilize it to set up the fat-reducing animal model, the present invention namely finishes based on this.
The inventor in the water environment of zebra fish life, not affecting in the healthy situation of zebra fish existence, utilizes fluorescence and/or other fatty dye markers and/or dyeing zebra fish fats cell through a large amount of studies confirm that, fluorescence is high-visible.Slimming medicine is pressed the finite concentration gradient add in the zebra fish juvenile fish feeding liquid, observe change in fluorescence.When medicine had antiobesity action, fluorescence can weaken or disappear, and did not have fat-reducing effect if fluorescence changes proof.Particularly,
One aspect of the present invention relates to the colouring method of a kind of zebra fish yolk sac and/or adipocyte, and it may further comprise the steps:
1) in the culture fluid of giving birth to the zebra fish juvenile fish, add fatty dyestuff, continue to cultivate; Randomly, in culture fluid, add Synthetic inhibitor of melanin, for example 1-phenyl-2-thiocarbamide;
2) randomly, in fluorescence microscopy Microscopic observation zebra fish yolk sac and/or adipocyte dyeing situation; Randomly, before observation, zebra fish is anaesthetized.
Another aspect of the present invention relates to a kind of method of screening slimming medicine, and it may further comprise the steps:
1) in the culture fluid of giving birth to the zebra fish juvenile fish, add fatty dyestuff, continue to cultivate; Randomly, in culture fluid, add Synthetic inhibitor of melanin, for example 1-phenyl-2-thiocarbamide;
2) 1) culture fluid in add slimming medicine to be measured, set up simultaneously the contrast that does not add the medicine of lose weight, continue cultivation; Randomly, in culture fluid, add Synthetic inhibitor of melanin, for example 1-phenyl-2-thiocarbamide;
3) zebra fish is taken out, in fluorescence microscopy Microscopic observation yolk sac and/or adipocyte dyeing situation; Randomly, before observation, zebra fish is anaesthetized; If compared with the control, the fluorescence intensity that the yolk sac that is colored and/or adipocyte produce weakens or disappears, and judges that then slimming medicine is effective.
In the present invention, the age of a fish of described zebra fish juvenile fish was preferably 0 day-10 days for hatching rear 0 day-15 days, for example be 0 day (namely hatching the rear same day), 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.In one embodiment of the invention, hatched rear 0 day for the zebra fish juvenile fish.
In the present invention, the time of continuing behind the fatty dyestuff of described adding to cultivate is 1 day-5 days, for example is 1 day-3 days, particularly, for example is 1 day, 2 days or 3 days; In one embodiment of the invention, continue to cultivate 3 days.
In the present invention, described continuation incubation time can be 1 day-5 days according to different slimming medicine adjustment behind the adding slimming medicine, for example is 2 days-4 days, particularly, for example is 2 days, 3 days or 4 days; In one embodiment of the invention, continue to cultivate 3 days.
Because the age of a fish of zebra fish juvenile fish after 15 days, other adipose tissue can occur in the juvenile fish body and disturb observation, therefore when fluorescence microscopy Microscopic observation fat stains situation, the age of a fish of preferred zebra fish juvenile fish was less than or equal to 15 days.
In the present invention, described fatty dyestuff refers to can be to the material of adipose tissue or cell dyeing, be generally lipid-soluble dye, it includes but not limited to oil red O, Nile red, Nile blue and the Sudan's class, and described the Sudan class dyestuff is selected from soudan III, Sudan IV and sudan black b; Its consumption can be adjusted according to different kind of dyes, can be 1-100ng/ml, for example is 10-50ng/ml, such as 10,20,30,40 or 50ng/ml; In embodiments of the invention, described fatty dyestuff is oil red O and Nile red, and its consumption is 1-50ng/ml, for example is 20ng/ml.
In the present invention, described Synthetic inhibitor of melanin can check melanin forms, and for example can form by suppressing the tyrosine hydroxylase check melanin, for example 1-phenyl-2-thiocarbamide or 30% hydrogen peroxide.In one embodiment of the invention, described Synthetic inhibitor of melanin is 1-phenyl-2-thiocarbamide.
In the present invention, 1-phenyl-2-thiocarbamide is dissolved in 0.3 * Danieau solution or the acetone soln, to increase solvability.Its working concentration is 10-50mg/L, and in embodiments of the invention, its concentration is 30mg/L.
In the present invention, describedly refer to observe after the match at light field or ultraviolet light at the fluorescence microscopy Microscopic observation, perhaps light field and ultraviolet Optical Field Superposition are observed.
