CN102353663A - Method for carrying out quantitative evaluation to compound hepatotoxicity by using zebra fish - Google Patents
Method for carrying out quantitative evaluation to compound hepatotoxicity by using zebra fish Download PDFInfo
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Abstract
The invention relates to a method for carrying out a quantitative evaluation to compound hepatotoxicity by using zebra fish. The method comprises the following steps of zebra fish selection, compound treatment, qualitative analysis and/or quantitative analysis. The method of the present invention has the advantages of reliability, rapidity, high efficiency, high cost performance and the like, and realizes the high-throughput evaluation of compound hepatotoxicity in vivo. The method of the invention is capable of obviously raising the evaluation speed to the compound hepatotoxicity and has important meaning for accelerating large scale evaluation to the compound hepatotoxicity.
Description
Technical field
The present invention relates to compound test and drug toxicity and safety evaluatio field, be specifically related to a kind of simple, economy, high flux realizes utilizing the hepatotoxic method of zebra fish juvenile fish (larva) quantitative evaluation compound.
Background technology
Liver is parenchymatous organ maximum in the body, about 3% of adult's the about percentage of liveweight of liver weight.Liver has special anatomical position, institutional framework and physio-biochemical characteristics.Liver is not only as the vitals of human body material and energetic supersession in body.No matter chemical substance gets into body from which kind of approach, finally all arrives liver rapidly through blood circulation system, and especially the toxic chemical that absorbs from alimentary canal at first contacted with liver before getting into the body circulation.Liver is as the major organs of toxic chemical bio-transformation; Under certain condition; Toxic chemical particularly increases malicious exogenous chemical substances its toxic metabolite product after liver metabolism transforms through internal metabolism and can at first damage liver very easily to the liver effect of causing damage.Therefore liver is the important target organ [1] of toxic chemical.
Many chemical substances (comprising medicine) in the human environment all can be to the liver effect of causing damage in the doses level.Every chemical substance of liver damage that can cause all can be described as the hepatotoxicity wind agitation compound.The hepatic injury that chemical toxicant causes can be divided into acute liver damage and chronic liver injury.Acute liver damage generally is that not full-time certain hepatotoxicant of contact of more heavy dose of hepatotoxicant of short term contact or liver function causes the common necrosis of liver cells of pathological change, steatosis, cholestasis etc.Chronic liver injury can cause because of the long-term low dose hepatotoxicant, the also sequelae that can cause by an acute necrosis, and pathological change comprises fiberization, cirrhosis, canceration etc.
Hepatotoxicity wind agitation is the major reason of the exploitation of restriction chemicals, production and application.An investigation shows that 84 kinds of compounds stop further drug development because of clinical toxicity, and wherein 15 kinds (18%) is because hepatotoxicity wind agitation.Hepatotoxicity wind agitation also is the main cause that marketed drug reclaims because of safety problem.From 1961 to 1992, method, moral, English and the U.S. reclaimed 181 kinds of medicines altogether, and wherein 23 kinds is because hepatotoxicity wind agitation (13%) [2].
The hepatotoxic method of assessing compound has two big types, and one type is in vitro test, and another kind of is in vivo studies.Hepatotoxic in vitro test detection method has following several: 2. 3. 4. 5. 6. genetically engineered cell [3] of subcellular components of hepatic cell line of isolated liver cell of hepatic tissue section of liver perfusion 1. exsomatizes.The whole animal test is adopted in the in vivo studies evaluation.The most frequently used animal used as test is that secondly rat or mouse are hamster, cavy, rabbit, dog etc.The detection method of in vivo studies has following several types: 1. the serum zymetology detects 2. liver excretory function and detects 4. liver organization pathological examination [1] of detection that 3. liver chemical composition changes.
Though it is classical compound hepatotoxicity wind agitation evaluation method that research shows above certain methods, and a lot of drawbacks and limitation are arranged.The required expense of experiment in vitro is lower; But the cyclic transformation and the medicine loop distribution in vivo that have lacked biological integral; The whole biologically active that can not really reflect medicine; The common compound amount of in vivo studies is big, experimental period is long; Complicated operation; Cost an arm and a leg, poor specificity, and cannot carry out high flux screening.Therefore set up and a kind ofly can well simulate toxic chemical process in vivo, it is significant to screen the hepatotoxicity wind agitation compound quickly and easily again.
