CN102600156A - Establishment method and application of zebrafish multiple sclerosis model - Google Patents
Establishment method and application of zebrafish multiple sclerosis model Download PDFInfo
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- CN102600156A CN102600156A CN2012100160726A CN201210016072A CN102600156A CN 102600156 A CN102600156 A CN 102600156A CN 2012100160726 A CN2012100160726 A CN 2012100160726A CN 201210016072 A CN201210016072 A CN 201210016072A CN 102600156 A CN102600156 A CN 102600156A
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Abstract
The invention relates to an establishment method for a zebrafish multiple sclerosis model, and a method for screening therapeutic medicines for multiple sclerosis or evaluating compound-induced nerve myelin injuries by using the animal model. The establishment method of the zebrafish multiple sclerosis model mainly comprises the following steps of: zebrafish selection, compound treatment, whole zebrafish immunofluorescent staining, image analysis and/or microplate selection, and statistical analysis. The method disclosed by the invention has the advantages of being economic, simple and convenient, rapid, efficient, less in the dosage of compound and the like, can realize a purpose of screening the therapeutic medicines for multiple sclerosis with a high flux in vivo, and is significant for acceleration for the research and development process of the therapeutic medicines for multiple sclerosis, and therapy for patients with multiple sclerosis.
Description
Technical field
The invention belongs to drug screening (evaluation) field; Be specifically related to a kind of method for building up of easy, economic, high-throughout Brachydanio rerio multiple sclerosis model, and utilize the method for this animal model screening multiple sclerosis therapy medicine or the inductive neural myelin damage of assessing compound.
Background technology
Multiple sclerosis (multiple sclerosis; MS) be the autoimmune disease that becomes characteristics with central nervous system's white matter demyelinating disease; It possibly be the individual nerve immunity process that takes place with the environmental factors effect of inheritance susceptible; Because its sickness rate is higher, becomes the chronic course of disease and tend to youngster to suffer from, and become one of most important nervous system disease
[1]Epidemiology shows 400,000 routine patients to be arranged disease approximately in North America, and China has 6.5 ten thousand routine patients approximately, and the patient in the whole world surpasses 1,000,000 examples, and female patient is more than the male, is mainly in the crowd of 20-40 year age bracket
[2]The MS new drug development is one of key project of international main drugmaker new drug development.Although the present clinical multiple MS medicine that had comprises hormones, immunomodulating class, immunosuppressant class, statins etc., these medicines are limited to the curative effect of MS, and toxic and side effects is big, and expense is high, therefore press for the new MS medicine of research and development
[3]
MS pathology damage mechanism and the cell-mediated autoimmune response of T, the dysfunction of cholinergic nerve, the activation of enzymes such as cytosolic phospholipase A2, kallikrein and a large amount of free radical releases etc. are closely related
[4]Traditional medicine thinks that MS is by the cell-mediated autoimmune disease of T, and promptly human body is organized himself and started immune attack and cause morbidity.Reactive encephalomyelitis (the experimental allergic encephalomyelitis of the animal model experiment paraphilia of MS; EAE) confirm that also the inflammatory process that activated T cells has started MS causes pathological changes such as demyelination; And follow inflammatory cell to invade profit and inflammatory mediator release, produce a large amount of free radicals, cytokine and chemotactic factor isoreactivity material.These active substances can directly become the reason of morbidity or increase the weight of factor, and (central nervous system, CNS) integrity of blood vessel and tissue destroys and dysfunction to cause the central nervous system
[2-5]
The clinical manifestation of EAE and pathological change are extremely similar with acute multiple sclerosis, thereby become the best animal model of research MS immunopathology mechanism and experimental treatment thereof
[6]EAE is central nervous system's specific autoimmune disease of a kind of artificial induction, can in multiple animal strain, induce generation.EAE can induce generation through the homogenate of active immunity myeloid tissue; Or, also can produce through the induced t cell of passive injection myelin antigenic response activity through the emulsive myelin antigen protein of immune Freund's complete adjuvant such as MBP, proteolipin albumen or myelin oligodendrocyte glycoprotein
[7-14]Though EAE is the method for classical research MS, this model experiment cycle length, complicated operation, cost are high, specificity not by force, poor repeatability and can't realize the purpose of high-flux medicaments sifting.
The cell in vitro model that does not also have at present suitable research myelin.There is research once to adopt dorsal root ganglion (DOG) neuron and oligodendrocyte associating cultured method to explore the influence that somatomedin forms myelin in the myelin forming process
[15-16]But setting up of cell associating cultivating system is consuming time longer; Method is unstable, and poor repeatability does not still suit to carry out external high flux MS medicine screening; And cell in vitro lacks metabolic conversion and the intravital loop distribution of medicine at biological integral, can not reflect medicine truth in vivo
[17]Therefore set up the good aids drug of a kind of ability process in vivo, the animal model that can estimate and screen the multiple sclerosis therapy medicine again quickly and easily is significant.
Brachydanio rerio is a kind of model organism of novelty.With compare with in-vitro screening model in the traditional body; Live body Brachydanio rerio 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.Brachydanio rerio is a kind of vertebrates, with human gene's similarity up to about 85%, 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, and required expense is low
[18]The more important thing is that zebra fish model has inherent advantage
[18-19]: 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 at after fertilization 24 hours (24hpf), the main histoorgan original hase of Brachydanio rerio forms, can be research a large amount of samples and short experimental period are provided; 4. embryo and juvenile fish are transparent, external fertilization, and ectogenesis, but direct observation, and can analyze a plurality of tracts simultaneously; 5. the embryo has the yolk that nutrition can be provided, and does not need feeding in first week, the interaction of chemical compound and food component in the time of can avoiding compound treatment; 6. embryo's volume is 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 Brachydanio rerio body; Water-fast material, macromolecular substances and protein can carry out microinjection.Therefore Brachydanio rerio can be used as the interior vertebrates model of high flux body of good evaluation and screening of medicaments.
Research shows that the architectural characteristic and the cell lineage relation of the myelin oligodendrocyte of Brachydanio rerio have the comparability of height with mammal
[20-23]Form relevant gene (like the dm20 gene, mbp gene and P0 gene etc.) with myelin in the Brachydanio rerio body and have good homology with mammal
[23]Although some is different for these genes and mammal, the gene order of high conservative enough gives expression to functionally similar albumen (table 1)
[24]When Brachydanio rerio approximately grows 2dpf in the brain compact myelin form, before this, form three relevant big genes with myelin in the Brachydanio rerio oligodendrocyte and be co expression
[24]
The comparison of relevant major protein with myelin in table 1 Brachydanio rerio and the mammal
Do not see the report of Brachydanio rerio multiple sclerosis model at present both at home and abroad as yet.Most of authors utilize the 26S Proteasome Structure and Function, myelin forming process of Brachydanio rerio research myelin or carry out genetic analysis to forming the relevant factor with myelin
[25-30]Transgenic zebrafish is a focus of studying the myelin biological function now, and like plp:EGFP system and oligo2:EGFP system, the EGFP fluorescin can both be expressed by the oligodendrocyte system of these several kinds of transgenic zebrafishes
[17]Buckley etc.
