CN101968484A

CN101968484A - Method for screening mitochondria targeted compounds by using zebra fish

Info

Publication number: CN101968484A
Application number: CN2010102966969A
Authority: CN
Inventors: 彭恩泽
Original assignee: 彭恩泽
Current assignee: Nanjing Xinhuan Detection Technology Co., Ltd.
Priority date: 2010-09-29
Filing date: 2010-09-29
Publication date: 2011-02-09
Anticipated expiration: 2030-09-29
Also published as: CN101968484B

Abstract

The invention relates to the field of compound detection, in particular to a method for screening mitochondria targeted compounds by using zebra fish. The method mainly comprises the following steps of: zebra fish selection, compound treatment, dyeing treatment and quantitative/qualitative analysis, wherein the dyeing treatment is that the dyeing treatment is performed on the zebra fish, on which the compound treatment is performed, for 0.5 to 1 hour at the temperature of 28 DEG C by taking JC-1 or DASPEI as a staining agent. The method covers the shortage that mitochondrial damage cannot be detected in vivo in the prior art by using an in vivo experimental analysis method for screening the mitochondria targeted compounds by using the zebra fish, is convenient, swift, economic and practical, and has the characteristics of high flux and high specificity.

Description

A kind of method of utilizing zebra fish to screen Mitochondrially targeted compound

Technical field

The present invention relates to compound (chemicals) detection range, be specifically related to a kind of simple, economy, high flux is realized utilizing zebra fish to screen the method for Mitochondrially targeted compound.

Background technology

Mitochondria is an important organelle in the eukaryotic, participates in multiple important physical and biochemical processes such as tricarboxylic acid cycle, fatty acid metabolism, oxidative phosphorylation in the cell.Mitochondria plays important pivotal role in Apoptosis, a series of metabolic process and Apoptosis are closely related in the mitochondria, as the excessive generation of oxygen radical, the release of cromoci, the abnormal opening in mitochondrial membrane permeability conversion hole etc.Mitochondrial membrane has the different kinds of ions passage can mediate ion transport, and the adjusting of ion channel may influence the function of mitochondria even cell.Injury of mitochondria can directly cause mitochondriopathy on the one hand, and is closely bound up with nerve degenerative diseases, tumour, aging etc. on the other hand ^[1]

The main process of apoptotic pathways of mitochondria mediation is that cromoci, Smac albumen, apoptosis inducing factor and endonuclease discharge from mitochondria.Wherein short apoptosis subtribe (Bax) is the product of p53 activated transcription, thereby can promote or induce the release of mitochondria antiapoptotic factors to promote apoptosis.Under normal circumstances these albumen are the non-mitochondria components that are positioned at cell, in case cell is subjected to inducing of inducer of apoptosis, they just to the mitochondria transposition, are regulated and control apoptosis as the target cell device with mitochondria.During the inducer of apoptosis cell death inducing, the mitochondrial membrane permeability strengthens, and Intramitochondrial various albumen are released, and comprise cromoci.Cromoci enters in the tenuigenin with apoptotic proteins activity factor Apaf-1 and the halfcystine aspartate specific protease-9(Caspase-9) precursor forms apoptotic body, under the acting in conjunction of the deoxidation atriphos (dATP) that in tenuigenin, exists, activation Caspase-9 precursor, start the Caspase cascade, final inducing cell produces apoptosis ^[2-3]The importance of mitochondria in Apoptosis also is the relation between it and the Bcl-2 gene family.Albumen such as Bcl-2, Bax, the Bcl-xl etc. of a lot of Bcl-2 families are positioned on the mitochondrial membrane.

(Degenerative disease of the central nervous system ND) is one group of chronic progressive external the nervous system disease based on the primary neuronal degeneration to nerve degenerative diseases.Such disease mainly comprises Alzheimer disease (Alzheimer ＇ s disease, AD), Parkinson's (Parkinson ＇ s disease, PD), Huntington chorea (Huntington disease), dissimilar spinocerebellar ataxia (spinal cerebellar ataxias), multiple sclerosis (multiple sclerosis, MS), multiple ALS (amyotrophic lateral sclerosis) and spinal cord muscular atrophy (spinal muscular atrophy) etc.One of pathomechanism that nerve retrograde affection is common is the apoptosis of neurocyte, the medicine of the apoptosis process screening control nerve retrograde affection that therefore can mediate by the mitochondria of block nerves cell degenerative change ^[4-6]

The main path of mitochondria mediation apoptosis of tumor cells is by the mitochondrial membrane permeability changes, the release of the mediated apoptosis factor.Have experiment in vitro to prove, Mitochondrial DNA Mutation can weaken normal respiratory function, and (reactive oxygen species, ROS), thereby the active cell apoptosis reaches the damage to the karyogene group to discharge high-caliber active oxygen ^[7-9]Antineoplastic acts on mitochondria, can cause mitochondrial permeability conversion hole open, and mitochondrial membrane potential descends or disappears, and respiratory chain is taken off the coupling connection then, and glutathione exhausts that ROS generation and cromoci and inducible factor discharge, and inducing apoptosis of tumour cell.

Old and feeble evolution and mitochondrial function are closely related unusually ^[10]Free radical theory is one of representative causes of senescence.Mitochondria also is the important place that produces active oxygen as the center of cellular respiration and oxidation ^[11]Generation and the elimination of ROS is in mobile equilibrium in the normal body, but along with the increase of organismic age, this balance is broken, and antioxidation activity successively decreases, and the oxygen radical generation increases and accumulates.Too much oxygen radical can be attacked mitochondrial membrane by peroxidation, causes the mitochondrial membrane permeability changes, and mitochondrial membrane potential descends or disappears, final cell death inducing.