Zebra fish is a kind of model organism of novelty, being applied in the toxicity of compound evaluation experimental more and more widely.With compare with in-vitro screening model in the traditional body; Live body zebra fish screening model has many advantages, has overcome the drawback that original external model screening model experimental period in the shortcoming of absorption, distribution, metabolism and the checking of drainage link and conventional bulk is long, cost is high.Compare with nematode, fruit bat, zebra fish is a kind of vertebrate, and is similar with the human gene height, finds that through Britain expert's predictive genes the gene of zebra fish and the similarity of human gene are up to about 85%, and the experimental result comparability is strong.Compare with 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 big, and the credible result degree is high, required expense low [4].The more important thing is that zebra fish has inherent advantage [4,5]: 1) feeding cost is low, 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 behind fertilization 24h, the main histoorgan original hase of zebra fish forms, can be research a large amount of samples and short experimental period are provided; 4) embryo and juvenile fish are transparent, and be in vitro fertilization, ectogenesis, but Direct observation, and can analyze a plurality of tracts simultaneously; 5) embryo has the yolk that nutrition can be provided, and does not need feeding in first week, the interaction of compound and food component in the time of can avoiding compound treatment; 6) embryo is very little, and young fish length has only 1-4mm, can in 6,12,24,48 or 96 orifice plates of a standard, analyze; 7) administering mode is simple: water-soluble small-molecule substance can be directly in skin, the gill and digestive system get into the zebra fish body; Water-fast material, macromolecular substances and protein can carry out microinjection.Therefore zebra fish can be used as the hepatotoxic research material of good compound.
Utilize the hepatotoxic report of zebra fish research compound less at present, and concentrate on mostly and carry out genetic analysis aspect [6-11] forming the relevant factor with liver.McGra Patricia and Li Chunqi mention the hepatotoxicity wind agitation that can estimate medicine with zebra fish at one piece of review article; Zebra fish liver out-of-shape after the hepatotoxicity wind agitation drug treating, edge fog, hepatic tissue color burn become dark-brown or black, but this article is just mentioned with the hepatotoxic possibility of zebra fish qualitative evaluation compound [4].Sadler etc. utilize the mutant zebra fish to set up the model [12] of liver diseases such as estimating hepatomegaly, fatty liver, cholestasis.Amali etc. utilize thioacetamide to handle the zebra fish model [13] that zebra fish is set up screening anti-fatty liver medicine.Passeri etc. utilize the molecular mechanism [14] of the steatosis of zebra fish research alcohol-induced.Patent 20101070675.2 discloses a kind of method with model organism zebra fish screening liver injury medicament, and zebra fish is screened but this method utilization is grown up, and experimental period is long, required expense is higher, can not carry out high flux screening.
Summary of the invention
In order to overcome above-mentioned existing existing defective of evaluation hepatotoxicity wind agitation compound method and deficiency, the inventor has aimed to provide experiment analytical method in a kind of convenient and swift, economical and practical body that utilizes zebra fish quantitative screening hepatotoxicity wind agitation compound through research.
The present invention realizes through following technical scheme:
A kind ofly it is characterized in that, may further comprise the steps with the hepatotoxic method of zebra fish quantitative evaluation compound:
(1) zebra fish is chosen
Choose the zebra fish of after fertilization 2-7 days normal development, put into microwell plate,
(2) compound treatment
Remove the nutrient solution in the microwell plate, a plurality of experimental group are set: testing compound processed group, positive controls, solvent control group, blank group, each experimental group adds corresponding solution, constant temperature culture then respectively according to the specification of microwell plate;
(3) qualitative analysis and/or quantitative test.