Described light field refers to observe under the normal optical of fluorescence microscope, and described ultraviolet light field refers to observe under the ultraviolet excitation of fluorescence microscope, and described light field and ultraviolet Optical Field Superposition refer to observe under the normal optical stack of observing image under image and the ultraviolet excitation.
In the present invention, described fluorescence intensity weakens or disappears, refer to compare with the contrast with slimming medicine not, fluorescence intensity obviously weakens under the range estimation condition or disappears, perhaps by being calculated as below 50% of contrast, for example fluorescence intensity is 50%, 40%, 30%, 20%, 10% of contrast, or below 5%.
Wherein the computational methods of fluorescence intensity are for utilizing image analysis software that fluoroscopic image is analyzed, and calculate fluorescence intensity, for example utilize the CCD imaging software.In one embodiment of the invention, utilize NIKON, NIS-Elements BR software is analyzed.
Another aspect of the present invention relates to the purposes that dyeing of the present invention and observational technique are used for the screening slimming medicine.
Of the present inventionly relate in one aspect to again the purposes that zebra fish is used for the screening slimming medicine.
In the present invention, the composition of described culture fluid is the composition that is suitable for the zebra fish juvenile growth, is preferably the zebra fish special culture solution, for example is Holt Buffer of the present invention or E3 solution, and two kinds of solution all can doubly dilute use by 1-10.In embodiments of the invention, described culture fluid is Holt Buffer.
In the present invention, adopt anaesthetic anesthesia zebra fish, anaesthetic commonly used includes but not limited to Tricaine (tricaine) or barbiturates anaesthetic, and in one embodiment of the invention, anaesthetic is Tricaine.The working concentration of Tricaine is its typical concentrations, for example is the 0.016%-0.08% mass percent.In embodiments of the invention, its concentration is 0.048% mass percent.
In embodiments of the invention, utilize the method for zebra fish screening slimming medicine to be:
1) dyeing: squab zebra fish juvenile fish is cleaned with culture fluid, the dye liquor mother liquor that adds oil red O/ acetone soln (or Nile red/acetone soln), dilute with E3 (or Holt Buffer) solution by concentration gradient 1-50ng/ml, fully add 24 orifice plates behind the mixing.For the ease of observing, the PTU (1-Phenyl-2-thiourea, 1-phenyl-2-thiocarbamide) that can add 10-50mg/L prevents pigmentation.Change liquid every day once, for three days on end.
2) slimming medicine administration: press the slimming medicine mother liquor that finite concentration adds beforehand dilution in the dyeing liquor, fully mixing adds 24 orifice plates.Set up simultaneously the hole that does not add the medicine of losing weight to be contrast.For the ease of observing, the PTU that can add final concentration and be 10-50mg/L prevents pigmentation.Change liquid every day once, for three days on end.
3) dyeing and efficacy of medicine observing: zebra fish is taken out, with 0.016%-0.08% (mass percent) Tricaine anesthesia, in fluorescence microscopy Microscopic observation change in fluorescence.
4) result: give slimming medicine zebra fish yolk sac fluorescent weakening, and it is obvious not add the control group fluorescence of slimming medicine, illustrates that slimming medicine is effective.In order to make the easier observation of image, image can be taken pictures by CCD, carry out afterwards the image stack.
The beneficial effect of the invention
The present invention utilizes fatty dyestuff to dye to live body zebra fish adipocyte, judges slimming medicine to the effect of adipocyte by the variation of observing the adipocyte fluorescence intensity, has set up thus zebra fish slimming medicine evaluation model.The method just need not infer in a large number and can judge that it is quick, directly perceived, sensitive, efficient, and can carry out high flux screening to experimental result, the structure of estimating animal model for slimming medicine provides a brand-new approach.