The action target spot of discovering many medicine or poisonous substances is positioned at mitochondrial membrane or mitochondrial inner membrane compound, its pharmacology or toxicological effect mainly realize that by adjusting mitochondrial respiration-chain function, metabolic enzyme activity, membrane permeability it is synthetic that medicine or poisonous substance can damage structure of mitochondria, enzyme or DNA ^[12-14]Therefore, can come assessing compound toxicity and screening new drug by detecting injury of mitochondria.

The detection of injury of mitochondria comprises detection, the detection of membrane phospholipid, the detection of film potential and the damage check of mitochondrial DNA to mitochondrial membrane permeability conversion hole.The detection method of injury of mitochondria mainly is to be based upon on cellular level or the molecular level basis at present.The detection method of injury of mitochondria mainly contains following several in the apoptosis process ^[15]:

(1) Western blot:, measure albumen distributions such as cromoci, AIF in the different cell components by different Western blots.The method treatment capacity is relatively low, and needs the mitochondrial separation of subcellular structure.This method experimentation complicated operation is difficult to avoid non-specific influence, is easy to generate false positive or false negative result.

(2) enzyme linked immunosorbent assay (ELISA): utilize the particular commodity kit that subcellular fraction is distributed and measure, susceptibility is higher, but needs subcellular structure to separate.This method experimental implementation complexity causes cellular damage easily, and can not reflect the absorption,distribution,metabolism,excretion of medicine at biological integral, can not reflect medicine truth in vivo.

(3) cell transfecting: utilize green fluorescent protein (green-fluorescence protein, GFP) the cDNA transfectional cell of the intermembranous albumen of coding of mark though this method can be applicable to living cells, and dynamic observes, but process is comparatively complicated, and treatment capacity is low.

(4) immunofluorescence or Laser Scanning Confocal Microscope: through the cell of fixing and infiltrationization processing, anti-and two anti-reactions successively with one, corresponding two resist and carry out fluorescence labeling, fluorescence microscope.Can utilize active caspase antibody and check the carrying out that detects apoptotic process than dyeing.But the method sample throughput is lower, and the dynamics of different intermembranous albumen dispose procedures is different, easily produces false negative result.

(5) electronics/immunoelectron microscope: can observe the ultrastructure of apoptotic cell, thereby the Direct observation mitochondrial outer membrane breaks and local inner membrance hernia goes out.This method sample throughput is very low, is difficult to carry out quantitative test, and the saturatingization phenomenon that takes place can not detect outer film integrality and do not destroy the time.

(6) cell fluorescence is measured: (fluorescence activated cell sorting FACS) measures to utilize the fluorescent activation cell sorting system.At first use low concentration digitalis selectivity cell membrane is changed thoroughly, cause that the kytoplasm cromoci leaks outside and is eliminated.Through fixing, immune labeled and cell fluorescence is measured again.Normocellular cromoci maintains in the mitochondrial membrane space, presents hyperfluorescence, and spills from adventitia as cromoci, then fluorescence signal a little less than.The method can be used for the processing of great amount of samples amount and need not subcellular fraction separating, but experimentation is comparatively complicated, need continue to optimize in the processing procedure.

But no matter above-mentioned is that the cellular level or the injury of mitochondria of molecular level detect, and all belongs to experiment in vitro and detects, and have a lot of defectives:

(1) mode of cellular incubation is taked in the detection of cellular level, though effective, the required expense of this method is lower, cell in vitro has lacked the metabolic cycles conversion of biological integral and the loop distribution in the body, can not really reflect whole biologically active;

(2) detection of molecular level needs mitochondria is separated, and has lacked medicine absorption, distribution, metabolism and discharge process in vivo equally, can not reflect medicine truth in vivo;

(3) mitochondria can cause damage in various degree in detachment process, and purity is lower relatively;

(4) stripped mitochondria enzyme activity is lower, and biological function is relatively poor;

(5) be difficult to avoid non-specific influence, thereby produce false positive or false negative result;

(6) sample size is low, can not carry out high flux screening, is difficult to quantitative test;

(7) experimental repeatability is poor, and specificity is relatively poor, and susceptibility is relatively poor;

(8) experimental implementation complexity, experimental period is long;

(9) experimental expenses is higher.

Have not yet to see the interior experiment report of body that injury of mitochondria detects, utilize zebra fish detection line plastochondria Study on Damage also seldom.Zhang YZ etc. ^[18]Utilize Laser Scanning Confocal Microscope to detect mitochondria behavior in the zebra fish ovum generating process in conjunction with the JC-1 fluorescence colour, this research female zebra fish ovary of at first will growing up exsomatizes, after compound treatment, make early stage egg mother cell suspension, be placed on observational study under the Laser Scanning Confocal Microscope through the mitochondria fluorescent dyeing again.This research is the mitochondria behavior that is used for observing in the zebra fish ovum generating process, is not to be detection to injury of mitochondria, can not be used for the mitochondrial toxicity of assessing compound, can not be used for screening Mitochondrially targeted compound.Publication number is China's invention of CN1866025A, disclose a kind of via zebra fish embryo model and detect the method that anti-new vessels generates protein factor or short new vessels generation protein factor activity, but this invention is not a target mitochondria screening compounds.