Preferably, described step (1) zebra fish is chosen the zebra fish of fertilization after 2 days;
Preferably, in described step (2) compound treatment zebra fish 28 ℃ of following constant temperature culture;
Preferably, zebra fish was cultivated 72 hours in described step (2) compound treatment;
Preferably, described microwell plate is selected 6-384 hole microwell plate for use;
Preferably, the solution that uses in the positive controls in the described step (2) is phenixin;
Preferably, the solution that uses in the solvent control group in the described step (2) is 0.1% DMSO;
Preferably, the solution that uses in described step (2) the empty control group is breeding water;
Preferably, the qualitative analysis step is following in the described step (3):
A) remove breeding water in the microwell plate, add equal-volume 1 * MESAB (methane-sulforic acid) anesthesia zebra fish, after fixing with 3% methylcellulose glue, place again and observe the zebra fish liver under the dissecting microscope;
B) can observe transparent zebra fish liver in solvent control group and the blank group; Positive controls can be observed the zebra fish liver of dark-brown or black non transparent; Observe compound treatment group zebra fish liver, show dark-brown or black non transparent, can judge that then this compound has hepatotoxicity wind agitation like compound treatment group zebra fish liver;
Preferably, the quantitative test step is following in the described step (3):
Utilize image processing software to carry out image quantitative analysis; Selected whole liver area calculates liver lightness summation, and each concentration group is got the picture of 10 zebra fishs and analyzed; Hepar damnification rate computing formula is:
Detailed Description Of The Invention
It is a kind of with the hepatotoxic method of model organism zebra fish quantitative evaluation compound that the present invention provides, and concrete scheme is:
Reagent and instrument
Phenixin (CCl
4) available from Shanghai Ling Feng chemical reagent company limited.Other reagent are provided by the prosperous Science and Technology Ltd. of Beijing ancient cooking vessel state.
Dissecting microscope (SMZ645, Nikon company, Japan); Power focus continuous zoom fluorescent microscope (AZ100, Nikon company, Japan) image analysis software (NIS-Elements D3.1, Nikon company, Japan).
1 zebra fish is chosen
Get 4～5 couples of zebra fish parents mating, according to Westerfield[15] method hatching embryo.The zebra fish that will be in the optimization process stage places observation under the dissecting microscope, and the zebra fish of the normotrophic zebra fish after fertilization of picking 2 days (2dpf) moves in 6,12,24,48, the 96 or 384 hole microwell plates, the zebra fish of putting into varying number according to the microwell plate specification.
2 compound treatment
8 experimental group are set: 5 compound treatment groups, 1 positive controls, 1 solvent control group, 1 blank group.Remove nutrient solution in the microwell plate, add certain volume (deciding) concentration in the compound treatment group and be respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M testing compound solutions according to the microwell plate specification; Add equal-volume 5mM hepatotoxicity wind agitation compound phenixin (CCl in the positive controls
4); Phenixin is a kind of toxic chemical [16] that can cause human and rodent hepatic fibrosis-renal tubular ectasia syndrome, cirrhosis.Add equal-volume solvent (being generally 0.1%DMSO) in the solvent control group; Add the equal-volume breeding water in the blank group.In 28 ℃ of constant incubators, cultivated 72 hours.
3 qualitative analyses
Remove breeding water in the microwell plate, add equal-volume 1 * MESAB (methane-sulforic acid) anesthesia zebra fish, after fixing with 3% methylcellulose glue, place again and observe the zebra fish liver under the dissecting microscope.The zebra fish liver of normal development shows as abundant, the transparent organ of blood flow under dissecting microscope.The zebra fish hepatic tissue sex change that the hepatotoxicity wind agitation compound is induced shows as that penetrability is poor, the organ (Fig. 1) of dark-brown or black.Correlative study shows that the liver of zebra fish dark-brown or black is owing to the liver cell hydropic degeneration causes (Fig. 2).Can observe transparent zebra fish liver in solvent control group and the blank group; Positive controls can be observed the zebra fish liver of dark-brown or black non transparent; Observe compound treatment group zebra fish liver, show dark-brown or black non transparent, can judge that then this compound has hepatotoxicity wind agitation like compound treatment group zebra fish liver.
