CN102845335B - 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, particularly the colouring method of a kind of zebra fish yolk sac and/or adipocyte, and the zebra fish slimming medicine screening model utilizing the method to build.
Background technology
Fat one of hazards becoming human health, along with the raising of material life condition, the adiposis incidence of disease rises year by year.Not only affect figure and activity, and easily cause the diseases such as hyperlipidemia, atherosclerotic, hypertension, coronary heart disease, diabetes.More than 10% suffer from obesity in world population according to incompletely statistics, developing country is particularly serious.In China's urban population, obesity crowd is more than 30%, and more than 40%, and there is the trend of expansion in Beijing.So prevention and corntrol is fat, very urgent, 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 research is like a raging fire.But the animal model evaluating fat-reducing drug effect is very deficient, although there is high fat diet to make fat mouse model and transgenic mouse model, it is under undernatured state, and individual difference is very large, poor repeatability, experimental data is inferred by a large amount of, and error is very large.Exploitation one can be intuitively visible, and experimental result need not infer that quick, directly perceived, sensitive, efficient, the high-throughout evaluation model that just can judge is very necessary in a large number.
Zebra fish is the vertebra model organism that development in recent years is got up.Zebra fish receives much attention with the advantage of its uniqueness.Zebra fish oophyte is transparent, even if the advanced stage of growing, still can observe whole cells.Fluorescent staining or use other label that cell-line can be made to observe clearer.Can not only the growth destiny of each cell of tracing observation, also can be observed the embryonic development situations such as the cell movement of gastrul stage, the formation in brain district and heartbeat, dysplastic mutant is also easy to be identified simultaneously.Embryonic development period, pattern of body form change is little, full embryo tissue specimen manufacturing technology, and full embryo hybridization in situ technique, can implement on embryo.Due to embryo be fertilized in vitro, ectogenesis, therefore independent operation can be carried out to embryo parent is outer.And the mouse generally used belongs to placentalia, during embryo operation, can not parent be left, during experimental analysis, just need a large amount of inference, experimental result often with physical presence different.In addition, the embryonic development of zebra fish is very fast (hatching about 3d from by precise and penetrating), and the embryo in same parent come is synchronous growth, is easy to the fetal material same period collecting moment in a large number.
Therefore zebra fish has other biological unrivaled advantage, mainly comprise the following aspects: 1. zebra fish nucleotide sequence analysis completes substantially, central nervous system, internal organs, blood and vision system have very high autoploidy with people on a molecular scale; 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, have abundant research background as embryonic development model; 4. use zebra fish embryo to have no effect to parent, early stage health is transparent, directly can observe growth course; 5. zebrafish embryo and juvenile fish very responsive to medicine, denier will respond, and minimum detectable activity is in ng/L level; 6. simple and easy to do, only tested substance need be put into water or the fast injection of cultivation embryo; Consumption is little; Test period is short; Test sample book number can be very large, can as the high flux screening of drugs and toxicants, to guarantee the significance on statistical significance.For this reason, OECD (OECD) zebra fish has been classified as standard fish that healthy toxicity and environmental toxicity detect (
http:// www.oecd.org/).Zebra fish has been recommended as river toxicity test fingerling and has formulated corresponding standard (ISO07346) by International Standards Organization (ISO).
Because zebra fish has above-mentioned characteristic, if use it for drug screening, have a lot of advantage, but not yet have relevant report at present.
Summary of the invention
The nutrition of zebra fish juvenile fish is provided by the yolk sac of fertilized egg, there is a large amount of fat in yolk sac.Inventor is surprised to find that, just can be dyeed by fatty dyestuff.Dyestuff is dissolved in the solution of raising zebra fish juvenile fish and jointly hatches, its fat stains situation can be observed.Due to above-mentioned discovery, make to utilize it to set up fat-reducing animal model to become possibility, namely the present invention completes based on this.
Inventor confirms through large quantifier elimination, and in the water environment of zebra fish life, when not affecting zebra fish existence and being healthy, utilize fluorescence and/or other fatty dye markers and/or dyeing zebra fish fats cell, fluorescence is high-visible.Slimming medicine is added in zebra fish juvenile fish feeding liquid by finite concentration gradient, observes change in fluorescence.When medicine has antiobesity action, fluorescence can weaken or disappear, if fluorescence does not change proof do not have fat-reducing effect.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 comprises the following steps:
1) in the culture fluid of giving birth to zebra fish juvenile fish, add fatty dyestuff, continue to cultivate; Optionally, in culture fluid, add Synthetic inhibitor of melanin, such as 1-phenyl-2-thiocarbamide;
2) optionally, at fluorescence microscopy Microscopic observation zebra fish yolk sac and/or adipocyte staining conditions; Optionally, before observation, zebra fish is anaesthetized.
