CN102907357A - Building method and application of zebra fish hyperlipidemia model - Google Patents
Building method and application of zebra fish hyperlipidemia model Download PDFInfo
- Publication number
- CN102907357A CN102907357A CN2012103776034A CN201210377603A CN102907357A CN 102907357 A CN102907357 A CN 102907357A CN 2012103776034 A CN2012103776034 A CN 2012103776034A CN 201210377603 A CN201210377603 A CN 201210377603A CN 102907357 A CN102907357 A CN 102907357A
- Authority
- CN
- China
- Prior art keywords
- zebra fish
- room temperature
- under
- minutes
- microwell plate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention relates to a building method of a zebra fish hyperlipidemia model and application of the animal model to hyperlipidemia disease research and lipid-lowering drug screening. The building method of the zebra fish hyperlipidemia model mainly includes the steps of zebra fish selection, feeding of zebra fish by yolk powder, histochemical staining or fluorescent staining, image analysis and/or microwell plate analysis and statistical analysis. The building method has the advantages of simplicity, convenience, rapidity, economy, high efficiency, high throughput and the like, and the model can be used for hyperlipidemia disease research and lipid-lowering drug screening. The building method and application of the zebra fish bacterial infection model are of great significance to acceleration of research and development on lipid-lowering drugs and improvement on treatment of patients suffering from hyperlipidemia.
Description
Technical field
The invention belongs to living model and make up the field, be specifically related to a kind of method for building up and application thereof of zebra fish hyperlipemia model.
Background technology
Fat metabolism or running make one or more lipids of blood plasma be higher than normal disease to be called hyperlipidemia unusually.Hyperlipidemia is a kind of systemic disease, refers to that blood cholesterol (TC) and/or triglycerides (TG) are too high or HDL-C (HDL-C) is excessively low, and modern medicine is referred to as dyslipidemia
[1]
A large amount of epidemiology survey and studies show that, hyperlipidemia is the most important hazards of atherosclerotic, coronary heart disease and Cerebral Vascular Disease.Hyperlipidemia also is an important risk factor of hypertension, IGT, diabetes.Hyperlipidemia also can cause fatty liver, cirrhosis, cholelithiasis, pancreatitis, fundus hemorrhage, blind, peripheral vascular disease, limping, hyperuricemia
[1]Chinese residents nutrition and the investigation of health conditions carried out in China according to the Ministry of Public Health in recent years show, resident's total prevalence rate is 18.6% more than 18 years old, and national patient's total number of persons reaches 1.6 hundred million.Lipid lowerers can reduce incidence and the lethality of these diseases,, far-reaching influence positive to the control generation of cardiovascular and cerebrovascular disease.At present clinical practice and the fat-reducing medicament that is in development can be divided into Statins by its lipopenicillinase mechanism and chemical constitution, nicotinic acid class, shellfish spy class, cholic acid intercalating agent class, polyenoid class, Chinese medicine class etc.; But existing fat-reducing medicament exists or unsatisfactory curative effect, or the problem such as toxic and side effect is large or expense is high, and therefore developing new fat-reducing medicament seems very necessary
[2]
Researching and developing new fat-reducing medicament is focus and the difficult point of present international and domestic new drug development, and selecting desirable hyperlipemia animal model is the successful keys of fat-reducing medicament research and development.The external model that is used at present hyperlipidemia mainly is to utilize to participate in the cell that hyperlipidemia forms, macrophage for example, before hyperlipidemia complication atherosclerotic occured, macrophage can be gathered in artery, and this process is carried out under endothelium adhesion factor ICVM and ECVM promotion.Under low-density lipoprotein (LDL) effect, macrophage can become foam cells.Can utilize this process to carry out the screening of fat-reducing medicament.Studies show that at present statins can suppress the generation of this process.Except macrophage, the T lymphocyte, the NK cell, adipocyte, liver cell, mast cells etc. can be used for screening blood lipid-lowering medicine
[3]But setting up of cell culture system is consuming time longer, and method is unstable, poor repeatability, and cell in vitro lacks medicine in the metabolic conversion of biological integral and the loop distribution in the body, can not reflect medicine truth in vivo.
The animal that is used at present hyperlipemia model has rat, mouse, rabbit, Golden Hamster, cavy, cay, cercopithecus aethiops and rhesus macaque etc., and also useful quail, chicken, pigeon are set up hyperlipemia model for other.The method for building up of these animal hyperlipemia model mainly is to utilize the nutrition purposes, especially for feeding animals such as high lipid food such as cholesterol, lard, sodium taurocholate, yolk powder.Although above-mentioned modeling method effect is better, and is consuming time longer, cost is large, is unsuitable for high-flux fast screening
[3]Set up the good aids drug of a kind of energy process in vivo, can estimate quickly and easily with the animal model that screens the hyperlipidemia medicine again and have important using value.
Zebra fish is a kind of model organism of novelty.With in the traditional body and in-vitro screening model compare, live body zebra fish screening model has many advantages, has overcome original external model screening model length experimental period, complicated operation, drawback that cost is high in the shortcoming of absorption, distribution, metabolism and the checking of drainage link and conventional bulk.Zebra fish is a kind of vertebrate, with the similarity of human gene up to about 85%, the experimental result comparativity is strong.Compare with the mammal such as muroid, zebrafish embryo is transparent, a plurality of organs of observation analysis simultaneously, and experimental period is short, and sample size is large, and the credible result degree is high, and required expense is low
[4]The more important thing is, zebra fish model has inherent advantage
[4]: 1. feeding cost is low, and sexual maturation cycle is short; 2. fertility is strong, and a tail raun can produce 200～300 pieces of ovum at every turn; 3. growth rate is fast, and after fertilization 24 hours, the main histoorgan of zebra fish formed substantially, can be research a large amount of samples and shorter experimental period are provided; 4. embryo and juvenile fish are transparent, and be in vitro fertilization, and ectogenesis can directly be observed, and can analyze simultaneously a plurality of tracts; 5. the embryo has the yolk sac that nutrition can be provided, and does not need feeding in the first week, the interaction of compound and food component in the time of can avoiding compound treatment; 6. embryo's volume is little, and young fish length only has 1～4mm, can analyze in 6,12,24,48 or 96 orifice plates of a standard; 7. administering mode is simple: water-soluble small-molecule substance can be directly in skin, the gill and digestive system enter the zebra fish body; Water-fast material, macromolecular substances and protein can carry out microinjection.Therefore zebra fish can be used as good evaluation and the interior vertebrate model of the high flux body of screening medicine.
The metabolism that Chinese scholars has been used zebra fish research nutriment such as lipid, carbohydrate absorbs.
Deng
[5,6]Utilize the metabolism of zebra fish research lipid.Konstantin etc.
[7]Utilize zebra fish research lipid material in endovascular gathering and oxidation.Baek etc.
[8]Utilize high cholesterol diet feeding zebra fish, set up the high cholesterol model, but the high cholesterol model sets up length consuming time in this article, cholesterol analysis uses the zebra fish after the cracking, testing process is loaded down with trivial details, is prone to false positive or false negative, is unsuitable for the fat-reducing medicament screening.Clifton etc.
[9]Utilize the new fat absorption inhibitor of zebra fish fluorescent dye screening, the author mentions with yolk powder and medicine co-treatment zebra fish in article, carries out afterwards fat stains, observes the inhibition fat absorption effect of medicine; In this article, the author does not set up the zebra fish hyperlipemia model, and experimental result is also just observed qualitatively, does not set up the pharmacodynamics method for quantitatively evaluating.