JC-1(iodate tetrachloro is for tetraethylbenzene imidazoles carbocyanine) be a kind of carbon cyanogen compound cationoid fluorescent dye, can enter and combine with the mitochondrial inner membrane specificity in the living cells.JC-1 is good voltage-dependent fluorescent dye, is considered to the specific fluorescent dye of the most responsive the most reliable mitochondrial membrane potential at present.Its principle is: the NM film potential is higher, and JC-1 accumulates in the mitochondrial matrix and forms polymkeric substance, and the maximum emission wavelength during exciting light 488nm is 590nm, can produce red fluorescence; The lesion wire mitochondrial membrane potential reduces, and JC-1 can not accumulate in the mitochondrial matrix, and this moment, JC-1 was a monomer, and maximum emission wavelength is 530nm during exciting light 488nm, can produce green fluorescence ^[16-17]Therefore, can represent the variation of mitochondrial membrane potential by detecting red green fluorescence intensity.In addition, the present invention also can adopt mitochondria fluorescent dye DASPEI(2 in the living cells, 4-dimethylamino styryl-N-ethylpyridine iodide) as coloring agent, it can produce green fluorescence at exciting light 488nm during emission light 515nm.

Detection method after mitochondria dyes through JC-1 mainly is to adopt flow cytometer, fluorescence microplate reader or laser confocal microscope etc. ^[19-22]Instrument is analyzed and researched.But adopt the research of these methods still to be in the experiment in vitro stage at present, and be based upon on the cellular level basis.Though these methods are effective, but experimental implementation complexity, experimental expenses is higher, be difficult to avoid non-specific influence, thereby produce false positive or false negative result, the most important thing is that cell in vitro has lacked the metabolic cycles conversion of biological integral and the loop distribution in the body, can not really reflect whole biologically active.

Summary of the invention

In order to overcome above-mentioned existing defective of prior art injury of mitochondria detection method and deficiency, the inventor has aimed to provide a kind of convenient and swift, economical and practical zebra fish that utilizes and has screened experiment analytical method in the body of Mitochondrially targeted compound through research.

Specifically, technical scheme of the present invention is as follows:

Summary of the invention

A kind of method of utilizing zebra fish to screen Mitochondrially targeted compound mainly 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

A scheme: remove the breeding water in the microwell plate, then according to testing compound processed group, injury of mitochondria positive controls, solvent control group, blank group, specification according to microwell plate adds corresponding testing compound solution, injury of mitochondria derivant solution, solvent, breeding water respectively, microwell plate was in 28 ℃ of following constant temperature culture 6-72 h(hours then), wherein: testing compound solution concentration is 0.1-1000 μ M, perhaps

B scheme: remove the breeding water in the microwell plate; then according to testing compound combined treatment group; the injury of mitochondria model group; the chondriosome protective positive controls; the solvent control group; the blank group; the mixed solution that adds corresponding testing compound+injury of mitochondria derivant according to the specification of microwell plate respectively; injury of mitochondria derivant solution; the mixed solution of injury of mitochondria derivant+mitochondrion protecting agent; solvent; breeding water; microwell plate was in 28 ℃ of following constant temperature culture 6-72 h(hours then); wherein: testing compound solution concentration is 0.1-1000 μ M

(3) JC-1 or DASPEI dyeing is handled

The solvent control group is divided into two groups at random, one group of group that do not dye as a setting wherein, other one group with the processing of dyeing of other groups, step is as follows:

A, remove the liquid in the microwell plate, add 10 μ M JC-1 or 1mM DASPEI according to the specification of microwell plate;

B, place 28 ℃ of constant temperature to cultivate 0.5-1 h(hour microwell plate);

C, remove the liquid in the microwell plate, clean with breeding water;

Specification according to microwell plate in D, the microwell plate adds breeding water, cultivates 5min(minute in 28 ℃ of constant temperature);

E, repeated experiments operation D) 2-3 time;

F, zebra fish is changed in the new microwell plate, removes the liquid in the microwell plate, add breeding water according to the specification of microwell plate,

(4) quantitative test of multi-functional microwell plate analyser is or/and the fluorescent microscope qualitative analysis.

The present invention utilizes live body zebra fish fluorescence colour detection line plastochondria target compound.The principle of JC-1 is: the NM film potential is higher, and JC-1 accumulates in the mitochondrial matrix and forms polymkeric substance, and the maximum emission wavelength during exciting light 488nm is 590nm, can produce red fluorescence; The lesion wire mitochondrial membrane potential reduces, and JC-1 can not accumulate in the mitochondrial matrix, and this moment, JC-1 was a monomer, and maximum emission wavelength is 530nm during exciting light 488nm, can produce green fluorescence.Therefore, can represent the variation of mitochondrial membrane potential by detecting red green fluorescence intensity.In addition, the present invention also can adopt the interior mitochondria fluorescent dye DASPEI of living cells as coloring agent, and it can produce green fluorescence at exciting light 488nm during emission light 515nm.

As preferably, according to the method for utilizing zebra fish to screen Mitochondrially targeted compound of the present invention, wherein: described step (1) zebra fish is chosen the zebra fish of fertilization after 4 days; Microwell plate is in 28 ℃ of following constant temperature culture 24h in described step (2) compound treatment.Under this condition, the injury of mitochondria derivant can cause maximum injury of mitochondria.

As preferably, according to the method for utilizing zebra fish to screen Mitochondrially targeted compound of the present invention, wherein: the whole dyeing course need lucifuge operation that described step (3) dyeing is handled.The reason of lucifuge is to avoid fluorescence to see that light disappears.