Another aspect of the present invention relates to a kind of method of screening slimming medicine, and it comprises the following steps:
1) in the culture fluid of giving birth to zebra fish juvenile fish, add fatty dyestuff, continue to cultivate; Optionally, in culture fluid, add Synthetic inhibitor of melanin, such as 1-phenyl-2-thiocarbamide;
2) 1) culture fluid in add slimming medicine to be measured, set up the contrast not adding slimming medicine simultaneously, continue cultivate; Optionally, in culture fluid, add Synthetic inhibitor of melanin, such as 1-phenyl-2-thiocarbamide;
3) zebra fish is taken out, at fluorescence microscopy Microscopic observation yolk sac and/or adipocyte staining conditions; Optionally, before observation, zebra fish is anaesthetized; If compared with the control, the fluorescence intensity that the yolk sac be colored and/or adipocyte produce weakens or disappears, then judge that slimming medicine is effective.
In the present invention, the age of a fish of described zebra fish juvenile fish for hatching latter 0 day-15 days, be preferably 0 day-10 days, be such as 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, for zebra fish juvenile fish hatches latter 0 day.
In the present invention, described add fatty dyestuff after time of continuing to cultivate be 1 day-5 days, being such as 1 day-3 days, particularly, such as, is 1 day, 2 days or 3 days; In one embodiment of the invention, cultivation 3 days is continued.
In the present invention, continuation incubation time described after adding slimming medicine can adjust according to different slimming medicine, and can be 1 day-5 days, such as, be 2 days-4 days, particularly, such as, is 2 days, 3 days or 4 days; In one embodiment of the invention, cultivation 3 days is continued.
Due to zebra fish juvenile fish the age of a fish after 15 days, there will be other adipose tissue in 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 is less than or equal to 15 days.
In the present invention, described fatty dyestuff refers to can 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 adjust according to different kind of dyes, can be 1-100ng/ml, such as, be 10-50ng/ml, 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, such as, be 20ng/ml.
In the present invention, described Synthetic inhibitor of melanin can be formed by check melanin, such as, can be formed by suppressing tyrosine hydroxylase check melanin, such as 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 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, described referring at fluorescence microscopy Microscopic observation is observed after the match at light field or ultraviolet light, or light field and ultraviolet Optical Field Superposition is observed.
Described light field refers to be observed under the common light of fluorescence microscope, and described ultraviolet light field refers to observes under the ultraviolet excitation of fluorescence microscope, and described light field and ultraviolet Optical Field Superposition refer to be observed image under common light and observe superposing of image under ultraviolet excitation.
In the present invention, described fluorescence intensity weakens or disappears, refer to compared with not using the contrast of slimming medicine, fluorescence intensity obviously weakens or disappears under range estimation condition, or by be calculated as contrast less than 50%, such as fluorescence intensity is 50%, 40%, 30%, 20%, 10% of contrast, or less than 5%.
Wherein the computational methods of fluorescence intensity are analyzed fluoroscopic image for utilizing image analysis software, and calculate fluorescence intensity, such as, utilize 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 dyeing of the present invention and observational technique for screening the purposes of slimming medicine.
Another aspect of the invention relates to zebra fish for screening the purposes of slimming medicine.
In the present invention, the composition of described culture fluid is the composition being suitable for zebra fish juvenile growth, and be preferably zebra fish special culture solution, be such as Holt Buffer of the present invention or E3 solution, two kinds of solution all doubly can 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, conventional anaesthetic 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, such as, be 0.016%-0.08% mass percent.In embodiments of the invention, its concentration is 0.048% mass percent.
In embodiments of the invention, the method utilizing zebra fish to screen slimming medicine is:
1) dye: squab zebra fish juvenile fish culture fluid is cleaned, add the dye liquor mother liquor of oil red O/ acetone soln (or Nile red/acetone soln), dilute by concentration gradient 1-50ng/ml E3 (or Holt Buffer) solution, fully add 24 orifice plates after 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: the slimming medicine mother liquor adding beforehand dilution in dyeing liquor by finite concentration, fully mixing adds 24 orifice plates.Set up the hole not adding slimming medicine to be contrast simultaneously.For the ease of observing, can add final concentration is that the PTU of 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, anaesthetizes, in fluorescence microscopy Microscopic observation change in fluorescence with 0.016%-0.08% (mass percent) Tricaine.