Dried hen egg yolk is that to adopt Fresh Egg be raw material, through the sampling observation passport control examination of passports, washes egg, sterilization, sprays, dries up, beats eggs, separates, 10 multiple working procedures such as filtration, homogeneous, pasteurize, spraying, drying are made, and are the ideal substitutes of Fresh Egg.The content＞60% of fat in yolk powder, and yolk powder has preferably emulsibility, is the rational feed (Fig. 1) of setting up the zebra fish hyperlipemia model.Oil red O (Oil Red O) and Nile red (nile red) all have been proved to be fatty specificity dyestuff, can be specifically and fatty combination, fat is dyed redness (Fig. 2).With oil red O or with Nile red zebra fish is dyeed, the fat content in the zebra fish blood is higher, and then the blood after the dyeing is just redder; Fat content in the zebra fish blood is lower, and then the blood color relation is more shallow after the dyeing
[9,10]
Summary of the invention
For defective and the deficiency that overcomes above-mentioned prior art, the inventor after deliberation, aim to provide a kind of simple, fast, rapid screening and estimate the method for blood lipid-lowering medicine in economic, the high-throughout zebra fish model body.
The present invention is achieved by the following technical solutions:
1. the method for building up of a zebra fish hyperlipemia model is characterized in that, may further comprise the steps:
(1) zebra fish is chosen
The normotrophic zebra fish of picking is put into microwell plate;
(2) feed zebra fish with dried hen egg yolk
Remove the culture fluid in the microwell plate, a plurality of experimental group are set: the dried hen egg yolk feeding group of several variable concentrations and 1 blank group, each experimental group add respectively the corresponding solution of equal volume, then constant temperature culture according to the specification of microwell plate.
Preferably, also comprise following step:
(3) zebra fish histochemical stain or fluorescent staining;
(4) graphical analysis and/or microwell plate analysis;
(5) statistical analysis.
Preferably, the zebra fish that selects in the step (1) is after fertilization 6-7 days zebra fish;
Preferably, the zebra fish incubation time is 72 hours in the step (2);
Preferably, the solution that the blank group is used is the zebra fish breeding water, and this breeding water dissolved oxygen concentration is that 6-8mg/L, water temperature are that 28 ℃, pH are that 7.2-7.6, total hardness are 200-250mg/L;
Preferably, the temperature of zebra fish constant temperature culture is 28 ℃;
Preferably, zebra fish histochemical stain or fluorescent staining may further comprise the steps: 1) fixing, and 2) dehydration, 3) dyeing, 4) decolouring, 5) the PBST wash-out;
Preferably, the graphical analysis concrete steps are: after zebra fish histochemical stain or the fluorescent staining of live body zebra fish finish, take pictures and preserve picture at microscopically; Carry out qualitative evaluation by fat stains intensity in the observation zebra fish blood on the one hand; Utilize on the other hand software that zebra fish afterbody blood vessel is carried out graphical analysis, carry out quantitative assessment by the fat stains intensity of calculating zebra fish afterbody blood vessel;
Preferably, microwell plate analysis concrete steps are: after the zebra fish fluorescent staining, zebra fish is changed in 96 orifice plates, then every hole 1 tail places microwell plate fluorescence intensity under the multi-functional microwell plate analyzer;
Preferably, the statistical analysis step is: statistical procedures result with
Expression is relatively adopted the one-factor analysis of variance between many groups, relatively adopts Dunnett ' s T-check to carry out statistical procedures between two groups, and p＜0.05 is that otherness is remarkable.
Preferably, zebra fish histochemical stain or fluorescent staining concrete steps are as follows:
1) fixing
Remove the dried hen egg yolk culture fluid in the microwell plate, add 4% paraformaldehyde (PFA) zebra fish is fixed, need room temperature fix 4 hours (h) above or 4 ℃ fixedly spend the night;
2) dehydration
1. under the room temperature, at the 75%PBS/25% of 10ml propane diols, dewatered 10 minutes;
2. under the room temperature, at the 50%PBS/50% of 10ml propane diols, dewatered 10 minutes;
3. under the room temperature, at the 25%PBS/75% of 10ml propane diols, dewatered 10 minutes;
4. under the room temperature, at 100% propane diols of 10ml, dewatered 10 minutes;
3) dyeing
A: histochemical stain: dye with 1% oil red O stain liquid, 28 ℃ are spent the night;
B: fluorescent staining: dye with 10ng/mL Nile red dyeing liquor, 28 ℃ are spent the night;
4) decolouring
1. under the room temperature, at 100% propane diols of 10ml, decoloured 10 minutes;
2. under the room temperature, at the 25%PBS/75% of 10ml propane diols, decoloured 10 minutes;
3. under the room temperature, at the 50%PBS/50% of 10ml propane diols, decoloured 10 minutes;
4. under the room temperature, at the 75%PBS/25% of 10ml propane diols, decoloured 10 minutes
5. under the room temperature, at the 100%PBS of 10ml, decoloured 10 minutes;
5) PBST wash-out
With PBST wash-out 15min, repeat 4 times under the room temperature.
2. the zebra fish hyperlipemia model that obtains of aforesaid method for building up is used for the purposes of hyperlipidaemic conditions research.
3. the zebra fish hyperlipemia model that obtains of aforesaid method for building up is used for estimating or the purposes of screening blood lipid-lowering medicine.
Preferably, further comprising the steps of:
(3) compound treatment
Remove the dried hen egg yolk culture fluid in the microwell plate, a plurality of experimental group are set: several compound treatment groups, 1 model group, 1 positive controls, 1 solvent control group and 1 blank group, each experimental group adds respectively the corresponding solution of equal volume, then constant temperature culture according to the specification of microwell plate;
(4) zebra fish histochemical stain or fluorescent staining;
(5) graphical analysis and/or microwell plate analysis;
(6) statistical analysis.
Preferably, the solution that the compound treatment group adds in the step (3) is testing compound solution, and the solution that positive controls adds is blood lipid-lowering medicine solution, and the solution that adds in the solvent control group is 0.1% DMSO;
Preferably, zebra fish histochemical stain or fluorescent staining may further comprise the steps: 1) fixing, and 2) dehydration, 3) dyeing, 4) decolouring, 5) the PBST wash-out;
Preferably, the graphical analysis concrete steps are: after zebra fish histochemical stain or the fluorescent staining of live body zebra fish finish, take pictures and preserve picture at microscopically; Carry out qualitative evaluation by fat stains intensity in the observation zebra fish blood on the one hand; Utilize on the other hand software that zebra fish afterbody blood vessel is carried out graphical analysis, carry out quantitative assessment by the fat stains intensity of calculating zebra fish afterbody blood vessel;
Preferably, microwell plate analysis concrete steps are: after the zebra fish fluorescent staining, zebra fish is changed in 96 orifice plates, then every hole 1 tail places microwell plate fluorescence intensity under the multi-functional microwell plate analyzer;
Preferably, the statistical analysis step is: statistical procedures result with
Expression is relatively adopted the one-factor analysis of variance between many groups, relatively adopts Dunnett ' s T-check to carry out statistical procedures between two groups, and p＜0.05 is that otherness is remarkable.
Preferably, the zebra fish incubation time after the compound treatment is 72 hours.