[17,31]Utilizing normal oligo2:EGFP is that transgenic fluorescence Brachydanio rerio filters out 80 kinds of chemical compounds that can change myelin oligodendrocyte number, and the chemical compound that wherein can increase oligodendrocyte quantity can be used as myelin and forms promoter.Yet transgenic fluorescence Brachydanio rerio is the normal Brachydanio rerio of myelin, and the pathological change of demyelination is different fully during with MS, and therefore, it is not necessarily effective to the treatment of MS that the myelin that utilizes normal transgenic myelin fluorescence Brachydanio rerio to screen forms promoter.
(Ethidium bromide is a kind of super-sensitive fluorescent dye EB) to ethidium bromide, is used for observing the DNA of agarose and PAAG, and it can be embedded in the base molecule and cause mispairing.Reported literature ethidium bromide (EB) can bring out the neural demyelination of rodent
[17]We find to handle Brachydanio rerio with EB, also can induce Brachydanio rerio marrowbrain neurocyte myelin damage (see figure 1).The present invention at first utilizes EB to induce Brachydanio rerio nervus centralis demyelination, produces Brachydanio rerio MS model, utilizes the inductive neural myelin pathological changes of this model discrimination MS medicine or assessing compound then.
MBP (Myelin basic protein; MBP) be the distinctive memebrane protein of neural myelin by ripe oligodendrocyte synthesis secretion; Be to keep neuron and the stable important substance basis of aixs cylinder myelin 26S Proteasome Structure and Function; It is the index that reflection central nervous system material injury, particularly myelinoclasis change special sensitivity
[32]MBP is the key component (table 1) of Brachydanio rerio myelin also.The present invention adopts whole Brachydanio rerio immunofluorescence dyeing method (zebrafish whole mount immunofluorescent staining); In conjunction with quantitative image analysis and high flux microwell plate analytical technology; MS Brachydanio rerio myelin is carried out quantitatively; Assessing compound screens potential MS medicine to the influence that myelin forms.The principle of whole Brachydanio rerio immunofluorescence dyeing method is a specific antigen-antibody reaction.It is the anti-Brachydanio rerio neurospongium of rabbit sheath protein anti-(anti-MBP) that the present invention one resists.After compound treatment Brachydanio rerio a period of time; At first Brachydanio rerio is used Dent ' s fixative (methanol: DMSO=4: 1) fixing; It is anti-with after the intravital MBP of Brachydanio rerio combines that aquation sealing back adds anti-MBP one; Add again that the goat anti-rabbit igg (H+L) two of FITC labelling is anti-to be closed with anti-MBP one resistive connection; Utilize fluorescence microscope to take pictures then or utilize ELIASA to collect data, data are carried out statistical procedures, thereby filter out the medicine or the inductive neural myelin damage of assessing compound of multiple sclerosis.As can be seen from Figure 1, untreated fish group Brachydanio rerio myelin fluorescence is very strong, and EB processed group Brachydanio rerio myelin fluorescent weakening or disappearance.Popularization is come, if certain chemical compound can increase the inductive Brachydanio rerio myelin of EB fluorescence intensity, then this chemical compound can be used as potential multiple sclerosis curative.In addition, Brachydanio rerio multiple sclerosis model also can be used to the inductive myelin damage of assessing compound among the present invention, and its principle is that this kind chemical compound is induced Brachydanio rerio myelin fluorescent weakening or disappearance.
Summary of the invention
In order to overcome defective and the deficiency that above-mentioned prior art exists; The inventor is through research; Aim to provide a kind of method for building up of live body Brachydanio rerio multiple sclerosis model, a kind of method of utilizing this model discrimination multiple sclerosis therapy medicine and the inductive myelin damage of assessing compound is provided simultaneously.Advantages such as that method provided by the invention has is easy, quick, economic, efficient, high flux.
The present invention realizes through following technical scheme:
A kind of method for building up of live body Brachydanio rerio multiple sclerosis model is characterized in that, may further comprise the steps:
(1) Brachydanio rerio is chosen
The normotrophic Brachydanio rerio of picking is put into microwell plate;
(2) compound treatment
Remove the culture fluid in the microwell plate, a plurality of experimental grouies are set: the derivant processed group of several variable concentrations and 1 blank group, each experimental group adds the corresponding solution of equal volume, constant temperature culture then respectively according to the specification of microwell plate;
(3) whole Brachydanio rerio immunofluorescence dyeing;
(4) graphical analysis and/or microwell plate analysis;
(5) statistical analysis.
Preferably, the derivant in the said step (2) is ethidium bromide (EB).
A kind of method of estimating or screening the multiple sclerosis therapy medicine is characterized in that, may further comprise the steps:
(1) Brachydanio rerio is chosen
The normotrophic Brachydanio rerio of picking is put into microwell plate;
(2) compound treatment
Remove the culture fluid in the microwell plate; A plurality of experimental grouies are set: several chemical compound combined treatment groups, 1 model group, 1 positive controls, 1 solvent control group and 1 blank group; Each experimental group adds the corresponding solution of equal volume, constant temperature culture then respectively according to the specification of microwell plate;
(3) whole Brachydanio rerio immunofluorescence dyeing;
(4) graphical analysis and/or microwell plate analysis;
(5) statistical analysis.
Preferably; The solution that chemical compound combined treatment group adds in the step (2) is the mixed solution of EB and testing compound; The solution that model group adds is EB solution; The solution that positive controls adds is the mixed solution of EB and multiple sclerosis therapy medicine, and the solution that adds in the solvent control group is 0.1% DMSO.
The method of the inductive myelin damage of a kind of assessing compound is characterized in that, may further comprise the steps:
(1) Brachydanio rerio is chosen
The normotrophic Brachydanio rerio of picking is put into microwell plate;
(2) compound treatment
Remove the culture fluid in the microwell plate; A plurality of experimental grouies are set: several testing compound processed group, 1 myelin damage positive controls, 1 solvent control group and 1 blank group; Each experimental group adds the corresponding solution of equal volume, constant temperature culture then respectively according to the specification of microwell plate;
(3) whole Brachydanio rerio immunofluorescence dyeing;
(4) graphical analysis and/or microwell plate analysis;
(5) statistical analysis.
Preferably, the solution that the testing compound processed group adds in the step (2) is testing compound solution, and the solution that adds in the myelin damage positive controls is EB solution, and the solution that adds in the solvent control group is 0.1% DMSO.
Preferably, in all methods of the present invention, Brachydanio rerio is the Brachydanio rerio of 2dpf in the step (1).
Preferably, in all methods of the present invention, the Brachydanio rerio incubation time is 96 hours (h) in the step (2).
Preferably, in all methods of the present invention, the solution that step (2) empty matched group uses is the Brachydanio rerio breeding water.
Preferably, in all methods of the present invention, the whole Brachydanio rerio immunofluorescence dyeing of step (3) may further comprise the steps:
1) fixing;
2) aquation;
3) add confining liquid, jiggle under the room temperature;
4) Brachydanio rerio and the anti-Brachydanio rerio neurospongium of rabbit sheath protein antibody (anti-MBP) are left standstill incubated overnight for common 4 ℃;
5) PBST cleans;
6) goat anti-rabbit igg two of adding FITC labelling is anti-, incubated at room, lucifuge;
7) PBST cleans.