As preferably, according to the method for utilizing zebra fish to screen Mitochondrially targeted compound of the present invention, wherein: described step (1) zebra fish is chosen the employing dissecting microscope.Preferred reason is can select normotrophic zebra fish more accurately under the dissecting microscope, is beneficial to next step compound treatment, helps testing result.

As preferably, according to the method for utilizing zebra fish to screen Mitochondrially targeted compound of the present invention, wherein: described microwell plate is selected the microwell plate in 6-96 hole for use.The reason of selecting these specification microwell plates is to discover, zebra fish of the present invention suits to analyze in 6,12,24,48 or 96 orifice plates of a standard.

As preferably, according to the method for utilizing zebra fish to screen Mitochondrially targeted compound of the present invention, wherein: after described step (2) adopted the A scheme to carry out compound treatment, the quantitative test of the multi-functional microwell plate analyser of step (4) was operated as follows:

Microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyser, exciting light is set to 488nm, (JC-1 is a coloring agent) or 515nm place (DASPEI is a coloring agent) gathers radiative fluorescence intensity at the 590nm place, calculates the injury of mitochondria rate by following formula:

。

As preferably, according to the method for utilizing zebra fish to screen Mitochondrially targeted compound of the present invention, wherein: after described step (2) adopted the B scheme to carry out compound treatment, the quantitative test of the multi-functional microwell plate analyser of step (4) was operated as follows:

Microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyser, and exciting light is set to 488nm, and (JC-1 is a coloring agent) gathers radiative fluorescence intensity at the 530nm place, calculates the injury of mitochondria protection ratio by following formula:

。

As preferably, according to the method for utilizing zebra fish to screen Mitochondrially targeted compound of the present invention, wherein: described step (4) fluorescent microscope qualitative analysis is operated as follows:

Remove the liquid in the microwell plate, adding concentration is 0.64mM methane-sulforic acid anesthesia zebra fish, be fixed on the concave-concave microslide with 3% methylcellulose glue after, place observation fluorescence color under the fluorescent microscope.The JC-1 principle is: under fluorescent microscope, JC-1 forms polymer in NM, produces red fluorescence; In the lesion wire plastochondria, exist, produce green fluorescence with monomeric form.The DASPEI principle is: under fluorescent microscope, produce bright green fluorescence in the NM, normal mitochondria deepening of green fluorescence or disappearance in the lesion wire plastochondria.

As preferably, according to the method for utilizing zebra fish to screen Mitochondrially targeted compound of the present invention, wherein: the solvent in described step (2) compound treatment is that volumetric concentration is 0.1% dimethyl sulfoxide (DMSO).

Detection method of the present invention has been avoided the defective of mitochondria damage detecting method on cellular level and the molecular level on the one hand, can truly reflect medicine metabolic cycles conversion and the interior loop distribution of body in vivo, comprise absorption, distribution, metabolism, the drainage of medicine, can really reflect the whole biologically active of medicine; The live body zebra fish has inherent advantage on the other hand ^[23-24]:

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 behind fertilization 24h, the main histoorgan original hase of zebra fish forms, can be

Research provides a large amount of samples and short experimental period;

4) embryo and juvenile fish are transparent, and be in vitro fertilization, ectogenesis, but Direct observation, and can analyze a plurality of devices simultaneously

The official system;

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 1-4 mm, can be in 6,12,24,48 or 96 of a standard

Analyze in the orifice plate;

7) administering mode is simple: water-soluble small-molecule substance can be directly in skin, the gill and digestive system enter the zebra fish body; Water-fast material, macromolecular substances and protein can carry out microinjection.

Detailed Description Of The Invention

Key instrument and reagent:

The iodate tetrachloro for tetraethylbenzene imidazoles carbocyanine (JC-1) fluorescent dye available from Dutch Leiden company (Molecular Probes Europe); 2,4-dimethylamino styryl-N-ethylpyridine iodide (2-[4-(Dimethylamino) styryl]-1-ethylpyridinium iodide, DASPEI), methane-sulforic acid (ethyl-m-aminobenzoate methanesulfonate, MESAB), methylcellulose glue (methyl cellulose), star spore rhzomorph (staurosporine) and Etoposide (etoposide) be all available from U.S. sigma company.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); Multi-functional microwell plate analyser (Mithras LB940, Berthold Technologies company, Germany).

1, determines the zebra fish optimization process stage

(1) zebra fish is chosen

The zebra fish of 2dpf, 3dpf, 4dpf, 5dpf, 6dpf, 7dpf placed under the dissecting microscope observe, the normotrophic zebra fish of picking moves into respectively in six 48 orifice plates, every hole 10 tails.(annotate: the dpf=day post fertilization among the present invention, Chinese is meant zebra fish after fertilization fate, is meant the zebra fish after fertilization two days as 2dpf.）

(2) compound treatment

6 experimental group (every group of zebra fish that is respectively 2dpf, 3dpf, 4dpf, 5dpf, 6dpf, 7dpf) are set, and each experimental group comprises 1 injury of mitochondria derivant processed group, 1 solvent control group, 1 blank group.(the dissolved oxygen DO 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 1mL 50 μ M star spore rhzomorphs in the injury of mitochondria derivant processed group; Adding 1mL concentration is 0.1% DMSO in the solvent control group; Add the 1mL breeding water in the blank group, in 28 ℃ of constant incubators, cultivate.