Preferably, zebra fish histochemical stain or fluorescent staining concrete steps are as follows:
1) fixing
Remove the dried hen egg yolk culture fluid in the microwell plate, add 4% paraformaldehyde (PFA) zebra fish is fixed, need room temperature fix 4 hours (h) above or 4 ℃ fixedly spend the night;
2) dehydration
5. under the room temperature, at the 75%PBS/25% of 10ml propane diols, dewatered 10 minutes;
6. under the room temperature, at the 50%PBS/50% of 10ml propane diols, dewatered 10 minutes;
7. under the room temperature, at the 25%PBS/75% of 10ml propane diols, dewatered 10 minutes;
8. under the room temperature, at 100% propane diols of 10ml, dewatered 10 minutes;
3) dyeing
A: histochemical stain: dye with 1% oil red O stain liquid, 28 ℃ are spent the night;
B: fluorescent staining: dye with 10ng/mL Nile red dyeing liquor, 28 ℃ are spent the night;
4) decolouring
6. under the room temperature, at 100% propane diols of 10ml, decoloured 10 minutes;
7. under the room temperature, at the 25%PBS/75% of 10ml propane diols, decoloured 10 minutes;
8. under the room temperature, at the 50%PBS/50% of 10ml propane diols, decoloured 10 minutes;
9. under the room temperature, at the 75%PBS/25% of 10ml propane diols, decoloured 10 minutes
10. under the room temperature, at the 100%PBS of 10ml, decoloured 10 minutes;
5) PBST wash-out
With PBST wash-out 15min, repeat 4 times under the room temperature.
Zebra fish hyperlipemia model provided by the invention is compared with animal hyperlipemia model in the past, and the present invention has following advantage:
1) in vivo an experiment material is the live body zebra fish, and as a kind of vertebrate, its screening model belongs to the body inner model, can truly reflect medicine absorption,distribution,metabolism,excretion in vivo, really reflects the whole biologically active of medicine.
2) high flux-zebra fish juvenile fish is very little, only has the 1-4 millimeter, can analyze with experimental period shortly to make zebra fish become a kind of ideal model that can carry out high-flux medicaments sifting in 6,12,24,48 orifice plates of a standard.
3) economy-required expense is low, expend greater than 10 dollars every every day take the screening experiment of monkey as the experiment carrier, expend greater than 1 dollar every every day take the screening experiment of mouse as the experiment carrier, and expend less than 0.01 dollar every every day take the screening experiment of zebra fish as the experiment carrier.
4) compound amount few-the detection compound consumption is few, usually only needs several milligrams, traditional screening experiment then needs the compound more than several milligrams.
5) easy-experimentation is simple to operate, and zebra fish just can carry out quantitatively and qualitative analysis after drug treating, dyeing, and the traditional experiment operating process is complicated, easily produces false positive results.
6) fast-and experimental period is short, can within a week, finish; And mouse often needs the time of several weeks to several months, and monkey often needs the time of several months to several years.Zebra fish was finished embryonic development at first 72 hours with interior.Most internals comprises cardiovascular system, intestines, liver and kidney, rapid shaping in 24-48 hour, and traditional experiment carrier mouse and monkey then needed respectively 21 days and 9 months can finish embryonic development.
7) efficient-zebrafish embryo and juvenile fish are transparent, can observe simultaneously a plurality of tracts, and experiment analytical method is simple, quick.
8) similarity of the gene of predictability-zebra fish and human gene is up to about 85% reliably, and its biological function is highly similar to mammal and the mankind, and the experimental result comparativity is strong, and predictability is good.
9) intuitive strong-embryo and juvenile fish are transparent, can directly place the stereomicroscopy Microscopic observation to contrast each experimental group zebra fish staining power.
10) high, the good reproducibility of stability-repeated experiments of the present invention is tens times, and it is basic identical that institute obtains experimental result.
Using value of the present invention
The advantages such as that the zebra fish hyperlipemia model has is reliable, quick, efficient, cheap, high performance-price ratio, this model can be used for hyperlipidaemic conditions research and screening blood lipid-lowering medicine.The present invention has significant to the research and development of accelerating blood lipid-lowering medicine and the treatment that improves hyperlipidemia patient.
Detailed Description Of The Invention
The object of the present invention is to provide a kind of construction method and application thereof of zebra fish hyperlipemia model.The advantages such as that method provided by the invention has is easy, quick, economic, efficient, high flux.
One, the invention provides a kind of method for building up of zebra fish hyperlipemia model, concrete steps are:
1. zebra fish is chosen
Get 4～5 couples of zebra fish parents mating, according to Westerfield
[10]The method hatched blastocyst.The zebra fish that will be in the optimization process stage places under the disecting microscope to be observed, and the normotrophic zebra fish of picking moves in 6,12,24,48, the 96 hole microwell plates, the zebra fish of putting into varying number according to the microwell plate specification.
2 dried hen egg yolks are fed zebra fish
5 experimental group are set: 4 dried hen egg yolk feeding groups, 1 be the feeding group not.Remove the breeding water in the microwell plate, dried hen egg yolk concentration is respectively 0.01%, 0.05%, 0.1% and 0.5%; Add isopyknic breeding water in the blank group.In 28 ℃ of constant incubators, cultivate.
3 zebra fish histochemical stain or fluorescent stainings
1) fixing
Remove the dried hen egg yolk culture fluid in the microwell plate, add 4% paraformaldehyde (PFA) zebra fish is fixed, need room temperature fix 4 hours (h) above or 4 ℃ fixedly spend the night.
2) dehydration
1. under the room temperature, at the 75%PBS/25% of 10ml propane diols, dewatered 10 minutes;
2. under the room temperature, at the 50%PBS/50% of 10ml propane diols, dewatered 10 minutes;
3. under the room temperature, at the 25%PBS/75% of 10ml propane diols, dewatered 10 minutes;
4. under the room temperature, at 100% propane diols of 10ml, dewatered 10 minutes.
3) dyeing
A: histochemical stain: dye with 1% oil red O stain liquid, 28 ℃ are spent the night;
B: fluorescent staining: dye with 10ng/mL Nile red dyeing liquor, 28 ℃ are spent the night.
4) decolouring
5) PBST wash-out
With PBST wash-out 15min, repeat 4 times under the room temperature.
4 graphical analyses
A: after the zebra fish histochemical stain finishes, utilize disecting microscope to observe, take pictures and preserve.Whether qualitatively judge the zebra fish hyperlipemia model by the blood fat staining power of observing each experimental group zebra fish of contrast on the one hand is successfully established; Carry out graphical analysis with image processing software on the other hand, calculate zebra fish afterbody blood fat staining power, judge quantitatively whether the zebra fish hyperlipemia model is successfully established.
B: the zebra fish fluorescent staining utilizes fluorescence microscope, takes pictures and preserves after finishing.Whether qualitatively judge the zebra fish hyperlipemia model by the blood fat fluorescence intensity of observing each experimental group zebra fish of contrast on the one hand is successfully established; Carry out image quantitative analysis with image processing software on the other hand, calculate zebra fish afterbody blood fat staining power, judge quantitatively whether the zebra fish hyperlipemia model is successfully established.
5 microwell plate analyses
After the zebra fish fluorescent staining, zebra fish is changed in 96 orifice plates over to every hole 1 tail.Then microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyzer.Exciting light is set to 485nm, gathers radiative fluorescence intensity at the 525nm place, and test repeats 3 times and gets its mean value, judges quantitatively whether the zebra fish hyperlipemia model is successfully established.
6 statistical analysis
The relative multiple that increases of blood fat that utilizes JMP8.0 software that above-mentioned graphical analysis and microwell plate are analyzed gained carries out statistical analysis.Statistical procedures result with
Expression is relatively adopted variance analysis between many groups, relatively adopts Dunnett ' s T-check to carry out statistical procedures between two groups, and p＜0.05 is that otherness is remarkable.Whether estimate the zebra fish hyperlipemia model according to the statistical procedures result is successfully established.
Two, the invention provides a kind of the evaluation and the method for screening blood lipid-lowering medicine, design is:
1 zebra fish is chosen
The zebra fish that will be in the optimization process stage places under the disecting microscope to be observed, and the normotrophic zebra fish of picking moves in 6,12,24,48,96 or 384 orifice plates, the zebra fish of putting into varying number according to the microwell plate specification.
2 dried hen egg yolks are fed zebra fish
7 experimental group are set, remove the breeding water in the microwell plate, add 0.5% dried hen egg yolk culture fluid, in 28 ℃ of constant incubators, cultivate.