Preferably; In all methods of the present invention; Step (4) graphical analysis concrete steps are: after whole Brachydanio rerio immunofluorescence dyeing finished, microscope was taken pictures and is preserved, and came qualitative evaluation through observing Brachydanio rerio myelin fluorescence intensity on the one hand; Utilize software to carry out graphical analysis on the other hand, calculate Brachydanio rerio myelin fluorescence intensity; Said step (4) microwell plate is analyzed concrete steps: behind the whole Brachydanio rerio immunofluorescence dyeing, Brachydanio rerio is changed in 96 orifice plates, every hole 1 tail places fluorescence intensity under the multi-functional microwell plate analyser with microwell plate then.
Preferably; In all methods of the present invention; Step (5) statistical analysis step is: statistical procedures result representes with
; Relatively adopt variance analysis between many groups; Relatively adopt Dunnett ' s T-check to carry out statistical procedures between two groups, p＜0.05 is that diversity is remarkable.
The method for building up of Brachydanio rerio multiple sclerosis model provided by the invention and application thereof not only can damage through graphical analysis qualitative and quantitative screening multiple sclerosis therapy medicine or the inductive myelin of assessing compound, and can be through realize the purpose of high flux screening and evaluation based on the ELIASA detection technique of microwell plate.Compare with model in the past, the present invention has following advantage:
1) in vivo-and experiment material is the live body Brachydanio rerio, as a kind of vertebrates, its screening model belongs to the body inner model, can truly reflect medicine absorption in vivo, distribution, metabolism and drainage, really reflects the whole biological activity of medicine.
2) high flux-Brachydanio rerio 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 Brachydanio rerio become a kind of ideal model that can carry out high throughput automated drug disposition screening.
3) 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 Brachydanio rerio.
4) compound amount few-the detection compound consumption is few, usually only needs several milligrams, traditional screening experiment then needs the chemical compound more than several milligrams.
5) easy-experimentation is simple to operate, and Brachydanio rerio 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.
6) fast-lack, can in 2～3 days, accomplish experimental period; And mouse often needs the time of several weeks to several months, and monkey often needs the time of several months to several years.Brachydanio rerio at first 72h with interior completion fetal development.Most internals comprises cardiovascular system, intestinal, liver and kidney, rapid shaping in 24-48h, and traditional experiment carrier mouse and monkey then needed 21 days and 9 months can accomplish fetal development respectively.
7) efficient-zebrafish embryo and juvenile fish are transparent, can observe a plurality of tracts simultaneously, and experiment analytical method is simple, quick.
8) gene of predictability-Brachydanio rerio and human gene's similarity is up to about 85% reliably, and its biological function is highly similar with the mammal and the mankind, and the experimental result comparability is strong, and predictability is good.
9) intuitive strong-embryo and juvenile fish are transparent, can directly place to observe fluorescence microscope under to contrast each experimental group Brachydanio rerio fluorescence intensity.
10) sensitivity height-the present invention can induce Brachydanio rerio to produce neural myelin damage with the ethidium bromide of low concentration (EB) very.
11) 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.
Use screening that the live body zebra fish model carries out the multiple sclerosis therapy medicine and evaluating drug effect and have advantages such as reliable, quick, efficient, cheap, high performance-price ratio, can realize the purpose of the interior high flux screening multiple sclerosis therapy medicine of body.The present invention is significant to the treatment of the research and development process of quickening the multiple sclerosis therapy medicine and multiple sclerosis patients.
Detailed Description Of The Invention
The object of the present invention is to provide a kind of method for building up of live body Brachydanio rerio multiple sclerosis model, a kind of method of utilizing this model discrimination multiple sclerosis therapy medicine and the inductive myelin damage of assessing compound is provided simultaneously.Advantages such as that method provided by the invention has is easy, quick, economic, efficient, high flux.
One, the present invention provides a kind of method for building up of live body Brachydanio rerio multiple sclerosis model, and design is:
1 Brachydanio rerio is chosen
Get 4～5 couples of Brachydanio rerio parents copulation, according to Westerfield
[33]Method hatching embryo.The Brachydanio rerio that will be in the optimization process stage places observation under the anatomic microscope, and the normotrophic Brachydanio rerio of picking moves in 6,12,24,48, the 96 or 384 hole microwell plates, the Brachydanio rerio of putting into varying number according to the microwell plate specification.
2 compound treatment
6 experimental grouies are set: 5 derivant processed group, 1 blank group.(the dissolved oxygen mass concentration is 6-8mg/L to remove breeding water in the microwell plate; Water temperature is 28 ℃; PH is 7.2-7.6; Total hardness is 200-250mg/L, down together), add certain volume (deciding) concentration in the derivant processed group and be respectively 10 μ M, 25 μ M, 50 μ M, 75 μ M, 100 μ M myelins damage derivant EB according to the microwell plate specification; Add isopyknic breeding water in the blank group.Cultivate in 28 ℃ of constant incubators according to the optimization process time span.
3 whole Brachydanio rerio immunofluorescence dyeings
1) fixing
After compound treatment finishes, utilize Dent ' s fixative (methanol: DMSO=4: 1) fixing embryo, room temperature 3 hours (h) above perhaps 4 ℃ spend the night, be transferred to then in 100% the methanol ,-20 ℃ of placements are no less than 1h.
2) aquation
1. sample is taken out from-20 ℃ refrigerator, room temperature is placed 5 minutes (min);
2. progressively aquation to 100%PBST (phosphate buffer that contains 0.1% tween);
I 75% methanol/25%PBST, room temperature, 5min jiggles
Ii 50% methanol/50%PBST, room temperature, 5min jiggles
3) add confining liquid, room temperature 3h jiggles;
4) Brachydanio rerio and the anti-Brachydanio rerio neurospongium of rabbit sheath protein antibody (anti-MBP) are left standstill incubated overnight for common 4 ℃;
5) PBST cleans 15min, jiggles, and repeats 4 times;
6) goat anti-rabbit igg (H+L) two of adding FITC labelling is anti-, incubated at room 2h, lucifuge;
7) PBST cleans 15min, jiggles, and repeats 4 times.
4 graphical analyses
After whole Brachydanio rerio immunofluorescence dyeing finishes, utilize three-dimensional fluorescence microscope to take pictures and preserve.On the one hand through observing the qualitative definite Brachydanio rerio multiple sclerosis model (see figure 2) of each experimental group Brachydanio rerio myelin fluorescence intensity of contrast; Utilize Nikon NIS-Elements D3.10 high vision process software to carry out image quantitative analysis on the other hand, calculate Brachydanio rerio myelin fluorescence intensity.Myelin damage ratio computing formula is following:
For example: blank group fluorescence intensity is 100000; Derivant processed group fluorescence intensity is respectively 80000,60000,40000,20000,10000, then gets according to computing formula: derivant processed group myelin damage ratio is respectively 20%, 40%, 60%, 80%, 90% (see figure 3).
5 microwell plate analyses
Or behind the whole Brachydanio rerio immunofluorescence dyeing, Brachydanio rerio 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 analyser.Exciting light is set to 495nm, and in the fluorescence intensity of 520nm place collection emitting light, the test repetition is got its meansigma methods 3 times.Myelin damage ratio computing formula is seen (a).