4 quantitative test
Utilize NIS-Elements D3.1 high vision process software to carry out image quantitative analysis.Selected whole liver area calculates liver lightness summation (Fig. 3), and after the sex change of zebra fish liver, the liver color becomes claims black, and transparent brightness reduces, thereby liver lightness summation can be used as the index of liver denaturation degrees.Each concentration group is got the picture of 10 zebra fishs and is analyzed.
Hepar damnification rate computing formula is:
For example: if compound treatment group lightness summation is for being respectively 1000,900,700,400,300; Positive controls lightness summation is 500; Group of solvents lightness summation is 1000; Then get according to computing formula: positive controls hepar damnification rate is 50%, and compound treatment group hepar damnification rate is respectively 0%, 10%, 30%, 60%, 70% (Fig. 4).
Experimental data
said that many groups were compared using analysis of variance between the two groups were compared using Stuendent's? t test was used for statistical analysis.But according to statistical procedures result quantitative evaluation compound hepatotoxicity wind agitation.
That live body zebra fish quantitative evaluation compound hepatotoxicity wind agitation model provided by the invention has is simple for production, quick, success ratio is high, good reproducibility and advantage such as reliable and stable, has reduced hepatotoxicity wind agitation modelling cost, has improved the reliability of experimental studies results.The present invention provides a kind of method for building up of live body zebra fish hepatonecrosis evaluation model, and a kind of method for building up of live body zebra fish fatty liver evaluation model is provided simultaneously.Through image is analyzed, the hepatotoxicity wind agitation of quantitative evaluation compound is the innovation part that the present invention shows unique characteristics.Compare advantages such as that the live body zebra fish hepatotoxicity wind agitation model that the present invention sets up has is easy, quick, economic, efficient, high flux with animal liver toxic model in the past.The present invention is significant to the screening of quickening the hepatotoxicity wind agitation compound.Specifically, the present invention has following advantage:
1) in vivo---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 in vivo, distribution, metabolism and drainage, really reflects the whole biologically active of medicine.
2) high flux---the zebra fish juvenile fish is very little, has only the 1-4 millimeter, can in 6,12,24,48,96 or 384 orifice plates of a standard, analyze with lacking experimental period to make zebra fish become a kind of ideal model that can carry out high throughput automated drug disposition screening.
3) quantitative---through graphical analysis, zebra fish liver denaturation degrees is quantized.
4) economy---required expense is low; With the monkey is that the screening experiment of testing carrier expends greater than 10 dollars every every day; The screening experiment that with the mouse is the experiment carrier expends greater than 1 dollar every every day, and is that the screening experiment of experiment carrier expends less than 0.01 dollar every every day with the zebra fish.
5) compound amount is few---and the detection compound consumption is few, only needs several milligrams usually, and traditional screening experiment then needs the compound more than several milligrams.
6) 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 complicated operating process is easy to generate false positive results.
7) quick---experimental period is short, can in 2～3 days, accomplish; And rodent often needs the time of several weeks to several months, and monkey often needs the time of several months to several years.Zebra fish at first 72 hours with interior completion embryonic development.Most internals comprises cardiovascular system, intestines, liver and kidney, rapid shaping in 24-48 hour, and traditional experiment carrier mouse and monkey then needed 21 days and 9 months can accomplish embryonic development respectively.
8) efficient---zebrafish embryo and juvenile fish are transparent, can observe a plurality of tracts simultaneously, and experiment analytical method is simple, quick.
9) predictability---the gene of zebra fish and the similarity of human gene are up to about 85%, and its biological function is highly similar with the mammal and the mankind, and the experimental result comparability is strong, and predictability is good reliably.
10) intuitive is strong---and embryo and juvenile fish are transparent, can directly place microscopically to observe zebra fish hepatonecrosis situation.
11) high, the good reproducibility of stability---repeated experiments of the present invention repeatedly, it is basic identical that institute obtains experimental result.
Description of drawings
The zebra fish liver sex change photo that Fig. 1 induces for The compounds of this invention.A is the zebra fish of normal development 5dpf;
B is CCl
4The liver sex change of inducing.Arrow mark part is a liver among the figure.
Fig. 2 is CCl of the present invention
4The liver tissue slices photo of handling.A is the liver organization h and E dyeing of the zebra fish of normal development; B is CCl
4The hepatic tissue h and E dyeing of the liver sex change of inducing.