(3) dyeing is handled

The solvent control group is divided into two groups at random, and wherein one group is not dyeed, as a setting group; With other one group of group of solvents, injury of mitochondria derivant processed group, the dyeing of blank group.Select for use JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

A, remove the liquid in the microwell plate, draw 1mL 10 μ M JC-1 in microwell plate with liquid-transfering gun;

B, place 28 ℃ of constant incubators to cultivate 1h microwell plate;

C, taking-up microwell plate remove the liquid in the microwell plate, use breeding water Rapid Cleaning 3 times;

Add the 1mL breeding water in D, the microwell plate, in 28 ℃ of constant incubators, cultivate 5min;

E, repeated experiments operation D) 2 time;

F, zebra fish is changed in the 48 new orifice plates, every hole 1 tail removes the liquid in the microwell plate, adds 350 μ L breeding waters.

(4) quantitative test

Microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyser.Exciting light is set to 488nm, gathers radiative fluorescence intensity at the 590nm place, and test repeats 3 times and gets its mean value.Injury of mitochondria rate computing formula is:

Experimental data with Expression is relatively adopted variance analysis between many groups, relatively adopts Student ' s t check to carry out statistical procedures between two groups.Statistical procedures result shows: the zebra fish injury of mitochondria rate of 2dpf, 3dpf, 4dpf, 5dpf, 6dpf, 7dpf is respectively (49.24 ± 2.37) %, (64.02 ± 7.67) %, (68.75 ± 4.18) %, (56.34 ± 3.74) %, (49.28 ± 3.48) %, (45.78 ± 3.34) %(sees Fig. 1), this shows the increase along with the zebra fish age, the injury of mitochondria that the injury of mitochondria derivant causes zebra fish takes the lead in increasing the back to be reduced, and the zebra fish injury of mitochondria rate of 4dpf is the highest.By variance analysis, the injury of mitochondria rate of injury of mitochondria derivant processed group is higher than the solvent control group, and difference has statistical significance (p＜0.05), and 4dpf zebra fish p value is minimum.

(5) qualitative analysis

Remove the liquid in the microwell plate, add 350 μ L concentration and be the methane-sulforic acid anesthesia zebra fish of 0.64mM, be fixed on the concave-concave microslide with 3% methylcellulose glue after, place observation fluorescence color under the fluorescent microscope.Under fluorescent microscope, JC-1 forms polymer in control group, produces red fluorescence; In the mitochondria damaging cells, exist, produce green fluorescence with monomeric form.Along with the increase at zebra fish age, deepening after injury of mitochondria derivant processed group green fluorescence brightens earlier, the zebra fish green fluorescence of 4dpf is the brightest.Therefore, qualitatively determine that zebra fish with 4dpf is as the compound optimization process stage.

2, determine compound optimization process time span

(1) zebra fish is chosen

The zebra fish of 4dpf placed under the dissecting microscope observe, the normotrophic zebra fish of picking moves into respectively in five 48 orifice plates, every hole 10 tails.

(2) compound treatment

Each microwell plate is provided with 1 injury of mitochondria derivant processed group, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, add 1mL 50 μ M star spore rhzomorphs in the injury of mitochondria derivant processed group; Adding 1mL concentration is 0.1% DMSO in the solvent control group; Add the 1mL breeding water in the blank group.Five microwell plates are put into 28 ℃ of constant incubators cultivate 6h, 12h, 24h, 48h, 72h respectively.

(3) dyeing is handled

The solvent control group is divided into two groups at random, and wherein one group is not dyeed, as a setting group; With other one group of group of solvents, injury of mitochondria derivant processed group, the dyeing of blank group, select for use JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

B, place 28 ℃ of constant incubators to cultivate 1h microwell plate;

E, repeated experiments operation D) 2 time;

(4) quantitative test

Experimental data with

Expression is relatively adopted variance analysis between many groups, relatively adopts Student ' s t check to carry out statistical procedures between two groups.Statistical procedures result shows: the zebra fish injury of mitochondria rate that the injury of mitochondria derivant is handled 6h, 12h, 24h, 48h, 72h is respectively (8.89 ± 1.96) %, (24.44 ± 1.17) %, (55.57 ± 2.10) %, (56.78 ± 1.93) %, (57.11 ± 1.68) %(sees Fig. 2), this shows the prolongation along with the processing time, and injury of mitochondria slows down after taking the lead in increasing gradually.By variance analysis, the injury of mitochondria rate that injury of mitochondria derivant processed group is handled 12h, 24h, 48h, 72h is higher than the solvent control group, difference has statistical significance (p＜0.05), and injury of mitochondria derivant processed group is handled no difference of science of statistics between 24h, 48h, the 72h, therefore selects SPT 24h as the compound optimization process time.The different compound optimization process times are also inequality, need the particular compound concrete analysis, and the present invention experimentizes according to the optimization process time span of injury of mitochondria derivant.

(5) qualitative analysis

Remove the liquid in the microwell plate, add 350 μ L concentration and be the methane-sulforic acid anesthesia zebra fish of 0.64mM, be fixed on the concave-concave microslide with 3% methylcellulose glue after, place observation fluorescence color under the fluorescent microscope.Under fluorescent microscope, JC-1 forms polymer in control group, produces red fluorescence; In the mitochondria damaging cells, exist, produce green fluorescence with monomeric form.Along with the prolongation in processing time, injury of mitochondria derivant processed group green fluorescence brightens gradually, handles the 24h green fluorescence intensity and reaches maximum, and 48h and 72h do not see obvious enhancing.Therefore, qualitative definite 24h is a compound optimization process time span.