4) result: give slimming medicine zebra fish yolk sac fluorescent weakening, and the control group fluorescence not adding slimming medicine is obvious, illustrates that slimming medicine is effective.In order to make image more easily observe, image can be taken pictures by CCD, carrying out imaging importing afterwards.
The beneficial effect of the invention
The present invention utilizes fatty dyestuff can dye to live body zebra fish adipocyte, judges the effect of slimming medicine to adipocyte, thereby establish zebra fish slimming medicine evaluation model by the change observing adipocyte fluorescence intensity.The method need not be inferred in a large number experimental result and just can judge, it is quick, directly perceived, sensitive, efficient, and can carry out high flux screening, and the structure for slimming medicine evaluation animal model provides a brand-new approach.
Accompanying drawing explanation
Fig. 1 basis of microscopic observation 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 superposes
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 superposes
Fig. 4 slimming medicine on the impact (oil red O stain) of zebra fish yolk sac adipocyte, wherein
A, B, C are the change in fluorescence after adding slimming medicine, A: light field, B: ultraviolet light field, C:A and B superposes
D, E, F are the positive control not adding slimming medicine, D: light field, E: ultraviolet light field, F:D and E superposes
The yellow capsule adipocyte of Fig. 5 Nile red dyeing zebra fish-egg, wherein
A, B, C are high concentration Nile red, A: light field, B: ultraviolet light field, C:A and B superposes
D, E, F are low concentration Nile red, D: light field, E: ultraviolet light field, F:D and E superposes
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 superposes
D, E, F are Nile red dyeing, and D: light field, E: ultraviolet light field, F:D and E superposes
Fig. 7 slimming medicine on the impact (Nile red dyeing) of zebra fish yolk sac adipocyte, wherein
A, B, C are the change in fluorescence after adding slimming medicine, A: light field, B: ultraviolet light field, C:A and B superposes
D, E, F are the positive control not adding slimming medicine, D: light field, E: ultraviolet light field, F:D and E superposes
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 for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturer suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can 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, mend to final volume with water;
Holt Buffer:NaCl 3.5g, KCl 0.05g, NaHCO
40.025g, CaCl
20.1g, mends to 1L with water;
Zebra fish anaesthetic Tricaine (sigma product), is dissolved in HoltBuffer solution with 0.4% mass percent and is mixed with 25 times of (25 ×) mother liquors;
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 use water mend to final volume;
Oil red O/ acetone soln, takes oil red O, is dissolved in acetone soln makes dye liquor mother liquor by 0.5mg/ml concentration;
PTU solution, is dissolved in 0.3 × Danieau liquid by PTU powder (sigma product) by the concentration of 0.001%-0.01%; Also PTU directly can be dissolved in acetone soln the mother liquor being mixed with 0.5mol/L, close for subsequent use under room temperature.
Nile red/acetone soln, takes Nile red, is dissolved in acetone soln is mixed with Nile red/acetone soln mother liquor by 0.5mg/ml concentration.
It is pure that agents useful for same is analysis.
Slimming medicine: imperial raw hall slim tea ((capital medicine) defends food card No. 110000-JSO046th, word (2006)), 3g/ bag, be soaked in 100ml about 90 DEG C hot water, after naturally cooling, every 1mlHolt Buffer culture fluid adds the tea that 1-100 μ l cools, and what adopt in the present invention is that 1ml culture fluid adds tea 10 μ l.
Fluorescence image analysis method: estimate and/or utilize NIKON, NIS-Elements BR software carries out the analysis of gray scale area scanning.
Under normal circumstances, fluorescent weakening degree varies, will carry out detection scanning, from totally weighing to every bar fish.The fish of taking out equal number before and after administration carries out detection scanning, compares after calculating mean value.Generally before non-administration, fluorescence intensity is minimum should reach 500pixels.
Embodiment 1: zebra fish yolk sac adipocyte is observed
The nutrition of zebrafish embryo and juvenile fish is provided by yolk sac.Find to there is a large amount of fat in yolk sac in experiment, high-visible under microscope.
Zebra fish fertilized egg obtains: select sexually matured zebra fish raun (zebra fish derives from Peking University's Life Science College), belly is expanded, to be sunken at the bottom of fish jar and to be reluctant that the raun moved is chosen, be put in zebra fish oopod with milter in 1: 1 ratio, 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, cultivate under 28.5 DEG C of conditions, usual cultivation 48-72 hour can be hatched, for experiment after hatching.