Fig. 3 measures process flow diagram for liver lightness summation of the present invention.
The hepar damnification rate figure that Fig. 4 induces for The compounds of this invention.
Fig. 5 is the embodiment of the invention 1 zebra fish different developmental phases hepar damnification rate figure.
Fig. 6 is the hepar damnification rate figure of 2 different disposal times of the embodiment of the invention.
The zebra fish liver sex change photo that Fig. 7 induces for the embodiment of the invention 3 tamosifens.A is 120 hours a zebra fish of normal development; B is 20 μ M tamosifen processed group, and the liver of observing zebra fish becomes black.Arrow mark part is a liver among the figure.
The hepar damnification rate figure that Fig. 8 induces for the embodiment of the invention 3 tamosifens.
The zebra fish liver sex change photo that Fig. 9 induces for the embodiment of the invention 4 trichloro biphenyls.A is 120 hours a zebra fish of normal development; B is 100 μ M trichloro biphenyl processed group, observes the black liver.Arrow mark part is a liver among the figure.
The hepar damnification rate figure that Figure 10 induces for the embodiment of the invention 4 trichloro biphenyls.
Figure 11 is the alcohol induced zebra fish liver sex change photo of the embodiment of the invention 5.A is 120 hours a zebra fish of normal development; B is 1.5% ethanol treatment groups, observes the black liver.Arrow mark part is a liver among the figure.
Figure 12 is the alcohol induced hepar damnification rate figure of the embodiment of the invention 5.
Embodiment
The selection of 1 used zebra fish stage of development of zebra fish hepatotoxicity wind agitation modelling of embodiment
1 zebra fish is chosen
The zebra fish of 0dpf, 1dpf, 2dpf, 3dpf, 4dpf is placed observation under the dissecting microscope, and the zebra fish of normotrophic each stage of development of picking moves into 6,12 respectively; 24; In 48, the 96 or 384 hole microwell plates, the zebra fish of putting into varying number according to the microwell plate specification.
2 compound treatment
5 experimental group (every group of zebra fish that is respectively 0dpf, 1dpf, 2dpf, 3dpf, 4dpf) are set, and each experimental group comprises 1 5mM phenixin (CCl
4) processed group, 1 solvent control group, 1 blank group.Add equal-volume 0.1%DMSO in the solvent control group; Add the equal-volume breeding water in the blank group, in 28 ℃ of constant incubators, cultivate 72h.
3 qualitative analyses
Remove breeding water in the microwell plate, add equal-volume 1 * MESAB (methane-sulforic acid) anesthesia zebra fish, after fixing with 3% methylcellulose glue, place again under the dissecting microscope and observe.The zebra fish that most of 2dpf, 3dpf, 4dpf handle under dissecting microscope observes the liver of dark-brown or black.But because the 2dpf drug treating time is the shortest and bring out the sex change of zebra fish liver effectively.Therefore in setting up zebra fish hepatotoxicity wind agitation model, select the zebra fish stage of development of 2dpf as the best.
4 quantitative test
Utilize NIS-Elements D3.1 high vision process software to carry out image quantitative analysis.Selected whole liver area, each concentration group of calculating liver lightness summation is got the picture of 10 zebra fishs and is analyzed.Hepar damnification rate computing formula is:
Experimental data
said that many groups were compared using analysis of variance between the two groups were compared using Stuendent's? t test was used for statistical analysis.Statistical procedures result shows: 0dpf, 1dpf, 2dpf, 3dpf, 4dpf zebra fishing line hepar damnification rate are respectively (4.24 ± 2.37) %, (6.02 ± 3.67) %, (30.75 ± 4.18) %, (28.34 ± 3.74) %, (27.28 ± 3.48) % (Fig. 5).This shows that zebra fish hepar damnification rate raise earlier and afterwards reduces along with the zebra fish stage of development, and 2dpf zebra fish hepar damnification rate is the highest.Through variance analysis, phenixin (CCl
4) the hepar damnification rate of processed group is higher than solvent control group, difference has statistical significance (P＜0.05).