3, based on above-mentioned zebra fish optimization process stage and compound optimization process time span, the present invention mainly estimates to injury of mitochondria with to two angles of chondriosome protective from compound

3.1 the mitochondrial Mitochondrially targeted compound of screening damage

(1) zebra fish is chosen

Get 4～5 couples of zebra fish parents mating, according to Westerfield ^[25]Method hatching embryo.The zebra fish that will be in the optimization process stage (being 4dpf) places observation under the dissecting microscope, and the normotrophic zebra fish of picking moves in 6,12,24,48 or 96 orifice plates, the zebra fish of putting into varying number according to the microwell plate specification.

(2) compound treatment

8 experimental group are set: 5 testing compound processed group, 1 injury of mitochondria positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, add the testing compound solution that certain volume (deciding according to the microwell plate specification) concentration is respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M respectively in 5 testing compound processed group; Add equal-volume injury of mitochondria derivant 50 μ M star spore rhzomorphs (staurosporine) or 50 μ M Etoposides (etoposide) in the positive controls; Add equal-volume solvent (be generally 0.1% DMSO) in the solvent control group; Add the equal-volume breeding water in the blank group.In 28 ℃ of constant incubators, cultivate 24h according to the optimization process time span.

(3) dyeing is handled

The solvent control group is divided into two groups at random, and wherein one group is not dyeed, as a setting group; With other one group of group of solvents, compound treatment group, positive controls, the dyeing of blank group, whole dyeing course needs the lucifuge operation, and step is as follows:

A, remove the liquid in the microwell plate, draw equal-volume 10 μ M JC-1 or 1mM DASPEI in microwell plate with liquid-transfering gun;

B, place 28 ℃ of constant incubators to cultivate 1h microwell plate;

Add the equal-volume breeding water in D, the microwell plate, in 28 ℃ of constant incubators, cultivate 5min;

E, repeated experiments operation D) 2-3 time;

F, zebra fish is changed in the 48 or 96 new orifice plates, every hole 1 tail removes the liquid in the microwell plate, adds 350 μ L(48 orifice plates) or 150 μ L(96 orifice plates) breeding water.

(4) quantitative test

Microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyser.Exciting light is set to 488nm, and (JC-1 is a coloring agent) or 515nm place (DASPEI is a coloring agent) gathers radiative fluorescence intensity at the 590nm place, and test repeats 3 times and gets its mean value.Injury of mitochondria rate computing formula is:

For example: if at exciting light 488nm, the group of solvents fluorescence intensity that collects during emission light 590nm is 550, compound treatment group fluorescence intensity is respectively 500,450,350,250,230, the positive controls fluorescence intensity is 200, background group fluorescence intensity is 50, then get according to computing formula: positive controls injury of mitochondria rate is 70%, and compound treatment group injury of mitochondria rate is respectively 10%, 20%, 40%, 60%, 64%(sees Fig. 3).

Experimental data with

Expression is relatively adopted variance analysis between many groups, relatively adopts Student ' s t check to carry out statistical procedures between two groups.But according to statistical procedures result quantitative evaluation toxicity of compound.

(5) qualitative analysis

Remove the liquid in the microwell plate, add 350 μ L(48 orifice plates) or 150 μ L(96 orifice plates) concentration is the methane-sulforic acid anesthesia zebra fish of 0.64mM, be fixed on the concave-concave microslide with 3% methylcellulose glue after, place observation fluorescence color under the fluorescent microscope.

JC-1 is as coloring agent: mitochondria normally can be observed red fluorescence in solvent control group and the blank group; Injury of mitochondria can be observed green fluorescence in the positive controls; Whether produce green fluorescence by observation compound treatment group and can judge that can this compound cause injury of mitochondria.DASPEI is as coloring agent: mitochondria normally can be observed bright green fluorescence in solvent control group and the blank group; Injury of mitochondria can be observed the green fluorescence deepening in the positive controls; Whether deepening can judge that can this compound cause injury of mitochondria by observing compound treatment group green fluorescence.But by observing contrast fluorescence color lightness qualitative evaluation toxicity of compound.

3.2 the mitochondrial Mitochondrially targeted compound of screening protection

(1) zebra fish is chosen

The zebra fish that will be in the optimization process stage places observation under the dissecting microscope, and the normotrophic zebra fish of picking moves in 6,12,24,48 or 96 orifice plates, the zebra fish of putting into varying number according to the microwell plate specification.

(2) compound treatment

9 experimental group are set: 5 testing compound combined treatment groups, 1 injury of mitochondria model group, 1 chondriosome protective positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, add injury of mitochondria derivant+0.1 μ M, 1 μ M, the 10 μ M of certain volume (deciding), the testing compound solution of 100 μ M, 1000 μ M in 5 testing compound combined treatment groups respectively according to the microwell plate specification; Add the injury of mitochondria derivant in the injury of mitochondria model group; Add injury of mitochondria derivant+mitochondrion protecting agent 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.In 28 ℃ of constant incubators, cultivate 24h according to the optimization process time span.

Testing compound combined treatment group of the present invention is except adopting above-mentioned mode of adding two kinds of materials (injury of mitochondria derivant+testing compound) simultaneously, and in addition, the testing compound combined treatment group of this step also can adopt following dual mode to handle:

First: after adding injury of mitochondria derivant 6～24h, 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; (being applicable to the screening curative)

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 the injury of mitochondria derivant again.(being applicable to the screening preventive medicine)

(3) dyeing is handled

The solvent control group is divided into two groups at random, and wherein one group is not dyeed, as a setting group; With other one group of group of solvents, testing compound combined treatment group, injury of mitochondria model group, positive controls, the dyeing of blank group, dyeing course is with step (3) JC-1 dyeing in the mitochondrial target mitochondria compound of 3.1 screening damages.