3) observational technique: after zebra fish juvenile fish hatches (usually at after fertilization 48-72 hour), by juvenile fish sucking-off, anaesthetize with 3 × Tricaine solution, at fluorescence microscopy Microscopic observation zebra fish juvenile fish yolk sac, just can see there is indistinct capsule balloon-shaped structure under 40 times of mirrors, comparatively 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) dye: the squab zebra fish juvenile fish culture fluid (Holt Buffer) obtained according to method described in embodiment 1 is cleaned, add oil red O/ acetone soln, its be by dye liquor mother liquor by concentration gradient 1-50ng/ml (concrete concentration is 1,5,10,20,30,40,50ng/ml) dilute with Holt Buffer solution, fully add 24 orifice plates after mixing.For the ease of observing, the PTU (sigma product) adding 30mg/L prevents pigmentation.Change liquid every day once, for three days on end.
2) dyeing is observed: taken out by zebra fish, 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 image more easily observe, being taken pictures by image by CCD, carrying out imaging importing afterwards, yolk sac place fluorescence clearly.See Fig. 2 and Fig. 3.
Embodiment 3: zebra fish juvenile fish adipocyte oil red O stain is used for slimming medicine evaluating drug effect
1) dye: colouring method is with embodiment 2 step 1), stin of thickness is 20ng/ml.
2) slimming medicine administration: the slimming medicine mother liquor adding beforehand dilution in dyeing liquor by finite concentration, fully mixing adds 24 orifice plates.Set up the hole not adding slimming medicine to be contrast simultaneously.For the ease of observing, 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: taken out by zebra fish, with 3 × Tricaine anesthesia, in fluorescence microscopy Microscopic observation change in fluorescence.
Result: give slimming medicine zebra fish yolk sac fluorescent weakening, and the control group fluorescence not adding slimming medicine is obvious, illustrates that slimming medicine is effective.In order to make image more easily observe, being taken pictures by image by CCD, carrying out imaging importing afterwards, seeing Fig. 4, the gray scale area scanning value wherein scheming B is 53pixels, and figure E is 556pixels.
Embodiment 4: utilize Nile red to zebra fish juvenile fish adipocyte dyeing and with oil red O stain effectiveness comparison
1) dye: the zebra fish juvenile fish culture fluid of being hatched by after fertilization is cleaned, by concentration gradient 1-50ng/ml (concrete concentration is 1,5,10,20,30,40,50ng/ml) add the mother liquor of Nile red/acetone soln and the mother liquor of oil red O/ acetone soln respectively, dilute with Holt Buffer solution, fully add 24 orifice plates after mixing.For the ease of observing, the PTU adding 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 image more easily observe, being taken pictures by image by CCD, carrying out imaging importing afterwards, result clearly.See Fig. 5, Fig. 6.
Embodiment 5: the Nile red dyeing of zebra fish juvenile fish adipocyte is used for slimming medicine evaluating drug effect
1) dye: according to embodiment 4 step 1) in the colouring method of Nile red/acetone dye, stin of thickness is 10ng/ml.
2) slimming medicine administration: the slimming medicine mother liquor adding beforehand dilution in dyeing liquor by finite concentration, dilutes with Holt Buffer solution, and fully mixing adds 24 orifice plates.Set up the hole not adding slimming medicine to be contrast simultaneously.For the ease of observing, the PTU adding 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: taken out by zebra fish, with 3 × Tricaine anesthesia, in fluorescence microscopy Microscopic observation change in fluorescence.
Result: give slimming medicine zebra fish yolk sac fluorescent weakening, and the control group fluorescence not adding slimming medicine is obvious, illustrates that slimming medicine is effective.In order to make image more easily observe, being taken pictures by image by CCD, carrying out imaging importing afterwards, seeing Fig. 7, the gray scale area scanning value wherein scheming B is 0pixels, and 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 amendment 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 (25)
1. a colouring method for zebra fish yolk sac and/or adipocyte, it comprises the following steps:
1) in the culture fluid of giving birth to zebra fish juvenile fish, add fatty dyestuff, continue to cultivate;
2) at fluorescence microscopy Microscopic observation zebra fish yolk sac and/or adipocyte staining conditions; Before observation, zebra fish is anaesthetized.
2. the colouring method of claim 1, wherein in step 1) in add Synthetic inhibitor of melanin simultaneously.
3. the colouring method of claim 1, the age of a fish of wherein said zebra fish juvenile fish is for hatching latter 0 day-15 days.
4. the colouring method of claim 1, the age of a fish of wherein said zebra fish juvenile fish is for hatching latter 0 day-10 days.
5. the colouring method of claim 1, wherein step 1) described in continuation incubation time be 1 day-5 days.
6. the colouring method of claim 1, wherein step 1) described in continuation incubation time be 1-3 days.
7. the colouring method of claim 1, 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 oil red, S Ⅳ and sudan black b.