The selection in 2 zebra fish hepatotoxicity wind agitation model drug processing times of embodiment
1 zebra fish is chosen
The zebra fish of 2dpf is placed observation under the dissecting microscope, and the normotrophic zebra fish of picking moves into 6,12,24,48 respectively, in the 96 or 384 hole microwell plates, and the zebra fish of putting into varying number according to the microwell plate specification.
2 compound treatment
Each microwell plate is provided with 1 5mM phenixin (CCl
4) processed group, 1 solvent control group, 1 blank group.Add equal-volume 0.1%DMSO in the solvent control group; Add the equal-volume breeding water in the blank group, four microwell plates are put into 28 ℃ of constant incubators cultivate 12h, 24h, 48h, 72h, 96h respectively.
3 qualitative analyses
Remove breeding water in the microwell plate, add equal-volume 1 * MESAB (methane-sulforic acid) anesthesia zebra fish, after fixing with 3% methylcellulose glue, place again under the dissecting microscope and observe.Under dissecting microscope, handle 72,96 hours 5mM phenixin (CCl
4) the processed group zebra fish observes the liver of dark-brown or black.Therefore in setting up zebra fish hepatotoxicity wind agitation model, select 72 hours Times Created as best zebra fish hepatotoxicity wind agitation model.
4 quantitative test
Utilize MS-Elements D3.1 high vision process software to carry out image quantitative analysis.Selected whole liver area calculates liver lightness summation, and each concentration group is got the picture of 10 zebra fishs and analyzed.Hepar damnification rate computing formula is:
Experimental data
said that many groups were compared using analysis of variance between the two groups were compared using Stuendent's? t test was used for statistical analysis.Statistical procedures result shows: phenixin (CCl
4) the zebra fish hepar damnification rate of handling 24h, 48h, 72h, 96h is respectively (4.44 ± 1.17) %, (9.57 ± 2.10) %, (29.78 ± 1.93) %, (28.89 ± 1.96) % (Fig. 6); This shows the prolongation along with the processing time, and the hepar damnification rate increases gradually.Through variance analysis, 5mMCCl
4The hepar damnification rate that processed group is handled 72h is higher than solvent control group, and difference has statistical significance (P＜0.05), and 5mMCCl
4The processed group processed group is handled no difference of science of statistics between 24h, the 48h, therefore selects SPT 72h as the compound optimization process time.
Embodiment 3 zebra fish models are estimated use in medicament-induced hepatotoxicity
1 zebra fish is chosen
The zebra fish of 2dpf is placed observation under the dissecting microscope, and the normotrophic zebra fish of picking moves into 6,12,24,48 respectively, in the 96 or 384 hole microwell plates, and the zebra fish of putting into varying number according to the microwell plate specification.
2 drug treating
8 experimental group are set: 5 drug treating groups, 1 positive controls, 1 solvent control group, 1 blank group.Drug treating group concentration is respectively 0.1 μ M, 1 μ M, 5 μ M, 10 μ M, 20 μ M tamosifens (tamoxifen).Tamosifen is a kind of selective estrogen receptor modulators.Estrogenic some activity of interfere.Tamosifen is used to treat some breast cancer and oophoroma, is a kind of known medicine with hepatotoxicity [17]; 5mM phenixin (CCl
4) as positive controls; Add equal-volume 0.1%DMSO in the solvent control group; Add the equal-volume breeding water in the blank group.In 28 ℃ of constant incubators, cultivate 72h.
3 qualitative analyses
Remove breeding water in the microwell plate, add equal-volume 1 * MESAB (methane-sulforic acid) anesthesia zebra fish, after fixing with 3% methylcellulose glue, place again under the dissecting microscope and observe.20 μ M tamosifen processed group zebra fishs observe the liver (Fig. 7) of black under dissecting microscope, and it is downright bad therefore to estimate the tamosifen inducing hepatocyte qualitatively, is the hepatotoxicity wind agitation compound.