(4) quantitative test

Microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyser.Exciting light is set to 488nm, and (JC-1 is a coloring agent) gathers radiative fluorescence intensity at the 530nm place, and test repeats 3 times and gets its mean value.Injury of mitochondria protection ratio computing formula is:

。

For example: if at exciting light 488nm; the injury of mitochondria model group fluorescence intensity that collects during emission light 530nm is 450; testing compound combined treatment group fluorescence intensity is respectively 400,350,300,200,180; background group fluorescence intensity is 50; the positive controls fluorescence intensity is 170; then get according to computing formula: positive controls injury of mitochondria protection ratio is 70%, and testing compound combined treatment group injury of mitochondria protection ratio is respectively 12.5%, 25%, 37.5%, 62.5%, 67.5%(sees Fig. 4).

Experimental data with

Expression is relatively adopted variance analysis between many groups, relatively adopts Student ' s t check to carry out statistical procedures between two groups.But according to statistical procedures result quantitative screening new drug.

(5) qualitative analysis

Remove the liquid in the microwell plate, add 350 μ L(48 orifice plates) or 150 μ L(96 orifice plates) concentration is the methane-sulforic acid anesthesia zebra fish of 0.64mM, be fixed on the concave-concave microslide with 3% methylcellulose glue after, place observation fluorescence color under the fluorescent microscope again.Mitochondria normally can be observed red fluorescence in solvent control group and the blank group; Injury of mitochondria can be observed green fluorescence in the injury of mitochondria model group; Green fluorescence lightness in testing compound processed group and the injury of mitochondria model group is compared, can save injury of mitochondria if the testing compound processed group, can be judged this compound than green fluorescence deepening or disappearance in the injury of mitochondria model group.

Compared with prior art, the present invention has the following advantages:

The present invention has advantages such as easy, quick, economic, efficient, high flux as a kind of method of in vivo estimating environmental toxicity and screening new drug.Compare with traditional in-vitro screening model, 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 to 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 70%～80%, 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, credible result degree height, required expense is low ^[24]Specifically, the present invention has following advantage:

(1) in vivo-and 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, and predictability is good.

(2) high flux-zebrafish embryo is very little, juvenile fish has only the 1-4 millimeter, can be in 6,12 of a standard, analyzing in 24,48 or 96 orifice plates with experimental period short makes zebra fish become screening model in a kind of ideal body that can carry out the high flux full automation experiment.

(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 zebra fish.

(4) consumption few-the detection compound consumption is few, usually only needs several milligrams, traditional screening experiment then needs the compound more than several milligrams.

(5) easy-experimentation is simple to operate, and zebra fish just can place after drug treating, dyeing and carry out under multi-functional microwell plate analyser and the fluorescent microscope quantitatively and qualitative analysis, and the traditional experiment complicated operating process is easy to generate false positive results.

(6) fast-lack, can in 2～3 days, finish 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.Zebra fish was finished embryonic development at first 72 hours with interior.Most internals comprises cardiovascular system, intestines, liver and kidney, rapid shaping in 24-48 hour, and traditional experiment carrier mouse and monkey then needed 21 days and 9 months can finish embryonic development respectively.

(7) efficiently-and zebra fish is similar to the human gene height, and the experimental result comparability is strong.Gene sequencing through the Britain expert is found, the gene of zebra fish and the similarity of human gene are up to 70-80%, the physiology of zebra fish is very similar to mammal with the formation of metabolic system, therefore, zebra fish screening experiment result's comparability (comparable) is compared with mammal and can be reached more than 80%.

(8) predictability good-zebra fish is similar to the human gene height, drug screening predictability as a result is good.The gene of zebra fish and the similarity of human gene are up to 70-80%.Therefore, the zebra fish screening model can better be predicted the absorption,distribution,metabolism,excretion process of medicine in human body than other screening models, can really reflect the physiological environment of medicine in human body.

(9) susceptibility height-mitochondrial membrane potential is essential to keeping mitochondrial normal function, is the sensitive indicator of estimating mitochondrial function.Mitochondrial membrane potential reduces or disappears is event the earliest in the Apoptosis cascade reaction process, in case mitochondrial membrane potential drastic change, Apoptosis is with irreversible.Therefore, the present invention utilizes JC-1 or DASPRI fluorescence colour detection line mitochondrial membrane potential susceptibility very high.

(10) high, the good reproducibility of stability-repeated experiments of the present invention is tens times, and it is basic identical that institute obtains experimental result.

(11) high specificity-will be stronger than the experimental result specificity of non-target with the mitochondria that is sent to of medicine or toxicity target.

Description of drawings

Fig. 1 is the injury of mitochondria rate change curve of zebra fish different developmental phases of the present invention.

Fig. 2 is the injury of mitochondria rate change curve of different disposal time span during The compounds of this invention is handled.

Fig. 3 is the injury of mitochondria rate change curve of variable concentrations lesion wire plastochondria compound of the present invention.

Fig. 4 is the injury of mitochondria protection ratio change curve of variable concentrations protective wire plastochondria compound of the present invention.

Fig. 5 is the injury of mitochondria rate change curve of the embodiment of the invention 1 variable concentrations adriamycin.

Fig. 6 is the injury of mitochondria rate change curve of the embodiment of the invention 2 variable concentrations vinclozolins.

Fig. 7 is the injury of mitochondria rate change curve of the embodiment of the invention 3 variable concentrations Teniposides.