6 statistical analysis
Utilize JMP8.0 software that the myelin damage ratio of above-mentioned graphical analysis and microwell plate analysis gained is carried out statistical analysis.Statistical procedures result representes with
; Relatively adopt variance analysis between many groups; Relatively adopt Dunnett ' s T-check to carry out statistical procedures between two groups, p＜0.05 is that diversity is remarkable.Confirm Brachydanio rerio multiple sclerosis model according to the statistical procedures result.
Two: the present invention also provides a kind of method of estimating or screening the multiple sclerosis therapy medicine, and design is:
1 Brachydanio rerio is chosen
The Brachydanio rerio that will be in the optimization process stage places observation under the anatomic microscope, and the normotrophic Brachydanio rerio of picking moves in 6,12,24,48,96 or 384 orifice plates, the Brachydanio rerio of putting into varying number according to the microwell plate specification.
2 compound treatment
9 experimental grouies are set: 5 chemical compound combined treatment groups, 1 model group, 1 positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, add myelin damage derivant EB+0.1 μ M, 1 μ M, 10 μ M, the 100 μ M of certain volume (deciding), the testing compound solution of 1000 μ M in 5 chemical compound combined treatment groups respectively according to the microwell plate specification; Add derivant EB in the model group; Add derivant EB+ multiple sclerosis therapy 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.
The compounds of this invention combined treatment group is except adopting above-mentioned mode of adding two kinds of materials (derivant+testing compound) simultaneously, and in addition, the chemical compound combined treatment group of this step also can adopt following dual mode to handle:
First: after adding derivant 48-72h, add the testing compound solution that final concentration is respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M again; (be applicable to and estimate and screening multiple sclerosis therapy medicine)
Second: at first add after final concentration is respectively testing compound solution 6～24h of 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M, add derivant again.(be applicable to and estimate and screening multiple sclerosis preventive drug)
3 whole Brachydanio rerio immunofluorescence dyeings
Method is with the whole Brachydanio rerio immunofluorescence dyeing step part in the invention one.
4 microwell plate analyses
Behind the whole Brachydanio rerio immunofluorescence dyeing, Brachydanio rerio 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 analyser.Exciting light is set to 495nm, and in the fluorescence intensity of 520nm place collection emitting light, the test repetition is got its meansigma methods 3 times.Myelin recovery rate computing formula is following:
For example: blank group and solvent control group Brachydanio rerio myelin fluorescence intensity are 10000; The positive controls fluorescence intensity is 7000; The model group fluorescence intensity is 2000; Chemical compound combined treatment group fluorescence intensity is respectively 4000,5000,6000,7000,8000, then gets according to computing formula: positive controls myelin recovery rate is that 62.5%, 5 chemical compound combined treatment group myelin recovery rate is respectively 25%, 37.5%, 50%, 62.5%, 75% (like Fig. 4).
5 graphical analyses
Or behind the whole Brachydanio rerio immunofluorescence dyeing, utilize three-dimensional fluorescence microscope to take pictures and preserve.On the one hand through observing each experimental group Brachydanio rerio myelin fluorescence intensity qualitative evaluation of contrast and screening multiple sclerosis therapy medicine (see figure 5); Utilize Nikon NIS-Elements D3.10 high vision process software to carry out image quantitative analysis on the other hand, calculate Brachydanio rerio myelin fluorescence intensity.Myelin recovery rate computing formula is seen (b).
6 statistical analysis
Statistical procedures result representes with
; Relatively adopt variance analysis between many groups; Relatively adopt Dunnett ' s T-check to carry out statistical procedures between two groups, p＜0.05 is that diversity is remarkable.According to statistical procedures evaluation of result and screening multiple sclerosis therapy medicine.
Three: the present invention also provides the method for the inductive myelin damage of a kind of assessing compound, and design is:
1 Brachydanio rerio is chosen
The Brachydanio rerio that will be in the optimization process stage places observation under the anatomic microscope, and the normotrophic Brachydanio rerio of picking moves in 6,12,24,48,96 or 384 orifice plates, the Brachydanio rerio of putting into varying number according to the microwell plate specification.
2 compound treatment
8 experimental grouies are set: 5 testing compound processed group, 1 myelin damage positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, add certain volume (deciding) concentration in the testing compound processed group and be respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M testing compounds according to the microwell plate specification; Add myelin damage derivant EB in the myelin damage positive controls; The DMSO that adds equal-volume 0.1% 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.
3 whole Brachydanio rerio immunofluorescence dyeings
Method is with the whole Brachydanio rerio immunofluorescence dyeing step part in the invention one.
4 microwell plate analyses
Behind the whole Brachydanio rerio immunofluorescence dyeing, Brachydanio rerio 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 analyser.Myelin damage ratio computing formula is seen (a).
5 graphical analyses
Or behind the whole Brachydanio rerio immunofluorescence dyeing, utilize three-dimensional fluorescence microscope to take pictures and preserve.On the one hand through observing the contrast inductive myelin damage of each experimental group Brachydanio rerio myelin fluorescence intensity qualitative evaluation chemical compound (like Fig. 2); Utilize Nikon NIS-Elements D3.10 high vision process software to carry out graphical analysis on the other hand, calculate Brachydanio rerio myelin fluorescence intensity.Myelin damage ratio computing formula is seen (a).
6 statistical analysis
Statistical procedures result representes with
; Relatively adopt variance analysis between many groups; Relatively adopt Dunnett ' s T-check to carry out statistical procedures between two groups, p＜0.05 is that diversity is remarkable.According to the inductive myelin degree of injury of statistical procedures evaluation of result chemical compound.
Description of drawings
Fig. 1 is the inductive neural myelin damage collection of illustrative plates of EB of the present invention.A is a untreated fish group, and B is the EB processed group.
Fig. 2 is the qualitative definite Brachydanio rerio multiple sclerosis collection of illustrative plates of the present invention.A is the blank group, and Brachydanio rerio myelin fluorescence intensity is very strong; B is the derivant processed group, after derivant EB handles, and the damage of Brachydanio rerio myelin, fluorescence intensity weakens even disappears.
The inductive myelin damage ratio of EB that Fig. 3 relies on for concentration of the present invention.
Fig. 4 relies on the myelin recovery rate of testing compound concentration for the present invention.
Fig. 5 is the pharmacodynamics collection of illustrative plates of qualitative evaluation multiple sclerosis therapy medicine of the present invention.A is the blank group; B is a solvent control group; C is a model group, and Brachydanio rerio myelin fluorescence intensity significantly weakens; The positive matched group of D; E is a chemical compound combined treatment group.Brachydanio rerio among D and the E is after the multiple sclerosis therapy drug treating, and fluorescence intensity obviously strengthens.
Fig. 6 is the best stage of development collection of illustrative plates of the qualitative definite Brachydanio rerio of the present invention.A is the blank group, and Brachydanio rerio myelin fluorescence intensity is very strong; B is the 2dpf Brachydanio rerio, after derivant EB handles, and the damage of Brachydanio rerio myelin, fluorescence intensity disappears.