4 quantitative test
Utilize NIS-Elements D3.1 high vision process software to carry out image quantitative analysis.Selected whole liver area calculates liver lightness summation, and each concentration group is got the picture of 10 zebra fishs and analyzed.Hepar damnification rate computing formula is:
Experimental data
said that many groups were compared using analysis of variance between the two groups were compared using Stuendent's? t test was used for statistical analysis.Statistical procedures result shows: tamosifen processed group zebra fish hepar damnification rate is respectively (3.76 ± 1.06) %, (8.44 ± 1.27) %, (15.77 ± 2.10) %, (24.18 ± 1.63) % (46.53 ± 2.13) % (Fig. 8); This shows the increase along with tamosifen concentration, and the hepar damnification rate increases gradually.Through variance analysis, 20 μ M tamosifen (tamoxifen) processed group hepar damnification rates are higher than solvent control group, and difference has statistical significance (P＜0.05).
Embodiment 4 zebra fish models are estimated environmental compound trichloro biphenyl hepatotoxicity wind agitation
1 zebra fish is chosen
The zebra fish of 2dpf is placed observation under the dissecting microscope, and the normotrophic zebra fish of picking moves into 6,12,24,48 respectively, in the 96 or 384 hole microwell plates, and the zebra fish of putting into varying number according to the microwell plate specification.
2 compound treatment
8 experimental group are set: 5 compound treatment groups, 1 positive controls, 1 solvent control group, 1 blank group.Compound treatment group concentration is respectively 0.1 μ M, 1 μ M, 10 μ M, 50 μ M, 100 μ M trichloro biphenyls; Trichloro biphenyl is a kind of of polychlorinated biphenyl; Polychlorinated biphenyl is the general designation of the different biphenyl chlorine-containing compound of many chloride numbers, is extensively applied to industrial transformers and capacitor at twentieth century.Research shows that polychlorinated biphenyl is a kind of hepatotoxicity wind agitation compound [18]; 5mM phenixin CCl
4As positive controls; Add equal-volume 0.1%DMSO in the solvent control group; Add the equal-volume breeding water in the blank group.In 28 ℃ of constant incubators, cultivate 72h.
3 qualitative analyses
Remove breeding water in the microwell plate, add equal-volume 1 * MESAB (methane-sulforic acid) anesthesia zebra fish, after fixing with 3% methylcellulose glue, place again under the dissecting microscope and observe.Ring 100 μ M trichloro biphenyl processed group zebra fishs observe the liver (Fig. 9) of black under dissecting microscope, and therefore estimating trichloro biphenyl qualitatively is the hepatotoxicity wind agitation compound.
4 quantitative test
Utilize NIS-Elements D3.1 high vision process software to carry out image quantitative analysis.Selected whole liver area calculates liver lightness summation, and each concentration group is got the picture of 10 zebra fishs and analyzed.Hepar damnification rate computing formula is:
Experimental data
said that many groups were compared using analysis of variance between the two groups were compared using Stuendent's? t test was used for statistical analysis.Statistical procedures result shows: trichloro biphenyl processed group zebra fish hepar damnification rate is respectively (3.56 ± 1.06) %, (5.34 ± 1.37) %, (10.87 ± 2.10) %, (19.18 ± 1.53) % (35.43 ± 2.10) % (Figure 10); This shows the increase along with trichloro biphenyl concentration, and the hepar damnification rate increases gradually.Through variance analysis, 100 μ M trichloro biphenyl processed group hepar damnification rates are higher than solvent control group, and difference has statistical significance (P＜0.05).
1 zebra fish is chosen
The zebra fish of 2dpf is placed observation under the dissecting microscope, and the normotrophic zebra fish of picking moves into 6,12,24,48 respectively, in the 96 or 384 hole microwell plates, and the zebra fish of putting into varying number according to the microwell plate specification.
2 compound treatment
8 experimental group are set: 5 compound treatment groups, 1 positive controls, 1 solvent control group, 1 blank group.Compound treatment group concentration is respectively 0.1%, 0.25%, 0.5%, 1%, 1.5% (v/v) ethanol, and long-term excessive drinking can cause fatty liver [19]; 5mM phenixin CCl
4As positive controls; Add equal-volume 0.1%DMSO in the solvent control group; Add the equal-volume breeding water in the blank group.In 28 ℃ of constant incubators, cultivate 72h.