3 compound treatment
7 experimental group are set: 4 compound treatment groups, 1 positive controls, 1 solvent control group, 1 blank group.Microwell plate is put into the hyperlipemia model zebra fish in each hole, removes the breeding water in the microwell plate, and the compound solution concentration of 4 compound treatment groups is respectively 0.01 μ g/ml, 0.1 μ g/ml, 1 μ g/ml and 10 μ g/ml; Add the hyperlipidemia medicine in the positive controls; Adding equal-volume concentration is 0.1% DMSO in the solvent control group; Add isopyknic breeding water in the blank group.Cultivate in 28 ℃ of constant incubators according to the optimization process time span.
4 zebra fish histochemical stain or fluorescent stainings
Experimental technique is with the zebra fish histochemical stain in the summary of the invention one or fluorescent staining step.
5 graphical analyses
A: after the zebra fish histochemical stain finishes, utilize disecting microscope to observe, take pictures and preserve.Come qualitative evaluation and screening blood lipid-lowering medicine by the blood fat staining power of observing each experimental group zebra fish of contrast on the one hand; Carry out graphical analysis with image processing software on the other hand, calculate zebra fish afterbody blood fat staining power.
B: the zebra fish fluorescent staining utilizes fluorescence microscope, takes pictures and preserves after finishing.Come qualitative evaluation and screening blood lipid-lowering medicine by the blood fat fluorescence intensity of observing each experimental group zebra fish of contrast on the one hand; Carry out image quantitative analysis with image processing software on the other hand, calculate zebra fish afterbody blood fat staining power.
Estimate the lipid-lowering effect of testing compound according to the blood fat reduced rate of testing compound processed group zebra fish, blood fat reduced rate computing formula is seen (a).
6 microwell plate analyses
After the zebra fish fluorescent staining, zebra fish is changed in 96 orifice plates over to every hole 1 tail.Then microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyzer.Exciting light is set to 485nm, gathers radiative fluorescence intensity at the 525nm place, and test repeats 3 times and gets its mean value, and blood fat reduced rate computing formula is seen (b).
7 statistical analysis
Statistical procedures result with
Expression is relatively adopted variance analysis between many groups, relatively adopts Dunnett ' s T-check to carry out statistical procedures between two groups, and p＜0.05 is that otherness is remarkable.According to statistical procedures evaluation of result and screening blood lipid-lowering medicine.
Description of drawings
Fig. 1 is zebra fish behind the feeding yolk powder.Arrow is depicted as the enteron aisle of zebra fish.A is feeding group not, and the zebra fish enteron aisle is transparent; B is the feeding group, and the zebra fish enteron aisle is opaque, the color blackening, and the atrament in the enteron aisle is yolk powder.
Fig. 2 is the zebra fish after dyeing.A is feeding group not, and fat content seldom is not dyed to redness substantially in zebra fish enteron aisle and the blood; B is the feeding group, behind the feeding yolk powder in zebra fish enteron aisle and the blood lipid content significantly increase, dyeing rear intestinal and blood all are dyed to redness.
Fig. 3 is the zebra fish dyeing picture behind the feeding variable concentrations dried hen egg yolk.A is feeding group not, and B is that 0.01% yolk powder is fed zebra fish, and C is that 0.05% yolk powder is fed zebra fish, and D is that 0.1% yolk powder is fed zebra fish, and E is that 0.5% yolk powder is fed zebra fish.Along with the increase of yolk powder concentration, the zebra fish staining power increases gradually.
Fig. 4 is the zebra fish blood fat oil red O stain intensity block diagram behind the variable concentrations dried hen egg yolk feeding, and along with the rising of dried hen egg yolk concentration, the dyeing of zebra fish blood fat obviously increases.
Fig. 5 is the zebra fish blood fat staining power block diagram behind the identical dried hen egg yolk concentration feeding different time, and along with the increase of feeding time, zebra fish blood fat staining power increases gradually.
Fig. 6 is the zebra fish oil red O stain picture after Lovastatin is processed.Figure A is model group, and figure B is the solvent control group, and figure C is the Lovastatin of 0.01 μ g/ml, and figure D is the Lovastatin of 0.05 μ g/ml, and figure E is the Lovastatin of 0.1 μ g/ml, and figure F is the Lovastatin of 0.2 μ g/ml.Along with the increase of Lovastatin concentration, zebra fish afterbody blood fat staining power weakens gradually.
Fig. 7 is the zebra fish Nile red dyeing picture after Lovastatin is processed.Figure A is model group, and figure B is the solvent control group, and figure C is the Lovastatin of 0.01 μ g/ml, and figure D is the Lovastatin of 0.05 μ g/ml, and figure E is the Lovastatin of 0.1 μ g/ml, and figure F is the Lovastatin of 0.2 μ g/ml.Along with the increase of Lovastatin concentration, zebra fish afterbody blood fat staining power weakens gradually.
Fig. 8 is the zebra fish blood fat reduced rate block diagram (oil red O stain) after Lovastatin is processed.Along with the increase of Lovastatin concentration, zebra fish blood fat reduced rate improves gradually.Compare with the solvent control group, 0.05 μ g/ml, 0.1 μ g/ml and 0.2 μ g/ml Lovastatin processed group all can significantly reduce zebra fish blood fat, *: p＜0.05, * *: p＜0.01.
Fig. 9 is the zebra fish blood fat reduced rate block diagram (Nile red dyeing) after Lovastatin is processed.Along with the increase of Lovastatin concentration, zebra fish blood fat reduced rate improves gradually.Compare with the solvent control group, 0.05 μ g/ml, 0.1 μ g/ml and 0.2 μ g/ml Lovastatin processed group all can significantly reduce zebra fish blood fat, *: p＜0.05, * *: p＜0.01.
Figure 10 is the zebra fish blood fat reduced rate block diagram (microwell plate analysis) after Lovastatin is processed.Along with the increase of Lovastatin concentration, zebra fish blood fat reduced rate improves gradually.Compare with the solvent control group, 0.05 μ g/ml, 0.1 μ g/ml and 0.2 μ g/ml Lovastatin processed group all can significantly reduce zebra fish blood fat, *: p＜0.05, * *: p＜0.01.
Figure 11 is the zebra fish Nile red dyeing picture after Simvastatin is processed.Figure A is model group, and figure B is the solvent control group, the figure positive contrast of C (Lovastatin of 0.05 μ g/ml), and figure D is the Simvastatin of 0.1 μ g/ml, and figure E is the Simvastatin of 0.5 μ g/ml, and figure F is the Simvastatin of 1 μ g/ml.Along with the increase of Simvastatin concentration, zebra fish afterbody blood fat staining power weakens gradually.
Figure 12 is the zebra fish blood fat reduced rate block diagram (oil red O stain) after Simvastatin is processed.Along with the increase of Simvastatin concentration, zebra fish blood fat reduced rate improves gradually.Compare with the solvent control group, 0.1 μ g/ml, 0.5 μ g/ml and 1 μ g/ml Simvastatin processed group all can significantly reduce zebra fish blood fat, *: p＜0.05, * *: p＜0.01.
Figure 13 is the zebra fish blood fat reduced rate block diagram (microwell plate analysis) after Bezafibrate is processed.Along with the increase of Bezafibrate concentration, zebra fish blood fat reduced rate improves gradually.Compare with the solvent control group, 5 μ g/ml and 10 μ g/ml Bezafibrate processed group all can significantly reduce zebra fish blood fat, *: p＜0.05.
Embodiment
Following examples are for the embodiment that further specifies zebra fish hyperlipemia model provided by the invention and purposes.Embodiment is in order to explain rather than to limit the scope of the invention by any way, and some change that those skilled in the art make within the scope of the claims and adjusting also should be thought and belongs to scope of the present invention.