Fig. 7 is the inductive Brachydanio rerio myelin of EB damage ratio during for different developmental phases of the present invention.* 2dpf compares with 4dpf with 3dpf, p＜0.05.
Fig. 8 is the Brachydanio rerio myelin damage ratio under the different disposal time span of the present invention.* 96h compares with 72h, p＜0.05; * 96h compares with 48h, p＜0.01; * * 96h compares with 12h with 24h, p＜0.001.
Fig. 9 is the whole Brachydanio rerio immunofluorescence dyeing collection of illustrative plates of Brachydanio rerio of the present invention after 75 μ M EB handle.A is the blank group; B is 75 μ M EB processed group, compares with the blank group, and fluorescence intensity significantly weakens.
Figure 10 relies on the Brachydanio rerio myelin damage ratio (graphical analysis) of EB concentration for the present invention.* 75 μ M compare p＜0.05 with 50 μ M; * 75 μ M compare p＜0.01 with 25 μ M with 10 μ M.
Figure 11 relies on the Brachydanio rerio myelin damage ratio (microwell plate analysis) of EB concentration for the present invention.* 75 μ M compare p＜0.05 with 50 μ M; * 75 μ M compare p＜0.01 with 25 μ M with 10 μ M.
Figure 12 is the pharmacodynamics collection of illustrative plates of qualitative evaluation T3/T4 of the present invention.A is the blank group; B is a solvent control group; C is a model group, and Brachydanio rerio myelin fluorescence intensity significantly weakens; D is a 10nM T3/T4 processed group, and Brachydanio rerio is after the T3/T4 treatment, and fluorescence intensity obviously strengthens.
The Brachydanio rerio myelin recovery rate that Figure 13 relies on for T3/T4 concentration of the present invention.* the 0.1nM group is compared p＜0.05 with solvent control group; * 1nM group is compared p＜0.01 with solvent control group; * * 10nM, 100nM and 1000nM compare with solvent control group, p＜0.001.
Figure 14 is the pharmacodynamics collection of illustrative plates of qualitative evaluation methyl meticortelone of the present invention.A is the blank group; B is a solvent control group; C is a model group, and Brachydanio rerio myelin fluorescence intensity significantly weakens; The positive matched group of D; E is 100 μ M methyl meticortelone processed group.Brachydanio rerio among D and the E is after the multiple sclerosis medicine is handled, and fluorescence intensity obviously strengthens.
The myelin recovery rate that Figure 15 relies on for methyl meticortelone concentration of the present invention.* 1 μ M group is compared p＜0.05 with solvent control group; * 10 μ M group is compared p＜0.01 with solvent control group; * * 100 μ M, 1000 μ M compare p＜0.001 with solvent control group with positive controls.
The myelin recovery rate that Figure 16 relies on for dexamethasone concentration of the present invention.* 1 μ M compares p＜0.05 with 10 μ M group with solvent control group; * 100 μ M compare p＜0.01 with 1000 μ M group with solvent control group; * * positive controls is compared with solvent control group, p＜0.001.
Figure 17 is the inductive myelin damage of qualitative evaluation LPC of the present invention collection of illustrative plates.A is the blank group; B is a solvent control group; C is a model group; D is 1000 μ M LPC processed group.Brachydanio rerio among C and the D is after myelin damage derivant is handled, and fluorescence intensity obviously weakens or disappears.
The Brachydanio rerio myelin damage ratio figure that Figure 18 relies on for LPC concentration of the present invention.* 1 μ M compares p＜0.05 with 10 μ M group with solvent control group; 100 μ M compare p＜0.01 with 1000 μ M group with solvent control group; * * myelin damage positive controls is compared p＜0.001 with solvent control group.
The specific embodiment
Following examples are in order to further specify the embodiment and the purposes of live body Brachydanio rerio multiple sclerosis model provided by the invention.Embodiment is in order to explain rather than limit by any way scope of the present invention, and some change that those skilled in the art are made within the scope of the claims and adjustment also should be thought and belong to scope of the present invention.
Reagent and instrument
Ethidium bromide (EB) and Levothyroxinnatrium sodium (T3/T4) are available from the brilliant pure Industrial Co., Ltd. in Shanghai, and other reagent provide by the prosperous Science and Technology Ltd. of Beijing ancient cooking vessel state.Anatomic microscope (SMZ645, Nikon company, Japan); Power focus continuous zoom fluorescence microscope (AZ100, Nikon company, Japan); Behavior analysis appearance (V3, ViewPoint Life Sciences, France).
1 Brachydanio rerio is chosen
When Brachydanio rerio grew 2dpf, myelin began to form in the brain
[17]The Brachydanio rerio of 2dpf, 3dpf, 4dpf placed under the anatomic microscope observe, the normotrophic Brachydanio rerio of picking moves into respectively in three 6 orifice plates, every hole 30 tails.(annotate: the dpf=day post fertilization among the present invention, Chinese is meant Brachydanio rerio after fertilization natural law, is meant the Brachydanio rerio after fertilization two days like 2dpf.)
2 compound treatment
3 experimental grouies (every group of Brachydanio rerio that is respectively 2dpf, 3dpf, 4dpf) are set, and each experimental group comprises 1 derivant processed group, 1 blank group.Remove the breeding water in the microwell plate, add the EB of 3mL 75 μ M in the derivant processed group; Add the 3mL breeding water in the blank group, in 28 ℃ of constant incubators, cultivate 96h.
3 whole Brachydanio rerio immunofluorescence dyeings
Method is with the whole Brachydanio rerio immunofluorescence dyeing step part in the invention one.
4 graphical analyses
After whole Brachydanio rerio immunofluorescence dyeing finishes, utilize three-dimensional fluorescence microscope to take pictures and preserve.Through observing the best stage of development of the qualitative definite Brachydanio rerio of each experimental group Brachydanio rerio myelin fluorescence intensity of contrast, utilize Nikon NIS-Elements D3.10 high vision process software to carry out graphical analysis on the other hand on the one hand, calculate Brachydanio rerio myelin fluorescence intensity.Myelin damage ratio computing formula is seen (a).
Blank group Brachydanio rerio myelin fluorescence staining is normal.Along with the increase at Brachydanio rerio age, derivant processed group Brachydanio rerio myelin degree of injury constantly reduces, the maximum (see figure 6) of 2dpf Brachydanio rerio myelin degree of injury.Statistical procedures result shows: the Brachydanio rerio myelin damage ratio of 2dpf, 3dpf, 4dpf is respectively (69.97 ± 3.69) %, (45.18 ± 2.98) %, (30.39 ± 1.23) % (see figure 7); This shows the increase along with the Brachydanio rerio age; The myelin damage ratio reduces gradually, and the Brachydanio rerio myelin damage ratio of 2dpf is the highest.Through variance analysis, the myelin damage ratio of derivant processed group is apparently higher than the blank group, and difference has statistical significance (p＜0.05); The Brachydanio rerio of 2dpf is compared p＜0.05 with the Brachydanio rerio myelin damage ratio of 3dpf and 4dpf.
1 Brachydanio rerio is chosen
The Brachydanio rerio of 2dpf placed under the anatomic microscope observe, the normotrophic Brachydanio rerio of picking moves into respectively in five 6 orifice plates, every hole 30 tails.