3 qualitative analyses
Remove breeding water in the microwell plate, add equal-volume l * MESAB (methane-sulforic acid) anesthesia zebra fish, after fixing with 3% methylcellulose glue, place again under the dissecting microscope and observe.1.5% (v/v) ethanol treatment groups zebra fish observes the liver (Figure 11) of dark-brown and black under dissecting microscope, and therefore estimating ethanol qualitatively is the hepatotoxicity wind agitation compound.
4 quantitative test
Utilize NIS-Elements D3.1 high vision process software to carry out image quantitative analysis.Selected whole liver area calculates liver lightness summation, and each concentration group is got the picture of 10 zebra fishs and analyzed.Hepar damnification rate computing formula is:
Experimental data
said that many groups were compared using analysis of variance between the two groups were compared using Stuendent's? t test was used for statistical analysis.Statistical procedures result shows: environmental compound processed group zebra fish hepar damnification rate is respectively (3.56 ± 1.06) %, (4.34 ± 1.37) %, (14.87 ± 2.10) %, (25.18 ± 1.53) % (35.43 ± 2.10) % (Figure 12); This shows the increase along with concentration of alcohol, and the hepar damnification rate increases gradually.Through variance analysis, 1.5% (v/v) ethanol treatment groups hepar damnification rate is higher than solvent control group, and difference has statistical significance (P＜0.05).
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Claims (10)
1. one kind with the hepatotoxic method of zebra fish quantitative evaluation compound, it is characterized in that, may further comprise the steps:
(1) zebra fish is chosen
Choose the zebra fish of after fertilization 2-7 days normal development, put into microwell plate;
(2) compound treatment
Remove the nutrient solution in the microwell plate, a plurality of experimental group are set: testing compound processed group, positive controls, solvent control group, blank group, each experimental group adds corresponding solution, constant temperature culture then respectively according to the specification of microwell plate;
(3) qualitative analysis and/or quantitative test.
2. the hepatotoxic method of assessing compound according to claim 1 is characterized in that: described step (1) zebra fish is chosen 2 days zebra fish of after fertilization.
3. the hepatotoxic method of assessing compound according to claim 1 is characterized in that: zebra fish is in 28 ℃ of following constant temperature culture in described step (2) compound treatment.
4. the hepatotoxic method of assessing compound according to claim 1 is characterized in that: zebra fish was cultivated 72 hours in described step (2) compound treatment.
5. the hepatotoxic method of assessing compound according to claim 1 is characterized in that: described microwell plate is selected 6-384 hole microwell plate for use.
6. the hepatotoxic method of assessing compound according to claim 1 is characterized in that: the solution that uses in the positive controls in the described step (2) is phenixin.
7. the hepatotoxic method of assessing compound according to claim 1 is characterized in that: the solution that uses in the solvent control group in the described step (2) is 0.1% DMSO.
8. the hepatotoxic method of assessing compound according to claim 1 is characterized in that: the solution that uses in described step (2) the empty control group is the zebra fish breeding water.
9. the hepatotoxic method of assessing compound according to claim 1 is characterized in that: the qualitative analysis step is following in the described step (3):
A) remove breeding water in the microwell plate, add equal-volume 1 * MESAB (methane-sulforic acid) anesthesia zebra fish, after fixing with 3% methylcellulose glue, place again and observe the zebra fish liver under the dissecting microscope;
B) can observe transparent zebra fish liver in solvent control group and the blank group; Positive controls can be observed the zebra fish liver of dark-brown or black non transparent; Observe compound treatment group zebra fish liver, show dark-brown or black non transparent, can judge that then this compound has hepatotoxicity wind agitation like compound treatment group zebra fish liver.
10. the hepatotoxic method of assessing compound according to claim 1 is characterized in that: the quantitative test step is following in the described step (3):
Utilize image processing software to carry out image quantitative analysis; Selected whole liver area calculates liver lightness summation, and each concentration group is got the picture of 10 zebra fishs and analyzed; Hepar damnification rate computing formula is:
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