Reagent and instrument
Oil red O (Sigma company, the U.S.), Nile red (lark prestige company, China), dried hen egg yolk (Advaced Hatchery Technology company, the U.S.) (moisture :≤4.0%, lipid: 〉=60% (neutral fat 62%～65%, phosphatidase 13 0%～35%, sterol 4%～5%), protein: 〉=30%, free fatty acid :≤4.5%), other reagent provide by the prosperous Science and Technology Ltd. of Beijing ancient cooking vessel state.Disecting microscope (SZX7, Olympus company, Japan), fluorescence microscope (AZ100, Nikon company, Japan).
1 zebra fish is chosen
When zebra fish grows 3dpf, begin to swallow [11].The zebra fish of 6dpf placed under the disecting microscope observe, the normotrophic zebra fish of picking moves into respectively in 6 orifice plates, every hole 30 tails (are annotated: the dpf=day post fertilization among the present invention, Chinese refers to zebra fish after fertilization fate, refers to the zebra fish after fertilization 3 days such as 3dpf).
2 dried hen egg yolks are fed zebra fish
5 experimental group are set: 4 dried hen egg yolk feeding groups, 1 be the feeding group not.Remove the breeding water in the microwell plate, dried hen egg yolk concentration is respectively 0.01%, 0.05%, 0.1% and 0.5%; Add isopyknic breeding water in the blank group.In 28 ℃ of constant incubators, cultivated 24 hours.
3 zebra fish oil red O stains
Method is with the zebra fish histochemical stain step part in the invention one.
4 graphical analyses
After the zebra fish histochemical stain finishes, utilize disecting microscope to observe, take pictures and preserve.Whether qualitatively judge the zebra fish hyperlipemia model by the blood fat staining power of observing each experimental group zebra fish of contrast on the one hand is successfully established; Carry out graphical analysis with image processing software on the other hand, calculate zebra fish afterbody blood fat staining power, judge quantitatively whether the zebra fish hyperlipemia model is successfully established.
Blank group zebra fish is owing to fat content in the blood is very low, so afterbody can not caught color basically.Along with yolk powder concentration increases, zebra fish blood fat staining power constantly increases, 0.5% yolk powder blood fat staining power the strongest (Fig. 3).Statistical procedures result shows: 0.01%, the zebra fish afterbody blood fat staining power of 0.05%, 0.1% and 0.5% yolk powder is respectively 109.0 ± 10.5,512.3 ± 50.8,2232.0 ± ± 280.8 and 2369.1 ± 152.3, this shows the increase along with yolk powder concentration, zebra fish blood fat staining power increases gradually, the zebra fish blood fat staining power the highest (Fig. 4) that 0.5% yolk powder is fed.The zebra fish that 0.5% yolk powder is fed is compared p＜0.05 with 0.01% with the zebra fish blood fat staining power that 0.05% yolk powder is fed.
Embodiment 2 determines yolk powder nursing Best Times length
1 zebra fish is chosen
The zebra fish of 6dpf placed under the disecting microscope observe, the normotrophic zebra fish of picking moves into respectively in 6 orifice plates, every hole 30 tails.
2 dried hen egg yolks are fed zebra fish
3 experimental group are set: each organizes the dried hen egg yolk of equal feeding 0.5%, respectively at cultivating 24 hours, 48 hours and 72 hours in 28 ℃ of constant incubators.
3 zebra fish oil red O stains
Method is with the zebra fish histochemical stain step part in the invention one.
4 graphical analyses
Image analysis method is with embodiment 1.Feed the increase of time along with dried hen egg yolk, zebra fish blood fat staining power constantly increases, and dried hen egg yolk is fed zebra fish blood fat staining power maximum after 72 hours.Statistical procedures result shows: dried hen egg yolk was fed after 24,48 and 72 hours, zebra fish afterbody blood fat staining power is respectively 2036.1 ± 232.4,4689.5 ± 498.3 and 5938 ± 519.5, this shows the increase along with feeding time, zebra fish blood fat staining power increases gradually, feeds zebra fish blood fat staining power maximum (Fig. 5) after 72 hours.Dried hen egg yolk is fed zebra fish blood fat staining power and yolk powder after 72 hours and is fed 24 and compare with 48 hours zebra fish blood fat staining power, has significant difference (p＜0.05).By best dried hen egg yolk concentration and the best incubation time that embodiment 1 and embodiment 2 determine, will be with 72 hours zebra fish of 0.5% dried hen egg yolk feeding as the hyperlipidemia zebra fish model.
Sensitivity and the reliability of embodiment 3 checking zebra fish hyperlipemia model
Based on the best feeding concentration of the dried hen egg yolk in embodiment 1 and 2 and best incubation time length, the present embodiment is estimated the lipid-lowering effect of known hyperlipidemia medicine [2] Lovastatin (Lovastatin) with the zebra fish hyperlipemia model, verify sensitivity and the reliability of zebra fish hyperlipemia model with this.
1 zebra fish is chosen
The zebra fish of 6dpf placed under the disecting microscope observe, the normotrophic zebra fish of picking moves into respectively in 6 orifice plates, every hole 30 tails.
2 dried hen egg yolks are fed zebra fish
6 experimental group are set, remove the breeding water in the microwell plate, add 0.5% dried hen egg yolk culture fluid, in 28 ℃ of constant incubators, cultivated 72 hours.
3 compound treatment
6 experimental group are set: 4 Lovastatin processed group, 1 solvent control group, 1 blank group.Add the Lovastatin that concentration is respectively 0.01 μ g/ml, 0.05 μ g/ml, 0.1 μ g/ml and 0.2 μ g/ml in the Lovastatin processed group, add 0.1%DMSO in the solvent control group, add breeding water in the blank group, in 28 ℃ of constant incubators, process 72h.
4 zebra fish histochemical stain or fluorescent stainings
Experimental technique is with the zebra fish histochemical stain in the summary of the invention one or fluorescent staining step.
5 graphical analyses
A: after the zebra fish histochemical stain finishes, utilize the disecting microscope observation, take pictures and preserve (Fig. 6).
B: the zebra fish fluorescent staining utilizes fluorescence microscope, takes pictures and preserves (Fig. 7) after finishing.
Come on the one hand the lipid-lowering effect of qualitative evaluation Lovastatin by the blood fat staining power of observing each experimental group zebra fish of contrast; Utilize on the other hand Nikon NIS-Elements D3.10 high vision process software to carry out image quantitative analysis, calculate zebra fish afterbody blood fat staining power, calculate the blood fat reduced rate of respectively organizing Lovastatin, the lipid-lowering effect of quantitative assessment Lovastatin according to formula (a).
Statistical procedures result shows: (1) zebra fish histochemical stain (oil red O stain): the zebra fish blood fat reduced rate of 4 Lovastatin processed group is respectively (9.4 ± 3.5) %, (22.8 ± 5.1) %, (37.5 ± 4.5) % and (43.9 ± 6.8) % (Fig. 8); (2) zebra fish fluorescent staining (Nile red dyeing): the zebra blood fat reduced rate of 4 Lovastatin processed group is respectively (12.1 ± 5.5) %, (25.4 ± 7.5) %, (40.1 ± 8.3) % and (45.5 ± 9.3) % (Fig. 9).This shows the increase along with Lovastatin concentration for the treatment of and processing time, and the blood fat reduced rate increases gradually.By variance analysis, no matter be to adopt histochemical stain or fluorescent staining, 0.05 μ g/ml, 0.1 μ g/ml compare with the solvent control group with 0.2 μ g/ml Lovastatin processed group, can both significantly reduce zebra fish blood fat (p＜0.05, p＜0.01); 0.01 μ g/ml Lovastatin processed group is compared with the solvent control group, although the zebra fish blood fat also has reduction, and not statistically significant (p＞0.05).