Description of drawings
Fig. 1 microscopically is observed zebra fish yolk sac adipocyte, wherein
A：40×，B：100×，C：200×
Fig. 2 low concentration oil red O stain zebra fish yolk sac adipocyte, wherein
A: light field, B: ultraviolet light field, C:A and B stack
Fig. 3 high concentration oil red O stain zebra fish yolk sac adipocyte, wherein
A: light field, B: ultraviolet light field, C:A and B stack
Fig. 4 slimming medicine is on the impact (oil red O stain) of zebra fish yolk sac adipocyte, wherein
A, B, C are the change in fluorescence behind the adding slimming medicine, A: light field, B: ultraviolet light field, C:A and B stack
D, E, F be not for adding the positive control of slimming medicine, D: light field, E: ultraviolet light field, F:D and E stack
The yellow capsule adipocyte of Fig. 5 Nile red dyeing zebra fish-egg, wherein
A, B, C are the high concentration Nile red, A: light field, B: ultraviolet light field, C:A and B stack
D, E, F are the low concentration Nile red, D: light field, E: ultraviolet light field, F:D and E stack
Fig. 6 Nile red and oil red O compare zebra fish yolk sac adipocyte Color under same concentrations, wherein
A, B, C are oil red O stain, A: light field, B: ultraviolet light field, C:A and B stack
D, E, F are Nile red dyeing, D: light field, E: ultraviolet light field, F:D and E stack
Fig. 7 slimming medicine is on the impact (Nile red dyeing) of zebra fish yolk sac adipocyte, wherein
A, B, C are the change in fluorescence behind the adding slimming medicine, A: light field, B: ultraviolet light field, C:A and B stack
D, E, F be not for adding the positive control of slimming medicine, D: light field, E: ultraviolet light field, F:D and E stack
Wherein the multiplication factor of Fig. 2-Fig. 7 is 25 times.
Embodiment
Below in conjunction with embodiment embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person among the embodiment carries out according to the condition of normal condition or manufacturer's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Solution preparation:
E3:5mmol/L NaCl, 0.17mmol/L KCl, 0.33mmol/L CaCl
2And 0.33mmol/LMgSO
4, water is mended to final volume;
Holt Buffer:NaCl 3.5g, KCl 0.05g, NaHCO
40.025g, CaCl
20.1g water is mended to 1L;
Zebra fish anaesthetic Tricaine (sigma product) is dissolved in 0.4% mass percent and is mixed with 25 times of (25 *) mother liquors in the Holt Buffer solution;
0.3 * Danieau solution: 17mmol/L NaCl, 2mmol/L KCl, 0.12mmol/LMgSO
4, 1.8mmol/L Ca (NO
3) 2,1.5mmol/L HEPES water is mended to final volume;
Oil red O/ acetone soln takes by weighing oil red O, is dissolved in by 0.5mg/ml concentration and makes the dye liquor mother liquor in the acetone soln;
PTU solution is dissolved in the concentration of PTU powder (sigma product) by 0.001%-0.01% in 0.3 * Danieau liquid; Also PTU directly can be dissolved in the mother liquor that is mixed with 0.5mol/L in the acetone soln, sealing is for subsequent use under the room temperature.
Nile red/acetone soln takes by weighing Nile red, is dissolved in by 0.5mg/ml concentration and is mixed with Nile red/acetone soln mother liquor in the acetone soln.
It is pure that agents useful for same is analysis.
Slimming medicine: drive and give birth to hall slim tea ((capital medicine) defends food card word (2006) 110000-JSO046 numbers), the 3g/ bag, be soaked in 90 ℃ of left and right sides hot water of 100ml, every 1mlHolt Buffer culture fluid adds the tea of 1-100 μ l cooling after naturally cooling off, and what adopt among the present invention is that the 1ml culture fluid adds tea 10 μ l.
The fluoroscopic image analytical method: estimate and/or utilize NIKON, NIS-Elements BR software carries out the analysis of gray scale area scanning.
Generally, the fluorescent weakening degree varies will detect scanning to every fish, from totally weighing.The fish of taking out equal number before and after the administration detects scanning, compares after the calculating mean value.Generally the fluorescence intensity minimum should reach 500pixels before administration not.
Embodiment 1: zebra fish yolk sac adipocyte is observed
The nutrition of zebrafish embryo and juvenile fish is provided by yolk sac.Find in the experiment to have a large amount of fat in the yolk sac, microscopically is high-visible.
Zebra fish fertilized egg is obtained: select sexually matured zebra fish raun (zebra fish derives from Peking University's Life Science College), expand, be sunken to belly at the bottom of the fish jar and be reluctant that the raun that moves chooses, be put in the zebra fish oopod in 1: 1 ratio with milter, the morning pumping board, allow its fertilization of freely knocking into the back, fertilized egg was taken out in after fertilization 4-6 hour, washing, be put in the Holt Buffer of 24 orifice plates, 28.5 cultivate under ℃ condition, usually cultivate and to hatch in 48-72 hour, after hatching, be used for experiment.
3) observational technique: after the zebra fish juvenile fish hatches (usually at after fertilization 48-72 hour), with the juvenile fish sucking-off, anaesthetize with 3 * Tricaine solution, at fluorescence microscopy Microscopic observation zebra fish juvenile fish yolk sac, just can see under 40 times of mirrors indistinct vesica shape structure is arranged, more obvious under 100 times of mirrors, very clear under 200 times of mirrors.See Fig. 1.