2 compound treatment
5 experimental grouies are set, and each experimental group comprises 1 EB processed group, 1 blank group.Remove the breeding water in the microwell plate, add the EB of 3mL 75 μ M in the EB processed group; Add isopyknic breeding water in the blank group.Five microwell plates are put into 28 ℃ of constant incubators cultivate 12h, 24h, 48h, 72h, 96h respectively.
3 whole Brachydanio rerio immunofluorescence dyeings
Method is with the whole Brachydanio rerio immunofluorescence dyeing step part in the invention one.
4 microwell plate analyses
Behind the whole Brachydanio rerio immunofluorescence dyeing, Brachydanio rerio 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 analyser.Exciting light is set to 495nm, and in the fluorescence intensity of 520nm place collection emitting light, the test repetition is got its meansigma methods 3 times.Myelin damage ratio computing formula is seen (a).
Statistical procedures result shows: the myelin damage ratio that EB handles 12h, 24h, 48h, 72h, 96h is respectively (10.25 ± 1.33) %, (21.18 ± 1.39) %, (31.45 ± 3.21) %, (48.58 ± 2.59) %, (69.97 ± 1.67) % (see figure 8); This shows the prolongation along with the processing time; The inductive Brachydanio rerio myelin of EB damage ratio constantly increases; The Brachydanio rerio myelin damage ratio that the derivant processed group is handled behind the 96h is compared with the blank group, and difference has statistical significance (p＜0.05); 96h compares with 72h, 48h, 24h and 12h, and difference has statistical significance (p＜0.05, p＜0.01 or p＜0.001).Therefore, select to handle 96h as chemical compound optimization process time span.Different chemical compound optimization process time spans are also inequality, need the particular compound concrete analysis.
The method for building up of embodiment 3 live body Brachydanio rerio multiple sclerosis models
Based on best stage of development of the Brachydanio rerio in embodiment 1 and 2 and chemical compound optimization process time span, set up live body Brachydanio rerio multiple sclerosis model through the concentration of optimizing EB.Design is following:
1 Brachydanio rerio is chosen
The Brachydanio rerio of 2dpf is placed observation under the anatomic microscope, and the normotrophic Brachydanio rerio of picking moves into respectively in 12 orifice plates, every hole 20 tails.
2 compound treatment
6 experimental grouies are set: 5 derivant processed group, 1 blank group.Remove the breeding water in the microwell plate, add the EB that 2mL concentration is respectively 10 μ M, 25 μ M, 50 μ M, 75 μ M, 100 μ M in the derivant processed group; Add isopyknic breeding water in the blank group, in 28 ℃ of constant incubators, cultivate 96h.
3 whole Brachydanio rerio immunofluorescence dyeings
Method is with the whole Brachydanio rerio immunofluorescence dyeing step part in the invention one.
4 graphical analyses
Behind the whole Brachydanio rerio immunofluorescence dyeing, utilize three-dimensional fluorescence microscope to take pictures and preserve (see figure 9).Utilize Nikon NIS-Elements D3.10 high vision process software to carry out graphical analysis, calculate Brachydanio rerio myelin fluorescence intensity.Myelin damage ratio computing formula is seen (a).
Statistical procedures result shows: the Brachydanio rerio myelin damage ratio of 5 derivant processed group is respectively (20.37 ± 2.35) %, (33.15 ± 3.02) %, (51.02 ± 2.15) %, (64.16 ± 3.23) %, (65.12 ± 2.12) % (see figure 10).This shows the increase along with EB concentration, slows down gradually after the myelin damage takes the lead in increasing.Through variance analysis; Concentration is that the derivant processed group myelin damage ratio of 75 μ M and 100 μ M is significantly higher than solvent control group; Difference has statistical significance (p＜0.05); And no difference of science of statistics (p＞0.05) between the derivant processed group of 75 μ M and 100 μ M, 75 μ M compare with 50 μ M, 25 μ M and 10 μ M, and difference has statistical significance (p＜0.05 or p＜0.01).
5 microwell plate analyses
Behind the whole Brachydanio rerio immunofluorescence dyeing, Brachydanio rerio 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 analyser.Exciting light is set to 495nm, and in the fluorescence intensity of 520nm place collection emitting light, the test repetition is got its meansigma methods 3 times.Myelin damage ratio computing formula is seen (a).
Statistical procedures result shows: the Brachydanio rerio myelin damage ratio of 5 derivant processed group is respectively (21.01 ± 1.36) %, (31.22 ± 4.51) %, (49.89 ± 1.99) %, (70.17 ± 2.55) %, (71.44 ± 3.33) % (seeing Figure 11).This shows the increase along with EB concentration, slows down gradually after the myelin damage takes the lead in increasing.Through variance analysis; Concentration is that the derivant processed group myelin damage ratio of 75 μ M and 100 μ M is significantly higher than solvent control group; Difference has statistical significance (p＜0.05); And no difference of science of statistics (p＞0.05) between the derivant processed group of 75 μ M and 100 μ M, 75 μ M compare with 50 μ M, 25 μ M and 10 μ M, and difference has statistical significance (p＜0.05 or p＜0.01).Graphical analysis is consistent with the microwell plate analysis result, therefore 75 μ MEB processed group is confirmed as Brachydanio rerio multiple sclerosis model group.
Embodiment 4 checking Brachydanio rerio multiple sclerosis models
Levothyroxinnatrium sodium (T3/T4) can be used for treating multiple sclerosis
[34]Present embodiment is the Brachydanio rerio multiple sclerosis model that utilizes known multiple sclerosis therapy medicine (T3/T4) checking embodiment 3 to set up.
1 Brachydanio rerio is chosen
The Brachydanio rerio of 2dpf is placed observation under the anatomic microscope, and the normotrophic Brachydanio rerio of picking moves into respectively in 24 orifice plates, every hole 10 tails.
2 compound treatment
8 experimental grouies are set: 5 chemical compound combined treatment groups, 1 model group, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, the EB+ concentration that adds the 1mL final concentration in the chemical compound combined treatment group and be 75 μ M is respectively the Levothyroxinnatrium sodium (T3/T4) of 0.1nM, 1nM, 10nM, 100nM, 1000nM; The EB that adds 1mL 75 μ M in the model group; Add 1mL0.1%DMSO in the solvent control group; Add the 1mL breeding water in the blank group, in 28 ℃ of constant incubators, handle 96h.
3 whole immunofluorescence dyeings
Method is with the whole immunofluorescence dyeing step part in the invention one.
4 graphical analyses
Behind the whole immunofluorescence dyeing, utilize three-dimensional fluorescence microscope to take pictures and preserve.On the one hand through observing each experimental group Brachydanio rerio myelin fluorescence intensity qualitative evaluation T3/T4 (seeing Figure 12) of contrast; Utilize Nikon NIS-Elements D3.10 high vision process software to carry out graphical analysis on the other hand, calculate Brachydanio rerio myelin fluorescence intensity.Myelin recovery rate computing formula is seen (b).Statistical procedures result shows: the Brachydanio rerio myelin recovery rate of 5 chemical compound combined treatment groups is respectively (22.11 ± 1.36) %, (34.22 ± 1.56) %, (57.63 ± 2.36) %, (58.16 ± 1.58) %, (58.03 ± 2.14) % (seeing Figure 13).This shows the increase along with T3/T4 concentration, and myelin slows down after recovering to take the lead in increasing gradually.Through variance analysis, 5 T3/T4 concentration group are compared with solvent control group, and difference has statistical significance (p＜0.05, p＜0.01 or p＜0.001).