6 microwell plate analyses
After the zebra fish fluorescent staining, zebra fish is changed in 96 orifice plates over to every hole 1 tail.Then microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyzer.Exciting light is set to 485nm, gathers radiative fluorescence intensity at the 525nm place, and test repeats 3 times and gets its mean value, and blood fat reduced rate computing formula is seen (b).
Statistical procedures result shows: the zebra blood fat reduced rate of 4 Lovastatin processed group is respectively (11.4 ± 6.1) %, (23.8 ± 7.5) %, (39.6 ± 8.3) % and (42.5 ± 9.3) % (Figure 10); This shows the increase along with Lovastatin concentration for the treatment of and processing time, and the blood fat reduced rate increases gradually.By variance analysis, 0.05 μ g/ml, 0.1 μ g/ml compare with the solvent control group with 0.2 μ g/ml Lovastatin processed group, can both significantly reduce zebra fish blood fat (p＜0.05, p＜0.01); 0.01 μ g/ml Lovastatin processed group is compared with the solvent control group, although the zebra fish blood fat also has reduction, and not statistically significant (p＞0.05).
Simvastatin (Simvastatin) can be used for treating hyperlipidemia [2].The present embodiment is the lipid-lowering effect that utilizes zebra fish hyperlipemia model quantitative assessment Simvastatin.
1 zebra fish is chosen
The zebra fish of 6dpf placed under the disecting microscope observe, the normotrophic zebra fish of picking moves into respectively in 6 orifice plates, every hole 30 tails.
2 dried hen egg yolks are fed zebra fish
6 experimental group are set, remove the breeding water in the microwell plate, add 0.5% dried hen egg yolk culture fluid, in 28 ℃ of constant incubators, cultivated 72 hours.
3 compound treatment
6 experimental group are set: 3 compound treatment groups, 1 positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, the compound treatment group adds respectively the Simvastatin that concentration is 0.1 μ g/ml, 0.5 μ g/ml and 1 μ g/ml; The Lovastatin that adds 0.05 μ g/ml in the positive controls; Add 0.1%DMSO in the solvent control group; Add breeding water in the blank group, in 28 ℃ of constant incubators, process 72h.
4 zebra fish histochemical stains
Experimental technique is with the integral zebra fish histochemical stain step in the summary of the invention one.
5 graphical analyses
After the zebra fish histochemical stain finishes, utilize disecting microscope to observe, take pictures and preserve.Come on the one hand the lipid-lowering effect (Figure 11) of qualitative evaluation Simvastatin by the blood fat staining power of observing each experimental group zebra fish of contrast; Utilize on the other hand Nikon NIS-Elements D3.10 high vision process software to carry out image quantitative analysis, calculate zebra fish afterbody blood fat staining power.Calculate the blood fat reduced rate of respectively organizing Simvastatin, the lipid-lowering effect of quantitative assessment Simvastatin according to formula (a).
Statistical procedures result shows: the zebra fish blood fat reduced rate of positive controls is (26.3 ± 4.1) %; The blood fat reduced rate of 3 Simvastatin processed group is respectively (19.5 ± 3.2) %, (25.7 ± 3.8) % and (33.5 ± 5.3) % (Figure 12); This shows the increase along with Simvastatin concentration, and the blood fat reduced rate raises gradually.By variance analysis, the Simvastatin processed group of 0.1 μ g/ml, 0.5 μ g/ml and 1 μ g/ml is compared with the solvent control group, can both significantly reduce zebra fish blood fat (p＜0.05, p＜0.01).
Bezafibrate (Bezafibrate) can be used for treating hyperlipidemia [2].The present embodiment is the lipid-lowering effect that utilizes zebra fish hyperlipemia model quantitative assessment Bezafibrate.
1 zebra fish is chosen
The zebra fish of 6dpf placed under the disecting microscope observe, the normotrophic zebra fish of picking moves into respectively in 6 orifice plates, every hole 30 tails.
2 dried hen egg yolks are fed zebra fish
6 experimental group are set, remove the breeding water in the microwell plate, add 0.5% dried hen egg yolk culture fluid, in 28 ℃ of constant incubators, cultivated 72 hours.
3 compound treatment
6 experimental group are set: 3 compound treatment groups, 1 positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, the compound treatment group adds respectively the Bezafibrate that concentration is 1 μ g/ml, 5 μ g/ml and 10 μ g/ml; The Lovastatin that adds 0.05 μ g/ml in the positive controls; Add 0.1%DMSO in the solvent control group; Add breeding water in the blank group, in 28 ℃ of constant incubators, process 72h.
4 zebra fish fluorescent stainings
Experimental technique is with the integral zebra fish fluorescent staining step in the summary of the invention one.
5 microwell plate analyses
After the zebra fish fluorescent staining, zebra fish is changed in 96 orifice plates over to every hole 1 tail.Then microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyzer.Exciting light is set to 485nm, gathers radiative fluorescence intensity at the 525nm place, and test repeats 3 times and gets its mean value, and blood fat reduced rate computing formula is seen (b).
Statistical procedures result shows: the zebra fish blood fat reduced rate of positive controls is (24.7 ± 3.9) %; The blood fat reduced rate of 3 Bezafibrate processed group is respectively (12.5 ± 7.2) %, (20.7 ± 5.8) % and (28.5 ± 7.3) % (Figure 13); This shows the increase along with Bezafibrate concentration, and the blood fat reduced rate raises gradually.By variance analysis, 5 μ g/ml compare with the solvent control group with 10g/ml Bezafibrate processed group, can both significantly reduce zebra fish blood fat (p＜0.05); 1 μ g/ml Bezafibrate processed group is compared with the solvent control group, although the zebra fish blood fat also has reduction, and not statistically significant (p＞0.05).
List of references
[1]Gupta?A，Sehgal?V，Mehan?S.HYPERLIPIDEMIA：An?Updated?Review[J].Intemational?Journal?of?Biopharmaceutical?&Toxicological?Research，2011，1(1)：81-89.
[2]Pahan?K.Lipid-lowering?drugs[J].Cell?Mol?Life?Sci.，2006，63(10)：1165-1178.
[3]Singh?V，Tiwari?RL，Dikshit?M，et.al.Models?to?study?atherosclerosis：a?mechanistic?insight[J].Curr?Vasc?Pharmacol.2009，7(1)：75-109.
[4]McGrath?P，Li?CQ.Zebrafish：a?predictive?model?for?assessing?drug-induced?toxicity[J].Drug?Discover?Today，2008，13(9)：394-401.
[5]
Salo?VT，Nyberg?L，et.al.Zebrafish：gaining?popularity?in?lipid?research[J].Biochem?J.，2010，429(2)：235-242.
[6]Hama?K，Provost?E，Baranowski?TC，et.al.In?vivo?imaging?of?zebrafish?digestive?organ?function?using?multiple?quenched?fluorescent?reporters[J].Am?J?Physiol?Gastrointest?Liver?Physiol.2009，296(2)：445-453.
[7]Stoletov?K，Fang?L，Choi?SH，et.al.Vascular?lipid?accumulation，lipoprotein?oxidation，and?macrophage?lipid?uptake?in?hypercholesterolemic?zebrafish[J].Circ?Res.2009104(8)：952-60.
[8]Baek?JS，Fang?L，Li?AC，et.al.Ezetimibe?and?simvastatin?reduce?cholesterol?levels?in?zebrafish?larvae?fed?a?high-cholesterol?diet[J].Cholesterol，2012，Article?ID?564705，doi：10.1155/2012/564705.
[9]Clifton?JD，Lucumi?E，Myers?MC，et?al.Identification?of?Novel?Inhibitors?of?Dietary?Lipid?Absorption?Using?Zebrafish.PLoS?ONE，2010，5(8)：e12386.doi：10.1371/joumal.pone.0012386.