Embodiment 2: utilize oil red O to dye to zebra fish juvenile fish adipocyte
1) dyeing: will clean with culture fluid (Holt Buffer) according to the squab zebra fish juvenile fish that embodiment 1 described method obtains, add oil red O/ acetone soln, it is that the dye liquor mother liquor is diluted with Holt Buffer solution by concentration gradient 1-50ng/ml (concrete concentration be 1,5,10,20,30,40,50ng/ml), fully adding 24 orifice plates behind the mixing.For the ease of observing, the PTU (sigma product) that adds 30mg/L prevents pigmentation.Change liquid every day once, for three days on end.
2) dyeing is observed: zebra fish is taken out, with the anesthesia of 3 * Tricaine (sigma product) solution, in fluorescence microscopy Microscopic observation change in fluorescence.
3) result: dyeing liquor high concentration (50ng/ml) is slightly darker than low concentration (20ng/ml) dyeing, and color is partially yellow.In order to make the easier observation of image, image is taken pictures by CCD, carry out afterwards the image stack, yolk sac place fluorescence is very obvious.Referring to Fig. 2 and Fig. 3.
Embodiment 3: zebra fish juvenile fish adipocyte is used for the slimming medicine evaluating drug effect with oil red O stain
1) dyeing: colouring method is with embodiment 2 steps 1), stin of thickness is 20ng/ml.
2) slimming medicine administration: press the slimming medicine mother liquor that finite concentration adds beforehand dilution in the dyeing liquor, fully mixing adds 24 orifice plates.Set up simultaneously the hole that does not add the medicine of losing weight to be contrast.For the ease of observing, the adding final concentration is that the PTU of 30mg/L prevents pigmentation.Change liquid every day once, for three days on end.Experimental group and control group respectively comprise 10-20 bar zebra fish.
3) dyeing and efficacy of medicine observing: zebra fish is taken out, with 3 * Tricaine anesthesia, in fluorescence microscopy Microscopic observation change in fluorescence.
Result: give slimming medicine zebra fish yolk sac fluorescent weakening, and it is obvious not add the control group fluorescence of slimming medicine, illustrates that slimming medicine is effective.In order to make the easier observation of image, image is taken pictures by CCD, carry out afterwards the image stack, see Fig. 4, the gray scale area scanning value of wherein scheming B is 53pixels, figure E is 556pixels.
Embodiment 4: utilize Nile red to compare to the dyeing of zebra fish juvenile fish adipocyte and with the oil red O stain effect
1) dyeing: the zebra fish juvenile fish that after fertilization is hatched is cleaned with culture fluid, add respectively the mother liquor of Nile red/acetone soln and the mother liquor of oil red O/ acetone soln by concentration gradient 1-50ng/ml (concrete concentration be 1,5,10,20,30,40,50ng/ml), dilute with Holt Buffer solution, fully add 24 orifice plates behind the mixing.For the ease of observing, the PTU that adds 30mg/L prevents pigmentation.Change liquid every day once, for three days on end.
2) coloration result: Nile red solution high concentration (50ng/ml) is slightly darker than low concentration (10ng/ml) color, but difference is not obvious.With oil red O solution and the Nile red solution-dyed of same concentrations, Nile red solution is darker than oil red O solution-dyed.In order to make the easier observation of image, image is taken pictures by CCD, carry out afterwards the image stack, the result is very obvious.See Fig. 5, Fig. 6.
Embodiment 5: zebra fish juvenile fish adipocyte is used for the slimming medicine evaluating drug effect with Nile red dyeing
1) colouring method of Nile red/acetone dyes dyeing: according to embodiment 4 steps 1), and stin of thickness is 10ng/ml.
2) slimming medicine administration: press the slimming medicine mother liquor that finite concentration adds beforehand dilution in the dyeing liquor, dilute with Holt Buffer solution, fully mixing adds 24 orifice plates.Set up simultaneously the hole that does not add the medicine of losing weight to be contrast.For the ease of observing, the PTU that adds 30mg/L prevents pigmentation.Change liquid every day once, for three days on end.Experimental group and control group respectively comprise 10-20 bar zebra fish.
4) dyeing and efficacy of medicine observing: zebra fish is taken out, with 3 * Tricaine anesthesia, in fluorescence microscopy Microscopic observation change in fluorescence.