Embodiment 5 estimates the pharmacodynamics of methyl meticortelone through graphical analysis
Methyl meticortelone can be used for treating multiple sclerosis
[35]Present embodiment is to utilize the therapeutic effect of zebra fish model quantitative assessment methyl meticortelone to multiple sclerosis.
1 Brachydanio rerio is chosen
The Brachydanio rerio of 2dpf is placed observation under the anatomic microscope, and the normotrophic Brachydanio rerio of picking moves in 48 orifice plates, every hole 3 tails.
2 compound treatment
9 experimental grouies are set: 5 chemical compound combined treatment groups, 1 model group, 1 positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, the EB+ concentration that adds 300 μ L final concentrations in the chemical compound combined treatment group and be 75 μ M is respectively the methyl meticortelone of 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M; The EB that adds 300 μ L, 75 μ M in the model group; The T3/T4 that adds 300 μ L 10nM in the positive controls; Add 300 μ L 0.1%DMSO in the solvent control group; Add 300 μ L breeding waters in the blank group, in 28 ℃ of constant incubators, handle 96h.
3 whole immunofluorescence dyeings
Method is with the whole immunofluorescence dyeing step part in the invention one.
4 graphical analyses
Behind the whole immunofluorescence dyeing, utilize three-dimensional fluorescence microscope to take pictures and preserve.Through observing each experimental group Brachydanio rerio fluorescence intensity qualitative evaluation methyl meticortelone (seeing Figure 14) of contrast, utilize Nikon NIS-Elements D3.10 high vision process software to carry out graphical analysis on the other hand on the one hand, calculate Brachydanio rerio myelin fluorescence intensity.Myelin recovery rate computing formula is seen (b).Statistical procedures result shows: the myelin recovery rate of positive controls is (49.74 ± 4.12) %; The myelin recovery rate of 5 chemical compound combined treatment groups is respectively (18.55 ± 3.22) %, (29.56 ± 1.79) %, (39.44 ± 2.33) %, (51.45 ± 2.41) %, (52.11 ± 2.33) % (seeing Figure 15).This shows the increase along with methyl meticortelone concentration, and myelin slows down after recovering to take the lead in increasing gradually.Through variance analysis, concentration is that 1 μ M, 10 μ M, 100 μ M and 1000 μ M group are compared with solvent control group, and difference has statistical significance (p＜0.05, p＜0.01 or p＜0.001).
Embodiment 6 is through the pharmacodynamics of microwell plate assay dexamethasone
Dexamethasone can be used for treating multiple sclerosis
[36]Present embodiment is to utilize the therapeutic effect of zebra fish model quantitative assessment dexamethasone to multiple sclerosis.
1 Brachydanio rerio is chosen
The Brachydanio rerio of 2dpf is placed observation under the anatomic microscope, and the normotrophic Brachydanio rerio of picking moves in 24 orifice plates, every hole 10 tails.
2 compound treatment
9 experimental grouies are set: 5 chemical compound combined treatment groups, 1 model group, 1 positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, the EB+ concentration that adds the 1mL final concentration in the chemical compound combined treatment group and be 75 μ M is respectively the dexamethasone of 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M; The EB that adds 1mL 75 μ M in the model group; The T3/T4 that adds 1mL10nM in the positive controls; Add 1mL 0.1%DMSO in the solvent control group; Add the 1mL breeding water in the blank group, in 28 ℃ of constant incubators, handle 96h.
3 whole immunofluorescence dyeings
Method is with the whole immunofluorescence dyeing step part in the invention one.
4 microwell plate analyses
Behind the whole immunofluorescence dyeing, Brachydanio rerio is changed in 96 orifice plates over to every hole 1 tail.Microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyser.Exciting light is set to 495nm, and in the fluorescence intensity of 520nm place collection emitting light, the test repetition is got its meansigma methods 3 times.Myelin recovery rate computing formula is seen (b).Statistical procedures result shows: the myelin recovery rate of positive controls is (52.24 ± 1.87) %; The myelin recovery rate of 5 chemical compound combined treatment groups is respectively (14.36 ± 2.45) %, (20.63 ± 1.85) %, (26.22 ± 2.66) %, (36.23 ± 1.78) %, (36.56 ± 3.25) % (seeing Figure 16).This shows the increase along with dexamethasone concentration, and myelin slows down after recovering to take the lead in increasing gradually.Through variance analysis, concentration is that 1 μ M, 10 μ M, 100 μ M and 1000 μ M group are compared with solvent control group, and difference has statistical significance (p＜0.05 or p＜0.01).
Embodiment 7 estimates the inductive myelin damage of L-LYSOLECITHIN SUNLECITHIN A (LPC)
L-LYSOLECITHIN SUNLECITHIN A (LPC) can dissolve myelin, causes body to produce focal demyelination pathological changes [37-39].Present embodiment is to utilize Brachydanio rerio multiple sclerosis model to come the qualitative and inductive myelin degree of injury of quantitative assessment LPC.
1 Brachydanio rerio is chosen
The Brachydanio rerio of 2dpf is placed observation under the anatomic microscope, and the normotrophic Brachydanio rerio of picking moves in 6 orifice plates, every hole 30 tails.
2 compound treatment
8 experimental grouies are set: 5 compound treatment groups, 1 myelin damage positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, add the L-LYSOLECITHIN SUNLECITHIN A (LPC) that 3mL concentration is respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M in the compound treatment group; The EB that adds 3mL 75 μ M in the myelin damage positive controls; Add 3mL0.1%DMSO in the solvent control group; Add the 3mL breeding water in the blank group, in 28 ℃ of constant incubators, handle 96h.
3 whole immunofluorescence dyeings
Method is with the whole immunofluorescence dyeing step part in the invention one.
4 graphical analyses
Behind the whole immunofluorescence dyeing, utilize three-dimensional fluorescence microscope to take pictures and preserve.Through observing the contrast inductive myelin damage of each experimental group Brachydanio rerio myelin fluorescence intensity qualitative evaluation LPC (seeing Figure 17), carry out graphical analysis on the other hand on the one hand, calculate Brachydanio rerio myelin fluorescence intensity.Myelin damage ratio computing formula is seen (a).Statistical procedures result shows: the myelin damage ratio of positive controls is (65.24 ± 3.88) %; The myelin damage ratio of 5 LPC processed group is respectively (13.78 ± 2.45) %, (21.56 ± 1.58) %, (26.42 ± 2.37) %, (30.75 ± 4.53) %, (34.63 ± 4.11) % (seeing Figure 18).This shows the increase along with LPC concentration, slows down gradually after the myelin damage takes the lead in increasing.Through variance analysis, concentration is that the LPC group of 1 μ M, 10 μ M, 100 μ M and 1000 μ M is compared with solvent control group, and difference has statistical significance (p＜0.05 or p＜0.01).