[10]Jones?Kevin，Alimov?Alexander，Rilo?Horacio，et.Al.A?high?throughput?live?transparent?animal?bioassay?to?identify?non-toxic?small?molecules?or?genes?that?regulate?vertebrate?fat?metabolism?for?obesity?drug?development.Nutr?Metab(Lond).2008；5：23.
[11]Westerfield?M.The?Zebrafish?Book：A?Guide?for?the?Laboratory?Use?of?Zebrafish[M].Eugene，Oregon：The?University?of?Oregon?Press，1993.
Claims (10)
1. the method for building up of a zebra fish hyperlipemia model is characterized in that, may further comprise the steps:
(1) zebra fish is chosen
The normotrophic zebra fish of picking is put into microwell plate;
(2) feed zebra fish with dried hen egg yolk
Remove the culture fluid in the microwell plate, a plurality of experimental group are set: the dried hen egg yolk feeding group of several variable concentrations and 1 blank group, each experimental group add respectively the corresponding solution of equal volume, then constant temperature culture according to the specification of microwell plate.
2. the method for claim 1 is characterized in that, also comprises following step:
(3) zebra fish histochemical stain or fluorescent staining;
(4) graphical analysis and/or microwell plate analysis;
(5) statistical analysis.
3. method for building up as claimed in claim 1 or 2 is characterized in that: preferred, the zebra fish that selects in the step (1) is after fertilization 4-7 days zebra fish;
Preferably, the zebra fish incubation time is 72 hours in the step (2);
Preferably, the solution that the blank group is used is the zebra fish breeding water, and this breeding water dissolved oxygen concentration is that 6-8mg/L, water temperature are that 28 ℃, pH are that 7.2-7.6, total hardness are 200-250mg/L;
Preferably, the temperature of zebra fish constant temperature culture is 28 ℃.
4. method for building up as claimed in claim 2 or claim 3 is characterized in that:
Preferably, zebra fish histochemical stain or fluorescent staining may further comprise the steps: 1) fixing, and 2) dehydration, 3) dyeing, 4) decolouring, 5) the PBST wash-out;
Preferably, the graphical analysis concrete steps are: after zebra fish histochemical stain or the fluorescent staining of live body zebra fish finish, take pictures and preserve picture at microscopically; Carry out qualitative evaluation by fat stains intensity in the observation zebra fish blood on the one hand; Utilize on the other hand software that zebra fish afterbody blood vessel is carried out graphical analysis, carry out quantitative assessment by the fat stains intensity of calculating zebra fish afterbody blood vessel;
Preferably, microwell plate analysis concrete steps are: after the fluorescent staining of live body zebra fish, zebra fish is changed in 96 orifice plates, then every hole 1 tail places microwell plate fluorescence intensity under the multi-functional microwell plate analyzer;
Preferably, the statistical analysis step is: statistical procedures result with
Expression is relatively adopted the one-factor analysis of variance between many groups, relatively adopts Dunnett ' s T-check to carry out statistical procedures between two groups, and p＜0.05 is that otherness is remarkable.
5. method as claimed in claim 4, it is characterized in that: zebra fish histochemical stain or fluorescent staining concrete steps are as follows:
1) fixing
Remove the dried hen egg yolk culture fluid in the microwell plate, add 4% paraformaldehyde (PFA) zebra fish is fixed, need room temperature fix 4 hours (h) above or 4 ℃ fixedly spend the night;
2) dehydration
1. under the room temperature, at the 75%PBS/25% of 10ml propane diols, dewatered 10 minutes;
2. under the room temperature, at the 50%PBS/50% of 10ml propane diols, dewatered 10 minutes;
3. under the room temperature, at the 25%PBS/75% of 10ml propane diols, dewatered 10 minutes;
4. under the room temperature, at 100% propane diols of 10ml, dewatered 10 minutes;
3) dyeing
A: histochemical stain: dye with 1% oil red O stain liquid, 28 ℃ are spent the night;
B: fluorescent staining: dye with 10ng/mL Nile red dyeing liquor, 28 ℃ are spent the night;
4) decolouring
1. under the room temperature, at 100% propane diols of 10ml, decoloured 10 minutes;
2. under the room temperature, at the 25%PBS/75% of 10ml propane diols, decoloured 10 minutes;
3. under the room temperature, at the 50%PBS/50% of 10ml propane diols, decoloured 10 minutes;
4. under the room temperature, at the 75%PBS/25% of 10ml propane diols, decoloured 10 minutes
5. under the room temperature, at the 100%PBS of 10ml, decoloured 10 minutes;
5) PBST wash-out
With PBST wash-out 15min, repeat 4 times under the room temperature.
6. the zebra fish hyperlipemia model that obtains of the described method for building up of claim 1-5 is used for the purposes of hyperlipidaemic conditions research.
7. the zebra fish hyperlipemia model that obtains of claim 1 or 3 described method for building up is used for estimating or the purposes of screening blood lipid-lowering medicine.
8. purposes as claimed in claim 7 is characterized in that: further comprising the steps of:
(3) compound treatment
Remove the dried hen egg yolk culture fluid in the microwell plate, a plurality of experimental group are set: several compound treatment groups, 1 model group, 1 positive controls, 1 solvent control group and 1 blank group, each experimental group adds respectively the corresponding solution of equal volume, then constant temperature culture according to the specification of microwell plate;
(4) zebra fish histochemical stain or fluorescent staining;
(5) graphical analysis and/or microwell plate analysis;
(6) statistical analysis.
9. method for building up as claimed in claim 8 is characterized in that:
Preferably, the solution that the compound treatment group adds in the step (3) is testing compound solution, and the solution that positive controls adds is blood lipid-lowering medicine solution, and the solution that adds in the solvent control group is 0.1% DMSO;
Preferably, zebra fish histochemical stain or fluorescent staining may further comprise the steps: 1) fixing, and 2) dehydration, 3) dyeing, 4) decolouring, 5) the PBST wash-out;
Preferably, the graphical analysis concrete steps are: after zebra fish histochemical stain or the fluorescent staining of live body zebra fish finish, take pictures and preserve picture at microscopically; Carry out qualitative evaluation by fat stains intensity in the observation zebra fish blood on the one hand; Utilize on the other hand software that zebra fish afterbody blood vessel is carried out graphical analysis, carry out quantitative assessment by the fat stains intensity of calculating zebra fish afterbody blood vessel;
Preferably, microwell plate analysis concrete steps are: after the fluorescent staining of live body zebra fish, zebra fish is changed in 96 orifice plates, then every hole 1 tail places microwell plate fluorescence intensity under the multi-functional microwell plate analyzer;
Preferably, the statistical analysis step is: statistical procedures result with
Expression is relatively adopted the one-factor analysis of variance between many groups, relatively adopts Dunnett ' s T-check to carry out statistical procedures between two groups, and p＜0.05 is that otherness is remarkable.
Preferably, the zebra fish incubation time after the compound treatment is 72 hours.