8. the colouring method of claim 7, wherein said fatty dyestuff is selected from oil red O and Nile red.
9. the colouring method of claim 2, wherein said Synthetic inhibitor of melanin is 1-phenyl-2-thiocarbamide.
10. the colouring method of claim 1, wherein said referring at fluorescence microscopy Microscopic observation is observed after the match at light field or ultraviolet light, or light field and ultraviolet Optical Field Superposition is observed.
11. 1 kinds of methods of screening slimming medicine, it comprises the following steps:
1) in the culture fluid of giving birth to zebra fish juvenile fish, add fatty dyestuff, continue to cultivate;
2) 1) culture fluid in add slimming medicine to be measured, set up the contrast not adding slimming medicine simultaneously, continue cultivate;
3) zebra fish is taken out, at fluorescence microscopy Microscopic observation yolk sac and/or adipocyte staining conditions; If compared with the control, the fluorescence intensity that the yolk sac be colored and/or adipocyte produce weakens or disappears, then judge that slimming medicine is effective.
The method of 12. claims 11, wherein in step 1) in add Synthetic inhibitor of melanin simultaneously.
The method of 13. claims 11, wherein in step 2) in add Synthetic inhibitor of melanin simultaneously.
The method of 14. claims 11, wherein step 3) in before observation, zebra fish is anaesthetized.
The method of 15. claims 11, the age of a fish of wherein said zebra fish juvenile fish is for hatching latter 0 day-15 days.
The method of 16. claims 11, the age of a fish of wherein said zebra fish juvenile fish is for hatching latter 0 day-10 days.
The method of 17. claims 11, wherein step 1) described in continuation incubation time be 1 day-5 days.
The method of 18. claims 11, wherein step 1) described in continuation incubation time be 1-3 days.
The method of 19. claims 11, wherein step 2) described in continuation incubation time be 1 day-5 days.
The method of 20. claims 11, wherein step 2) described in continuation incubation time be 2-4 days.
The method of 21. claims 11, 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 oil red, S Ⅳ and sudan black b.
The method of 22. claims 21, wherein said fatty dyestuff is selected from oil red O and Nile red.
The method of 23. claims 12 or 13, wherein said Synthetic inhibitor of melanin is 1-phenyl-2-thiocarbamide.
The method of 24. claims 11, wherein said referring at fluorescence microscopy Microscopic observation is observed after the match at light field or ultraviolet light, or light field and ultraviolet Optical Field Superposition is observed.
The colouring method of 25. any one of claim 1-10 is for screening the purposes of slimming medicine.
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| US5932418A (en) * | 1996-04-08 | 1999-08-03 | Naiad Systems, Inc. | Fish embryo screening test for genotoxic agents using three different developmental life stages |
| WO2004044241A2 (en) * | 2002-11-13 | 2004-05-27 | DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung | Use of fish larvae as a screening model |
| CN1866025A (en) * | 2006-06-01 | 2006-11-22 | 中国科学院海洋研究所 | Method for detecting activity of anti-angiogenesis protein factor or pro-angiogenesis protein factor via zebra fish embryo model and application method thereof |
| JP2008220329A (en) * | 2007-03-15 | 2008-09-25 | Mie Univ | Method for measuring ingestion amount of every fish individual |
-
2011
- 2011-06-27 CN CN201110174888.7A patent/CN102845335B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5932418A (en) * | 1996-04-08 | 1999-08-03 | Naiad Systems, Inc. | Fish embryo screening test for genotoxic agents using three different developmental life stages |
| WO2004044241A2 (en) * | 2002-11-13 | 2004-05-27 | DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung | Use of fish larvae as a screening model |
| CN1866025A (en) * | 2006-06-01 | 2006-11-22 | 中国科学院海洋研究所 | Method for detecting activity of anti-angiogenesis protein factor or pro-angiogenesis protein factor via zebra fish embryo model and application method thereof |
| JP2008220329A (en) * | 2007-03-15 | 2008-09-25 | Mie Univ | Method for measuring ingestion amount of every fish individual |
Non-Patent Citations (2)
| Title |
|---|
| Genetic Analysis of Digestive Physiology Using Fluorescent Phospholipid Reporters;Steven A.Farber等;《Science》;20010518;第292卷;第1385-1388页 * |
| Microsomal Triglyceride Transfer Protein Is Required for Yolk Lipid Utilization and Absorption of Dietary Lipids in Zebrafish Larvae;Amnon Schlegel等;《Biochemistry Accelerated publications》;20061201;第45卷(第51期);第15179-15187页 * |
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| CN102845335A (en) | 2013-01-02 |
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