Visible by above-mentioned preferred embodiment: live body zebra fish model provided by the invention can be easy, quick, economical, efficient, high flux, estimate and screen the multiple sclerosis therapy medicine exactly.Method step provided by the invention is simple, and is with low cost, and accuracy is high; Have good stability and reliability; The live body Brachydanio rerio can really be reflected the whole biological activity of medicine, comprises absorption, distribution, metabolism, the drainage of medicine, can realize high flux screening.
Although the inventor has done comparatively detailed elaboration to technical scheme of the present invention and has enumerated; Be to be understood that; For the those skilled in the art in this area, be obvious to the replacement scheme that the foregoing description modifies and/or flexible perhaps employing is equal to, all can not break away from the essence of spirit of the present invention; The term that occurs among the present invention is used for can not being construed as limiting the invention the elaboration of technical scheme of the present invention and understanding.
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Claims (10)
1. the method for building up of a live body Brachydanio rerio multiple sclerosis model is characterized in that, may further comprise the steps:
(1) Brachydanio rerio is chosen
The normotrophic Brachydanio rerio of picking is put into microwell plate;
(2) compound treatment
Remove the culture fluid in the microwell plate, a plurality of experimental grouies are set: the derivant processed group of several variable concentrations and 1 blank group, each experimental group adds the corresponding solution of equal volume, constant temperature culture then respectively according to the specification of microwell plate;
(3) whole Brachydanio rerio immunofluorescence dyeing;
(4) graphical analysis and/or microwell plate analysis;
(5) statistical analysis.
2. the method estimating or screen the multiple sclerosis therapy medicine is characterized in that, may further comprise the steps:
(1) Brachydanio rerio is chosen
The normotrophic Brachydanio rerio of picking is put into microwell plate;
(2) compound treatment
Remove the culture fluid in the microwell plate; A plurality of experimental grouies are set: several chemical compound combined treatment groups, 1 model group, 1 positive controls, 1 solvent control group and 1 blank group; Each experimental group adds the corresponding solution of equal volume, constant temperature culture then respectively according to the specification of microwell plate;
(3) whole Brachydanio rerio immunofluorescence dyeing;
(4) graphical analysis and/or microwell plate analysis;
(5) statistical analysis.
3. the method for the inductive myelin damage of assessing compound is characterized in that, may further comprise the steps:
(1) Brachydanio rerio is chosen
The normotrophic Brachydanio rerio of picking is put into microwell plate;
(2) compound treatment
Remove the culture fluid in the microwell plate; A plurality of experimental grouies are set: several testing compound processed group, 1 myelin damage positive controls, 1 solvent control group and 1 blank group; Each experimental group adds the corresponding solution of equal volume, constant temperature culture then respectively according to the specification of microwell plate;
(3) whole Brachydanio rerio immunofluorescence dyeing;
(4) graphical analysis and/or microwell plate analysis;
(5) statistical analysis.
4. according to the described method of claim 1-3; It is characterized in that: Brachydanio rerio is the Brachydanio rerio of 2dpf in the said step (1); The Brachydanio rerio incubation time is 96 hours in the said step (2), and the solution that said step (2) empty matched group uses is the Brachydanio rerio breeding water.
5. method according to claim 1 is characterized in that: the derivant in the said step (2) is ethidium bromide (EB).
6. according to each described method of claim 1-3, it is characterized in that: the whole Brachydanio rerio immunofluorescence dyeing of said step (3) may further comprise the steps:
1) fixing;
2) aquation;
3) add confining liquid, jiggle under the room temperature;
4) Brachydanio rerio and the anti-Brachydanio rerio neurospongium of rabbit sheath protein antibody (anti-MBP) are left standstill incubated overnight for common 4 ℃;
5) PBST cleans;
6) goat anti-rabbit igg two of adding FITC labelling is anti-, incubated at room, lucifuge;
7) PBST cleans.
7. method according to claim 2; It is characterized in that: the solution that chemical compound combined treatment group adds in the step (2) is the mixed solution of EB and testing compound; The solution that model group adds is EB solution; The solution that positive controls adds is the mixed solution of EB and multiple sclerosis therapy medicine, and the solution that adds in the solvent control group is 0.1% DMSO.
8. method according to claim 3; It is characterized in that: the solution that the testing compound processed group adds in the step (2) is testing compound solution; The solution that adds in the myelin damage positive controls is EB solution, and the solution that adds in the solvent control group is 0.1% DMSO.
9. according to each described method of claim 1-3; It is characterized in that: said step (4) graphical analysis concrete steps are: after whole Brachydanio rerio immunofluorescence dyeing finishes; Microscope is taken pictures and is preserved; Come qualitative evaluation through observing Brachydanio rerio myelin fluorescence intensity on the one hand, utilize software to carry out graphical analysis on the other hand, calculate Brachydanio rerio myelin fluorescence intensity; Said step (4) microwell plate is analyzed concrete steps: behind the whole Brachydanio rerio immunofluorescence dyeing; Brachydanio rerio is changed in 96 orifice plates; Every hole 1 tail; Then microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyser, the quantitative assessment chemical compound is to the influence or the inductive myelin damage of chemical compound of Remyelination.
10. according to each described method of claim 1-3, the neural myelin fluorescence staining of the whole Brachydanio rerio of said step (3) is used Cell immunohistochemical staining method, reactive dye fluorescent staining method or nonactive dye fluorescence colouring method.
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| CN102980981A (en) * | 2012-11-06 | 2013-03-20 | 江苏省中医药研究院 | Novel method for evaluation of osteoporosis prevention treatment drug effect |
| CN103966261A (en) * | 2014-03-21 | 2014-08-06 | 中国人民解放军总医院 | Transgenic zebrafish with specific myelin sheath removal capacity as well as preparation method and application thereof |
| CN103966261B (en) * | 2014-03-21 | 2016-08-17 | 中国人民解放军总医院 | The transgenic zebrafish and its preparation method and application of myelin tissue can be removed by specificity |
| CN107058320A (en) * | 2017-04-12 | 2017-08-18 | 南开大学 | The preparation and its application of IL7R gene delection zebra fish mutant |
| CN107058320B (en) * | 2017-04-12 | 2019-08-02 | 南开大学 | The preparation and its application of IL7R gene delection zebra fish mutant |
| CN110178757A (en) * | 2019-04-19 | 2019-08-30 | 广东医科大学附属医院 | A kind of zebra fish cerebral trauma model and its preparation method and application |
| CN110178757B (en) * | 2019-04-19 | 2021-08-27 | 广东医科大学附属医院 | Zebra fish brain trauma model and preparation method and application thereof |
| CN110463654A (en) * | 2019-08-15 | 2019-11-19 | 贵州中医药大学 | A method of establishing zebra fish angiogenesis Disorder Model |
| CN110463654B (en) * | 2019-08-15 | 2021-09-07 | 贵州中医药大学 | Method for establishing zebra fish angiogenesis obstacle model |
| CN116269227A (en) * | 2023-03-15 | 2023-06-23 | 山东省科学院生物研究所 | Method for rapidly and accurately evaluating nerve effect of external factors |
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