10. method as claimed in claim 9, it is characterized in that: zebra fish histochemical stain or fluorescent staining concrete steps are as follows:
1) fixing
Remove the dried hen egg yolk culture fluid in the microwell plate, add 4% paraformaldehyde (PFA) zebra fish is fixed, need room temperature fix 4 hours (h) above or 4 ℃ fixedly spend the night;
2) dehydration
5. under the room temperature, at the 75%PBS/25% of 10ml propane diols, dewatered 10 minutes;
6. under the room temperature, at the 50%PBS/50% of 10ml propane diols, dewatered 10 minutes;
7. under the room temperature, at the 25%PBS/75% of 10ml propane diols, dewatered 10 minutes;
8. under the room temperature, at 100% propane diols of 10ml, dewatered 10 minutes;
3) dyeing
A: histochemical stain: dye with 1% oil red, 0 dyeing liquor, 28 ℃ are spent the night;
B: fluorescent staining: dye with 10ng/mL Nile red dyeing liquor, 28 ℃ are spent the night;
4) decolouring
6. under the room temperature, at 100% propane diols of 10ml, decoloured 10 minutes;
7. under the room temperature, at the 25%PBS/75% of 10ml propane diols, decoloured 10 minutes;
8. under the room temperature, at the 50%PBS/50% of 10ml propane diols, decoloured 10 minutes;
9. under the room temperature, at the 75%PBS/25% of 10ml propane diols, decoloured 10 minutes
10. under the room temperature, at the 100%PBS of 10ml, decoloured 10 minutes;
5) PBST wash-out
With PBST wash-out 15min, repeat 4 times under the room temperature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103776034A CN102907357A (en) | 2012-09-27 | 2012-09-27 | Building method and application of zebra fish hyperlipidemia model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103776034A CN102907357A (en) | 2012-09-27 | 2012-09-27 | Building method and application of zebra fish hyperlipidemia model |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102907357A true CN102907357A (en) | 2013-02-06 |
Family
ID=47606122
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012103776034A Pending CN102907357A (en) | 2012-09-27 | 2012-09-27 | Building method and application of zebra fish hyperlipidemia model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102907357A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104542389A (en) * | 2014-12-23 | 2015-04-29 | 中国科学院苏州生物医学工程技术研究所 | Preparation method for non-alcoholic fatty liver disease zebra fish |
| CN109105665A (en) * | 2018-09-05 | 2019-01-01 | 山东大学 | A kind of protectant method of screening turbot feed |
| CN109906979A (en) * | 2019-04-17 | 2019-06-21 | 上海大学 | A kind of method of zebra fish high cholesterol model foundation |
| CN112655651A (en) * | 2021-01-13 | 2021-04-16 | 叶繁全 | Method for inducing zebra fish thrombus model by using sodium laurate |
| CN112715435A (en) * | 2021-01-13 | 2021-04-30 | 叶繁全 | Method for preparing zebra fish thrombus model by using ferric chloride |
| CN113243466A (en) * | 2021-07-01 | 2021-08-13 | 常州鼠一鼠二生物科技有限公司 | Zebra fish hyperlipidemia model feed and preparation method and application thereof |
| CN114199840A (en) * | 2021-12-06 | 2022-03-18 | 山东省科学院生物研究所 | Heart comforting tablet quality control method based on biological effect |
-
2012
- 2012-09-27 CN CN2012103776034A patent/CN102907357A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104542389A (en) * | 2014-12-23 | 2015-04-29 | 中国科学院苏州生物医学工程技术研究所 | Preparation method for non-alcoholic fatty liver disease zebra fish |
| CN104542389B (en) * | 2014-12-23 | 2017-01-25 | 中国科学院苏州生物医学工程技术研究所 | Preparation method for non-alcoholic fatty liver disease zebra fish |
| CN109105665A (en) * | 2018-09-05 | 2019-01-01 | 山东大学 | A kind of protectant method of screening turbot feed |
| CN109906979A (en) * | 2019-04-17 | 2019-06-21 | 上海大学 | A kind of method of zebra fish high cholesterol model foundation |
| CN112655651A (en) * | 2021-01-13 | 2021-04-16 | 叶繁全 | Method for inducing zebra fish thrombus model by using sodium laurate |
| CN112715435A (en) * | 2021-01-13 | 2021-04-30 | 叶繁全 | Method for preparing zebra fish thrombus model by using ferric chloride |
| CN113243466A (en) * | 2021-07-01 | 2021-08-13 | 常州鼠一鼠二生物科技有限公司 | Zebra fish hyperlipidemia model feed and preparation method and application thereof |
| CN114199840A (en) * | 2021-12-06 | 2022-03-18 | 山东省科学院生物研究所 | Heart comforting tablet quality control method based on biological effect |
| CN114199840B (en) * | 2021-12-06 | 2023-11-17 | 山东省科学院生物研究所 | Quality control method of Xinkeshu tablet based on biological effect |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102907357A (en) | Building method and application of zebra fish hyperlipidemia model | |
| Dona et al. | Precision high-throughput proton NMR spectroscopy of human urine, serum, and plasma for large-scale metabolic phenotyping | |
| CN101968484B (en) | Method for screening mitochondria targeted compounds by using zebra fish | |
| CN102353663A (en) | Method for carrying out quantitative evaluation to compound hepatotoxicity by using zebra fish | |
| Esmaeili et al. | Proteomic investigation of liver and white muscle in efficient and inefficient Chinook salmon (Oncorhynchus tshawytscha): Fatty acid metabolism and protein turnover drive feed efficiency | |
| CN105424666A (en) | Method for quickly and quantitatively evaluating blood vessel generation promotion function of chemical compounds on zebra fish | |
| CN102288750B (en) | Method for establishing zebra fish P-glycoprotein inhibitor screening model and application thereof | |
| Adeyemo et al. | The effect of graded levels of dietary methionine on the haematology and serum biochemistry of broilers. | |
| CN102023207B (en) | Method for carrying out enzyme-linked immunoadsorption detection on integral zebra fish and application thereof | |
| CN104122355B (en) | Method for evaluating kidney toxicity of compounds through detecting contents of creatinine in zebra fish tissues | |
| Venter et al. | Establishing sampling confidence parameters: Effect of sampling and transport conditions on haemocyte and metabolite profiles of Greenshell mussels | |
| Aru et al. | The plasma metabolome of Atlantic salmon as studied by 1H NMR spectroscopy using standard operating procedures: effect of aquaculture location and growth stage | |
| CN108267584B (en) | Method for detecting pancreatic lipase activity in zebra fish body and application | |
| CN103430890B (en) | The method for building up of ROS detection model and its application in a kind of zebra fish body | |
| CN103348939A (en) | Establishment and application of zebra fish allergy detection model | |
| CN109085331A (en) | A kind of detection method of 2,4-DNT toxicity | |
| CN104142384B (en) | It is a kind of to screen with the method for protecting or improving renal function reactive compound | |
| CN105169415B (en) | A method of screening has protection zebra fish liver function reactive compound | |
| CN111505260A (en) | Method for establishing heavy metal oral bioassability and toxicity detection system | |
| CN109730028A (en) | A method of Models of Nonalcoholic Fatty Liver Disease is established using mouse | |
| CN109906979A (en) | A kind of method of zebra fish high cholesterol model foundation | |
| Cao et al. | The cyan fluorescent protein (CFP) transgenic mouse as a model for imaging pancreatic exocrine cells | |
| Escribano et al. | Utility of the discriminant analysis to categorize untrained Spanish Pure Bred horses on the basis of biochemical and muscle variables before and after a standardized exercise test | |
| CN105999303A (en) | Method for screening active compounds capable of adjusting gastrointestinal motility of zebrafish | |
| Jadhao et al. | Understanding the histology and hematobiochemical aspects of Kadaknath an Indian black fowl with an appealing taste native to Dhar district Madhya Pradesh |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 1, A District, 5 building, 88 Jiangling Road, Binjiang District, Zhejiang, Hangzhou 310052, China Applicant after: HANGZHOU HUNTER BIOTECHNOLOGY CO., LTD. Address before: Hangzhou City, Zhejiang province 311231 - Xiaoshan economic and Technological Development Zone Land in Zhejiang Province high-tech agricultural demonstration park Applicant before: Hangzhou Hunter Biotechnology, Inc. |
|
| COR | Change of bibliographic data | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130206 |
|
| WD01 | Invention patent application deemed withdrawn after publication |