Result: give slimming medicine zebra fish yolk sac fluorescent weakening, and it is obvious not add the control group fluorescence of slimming medicine, illustrates that slimming medicine is effective.In order to make the easier observation of image, image is taken pictures by CCD, carry out afterwards the image stack, see Fig. 7, the gray scale area scanning value of wherein scheming B is 0pixels, figure E is 841pixels.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (9)
1. the colouring method of a zebra fish yolk sac and/or adipocyte, it may further comprise the steps:
1) in the culture fluid of giving birth to the zebra fish juvenile fish, add fatty dyestuff, continue to cultivate; Randomly, add simultaneously Synthetic inhibitor of melanin, for example 1-phenyl-2-thiocarbamide;
2) randomly, in fluorescence microscopy Microscopic observation zebra fish yolk sac and/or adipocyte dyeing situation; Randomly, before observation, zebra fish is anaesthetized.
2. method of screening slimming medicine, it may further comprise the steps:
1) in the culture fluid of giving birth to the zebra fish juvenile fish, add fatty dyestuff, continue to cultivate; Randomly, add simultaneously Synthetic inhibitor of melanin, for example 1-phenyl-2-thiocarbamide;
2) 1) culture fluid in add slimming medicine to be measured, set up simultaneously the contrast that does not add the medicine of lose weight, continue cultivation; Randomly, add simultaneously Synthetic inhibitor of melanin, for example 1-phenyl-2-thiocarbamide;
3) zebra fish is taken out, in fluorescence microscopy Microscopic observation yolk sac and/or adipocyte dyeing situation; Randomly, before observation, zebra fish is anaesthetized; If compared with the control, the fluorescence intensity that the yolk sac that is colored and/or adipocyte produce weakens or disappears, and judges that then slimming medicine is effective.
3. claim 1 or 2 method, the age of a fish of wherein said zebra fish juvenile fish was preferably 0 day-10 days for hatching rear 0 day-15 days.
4. claim 1 or 2 method, wherein step 1) described in the continuation incubation time be 1 day-5 days, for example be 1-3 days.
5. the method for claim 2, wherein step 2) described in the continuation incubation time be 1 day-5 days, for example be 2-4 days.
6. claim 1 or 2 method, wherein said fatty dyestuff is selected from oil red O, Nile red, Nile blue and the Sudan's class, and described the Sudan class dyestuff is selected from soudan III, Sudan IV and sudan black b, is preferably oil red O and Nile red.
7. claim 1 or 2 method wherein saidly refer to observe after the match at light field or ultraviolet light at the fluorescence microscopy Microscopic observation, perhaps light field and ultraviolet Optical Field Superposition are observed.
8. the method for claim 1 is used for the purposes of screening slimming medicine.
9. zebra fish is used for the purposes of screening slimming medicine.
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| CN103301480A (en) * | 2013-05-15 | 2013-09-18 | 杭州环特生物科技有限公司 | Method of creating zebra fish peristalsis model and screening prokinetic medicaments |
| CN104297222A (en) * | 2014-10-16 | 2015-01-21 | 漳州片仔癀药业股份有限公司 | Zebrafish embryo alcoholic liver detecting model and construction method and application of zebrafish embryo alcoholic liver detecting model |
| CN108651337A (en) * | 2018-05-15 | 2018-10-16 | 江西省水产科学研究所 | A kind of production Drifting egg fish early stage different developmental phases form microscopy observation method |
| CN112147288A (en) * | 2020-08-31 | 2020-12-29 | 南京新环检测科技有限公司 | Method for evaluating weight-losing function of health food |
| CN114807224A (en) * | 2022-04-25 | 2022-07-29 | 中国人民解放军军事科学院军事医学研究院 | Construction method and application of visual zebra fish bile acid metabolism model |
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| CN103301480B (en) * | 2013-05-15 | 2019-11-08 | 杭州环特生物科技股份有限公司 | It establishes zebra fish enterocinesia model and screening promotees the method for gastroenteritic power drug |
| CN104297222A (en) * | 2014-10-16 | 2015-01-21 | 漳州片仔癀药业股份有限公司 | Zebrafish embryo alcoholic liver detecting model and construction method and application of zebrafish embryo alcoholic liver detecting model |
| CN108651337A (en) * | 2018-05-15 | 2018-10-16 | 江西省水产科学研究所 | A kind of production Drifting egg fish early stage different developmental phases form microscopy observation method |
| CN112147288A (en) * | 2020-08-31 | 2020-12-29 | 南京新环检测科技有限公司 | Method for evaluating weight-losing function of health food |
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| CN114807224B (en) * | 2022-04-25 | 2023-09-29 | 中国人民解放军军事科学院军事医学研究院 | Construction method and application of visual zebra fish bile acid metabolism model |
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