Fig. 8 is the injury of mitochondria protection ratio change curve of the embodiment of the invention 4 variable concentrations Dipyridamoles.

Fig. 9 is the injury of mitochondria protection ratio change curve of the embodiment of the invention 5 variable concentrations tanshin polyphenolic acid Bs.

Embodiment

Below in conjunction with embodiment, be described more specifically content of the present invention.Should be appreciated that enforcement of the present invention is not limited to the following examples, all will fall into protection domain of the present invention any pro forma accommodation and/or the change that the present invention made.

In the present invention, if not refer in particular to, all equipment and raw material etc. all can be buied from market or the industry is commonly used.Do not specialize if having, the method that embodiment adopts is this area current techique.

Key instrument and reagent explanation:

Breeding water among the embodiment meets: the dissolved oxygen DO mass concentration is 6-8mg/L; Water temperature is 28 ℃; PH is 7.2-7.6; Total hardness is 200-250mg/L.

Embodiment 1The target mitochondria is estimated drug toxicity

Present embodiment is undertaken by following step:

(1) zebra fish is chosen

The zebra fish of 4dpf is placed observation under the dissecting microscope, and the normotrophic zebra fish of picking moves into respectively in 48 orifice plates, every hole 10 tails.

(2) compound treatment

8 experimental group are set: 5 drug treating groups to be measured, 1 injury of mitochondria positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, add 1mL concentration in 5 drug treating groups to be measured respectively and be respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M adriamycins ^[26]Solution; Adding 1mL concentration in the positive controls is the Etoposide solution of 50 μ M; Adding 1mL concentration is 0.1% DMSO in the solvent control group; Add the 1mL breeding water in the blank group.In 28 ℃ of constant incubators, cultivate 24h.

(3) dyeing is handled

The solvent control group is divided into two groups at random, and wherein one group is not dyeed, as a setting group; With other one group of group of solvents, drug treating group to be measured, positive controls, the dyeing of blank group, select for use JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

B, place 28 ℃ of constant incubators to cultivate 1h microwell plate;

E, repeated experiments operation D) 2 time;

(4) quantitative test

Experimental data with

Expression is relatively adopted variance analysis between many groups, relatively adopts Student ' s t check to carry out statistical procedures between two groups.But by statistical procedures result quantitative evaluation drug toxicity.

Calculate according to above-mentioned formula, statistical procedures result shows that positive controls injury of mitochondria rate is (69.11 ± 1.68) %, and 5 drug treating group injury of mitochondria rates to be measured are respectively (26.47 ± 0.69) %, (49.57 ± 1.07) %, (60.51 ± 2.01) %, (63.97 ± 1.36) %, (64.56 ± 2.57) % (see figure 5).Drug treating group to be measured is compared difference with the solvent control group have statistical significance (p＜0.05).

(5) qualitative analysis

Remove the liquid in the microwell plate, add 350 μ L concentration and be the methane-sulforic acid anesthesia zebra fish of 0.64mM, be fixed on the concave-concave microslide with 3% methylcellulose glue after, place observation fluorescence color under the fluorescent microscope.

Mitochondria is normally observed red fluorescence in solvent control group and the blank group; Injury of mitochondria is observed green fluorescence in the positive controls; Drug treating group injury of mitochondria to be measured is observed green fluorescence, can judge that adriamycin has caused injury of mitochondria.Therefore, but by observing contrast fluorescence color lightness qualitative evaluation drug toxicity.

Embodiment 2The target mitochondria is estimated the environmental toxicity compound

Present embodiment is undertaken by following step:

(1) zebra fish is chosen

The zebra fish of 4dpf is placed observation under the dissecting microscope, and the normotrophic zebra fish of picking moves in 48 orifice plates, every hole 10 tails.

(2) compound treatment

8 experimental group are set: 5 environmental toxicity compound treatment groups to be measured, 1 injury of mitochondria positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, add 1mL concentration in 5 environmental toxicity compound treatment groups to be measured respectively and be respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M environmental toxicity compound vinclozolins ^[27]Solution; Adding 1mL concentration in the positive controls is the star spore rhzomorph solution of 50 μ M; Adding 1mL concentration is 0.1% DMSO in the solvent control group; Add the 1mL breeding water in the blank group.In 28 ℃ of constant incubators, cultivate 24h.

(3) dyeing is handled

The solvent control group is divided into two groups at random, and wherein one group is not dyeed, as a setting group; With other one group of group of solvents, environmental toxicity compound treatment group to be measured, positive controls, the dyeing of blank group, select for use DASPEI as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

A, remove the liquid in the microwell plate, draw 1mL 1mM DASPEI in microwell plate with liquid-transfering gun;

B, place 28 ℃ of constant incubators to cultivate 0.5h microwell plate;

E, repeated experiments operation D) 3 time;

(4) quantitative test

Microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyser.Exciting light is set to 488nm, gathers radiative fluorescence intensity at the 515nm place, and test repeats 3 times and gets its mean value.Injury of mitochondria rate computing formula is:

Experimental data with

Expression is relatively adopted variance analysis between many groups, relatively adopts Student ' s t check to carry out statistical procedures between two groups.But by statistical procedures result quantitative evaluation environmental toxicity compound.

Calculate according to above-mentioned formula, statistical procedures result shows that positive controls injury of mitochondria rate is (69.11 ± 1.68) %, and 5 environmental toxicity compound treatment group injury of mitochondria rates to be measured are respectively (23.21 ± 2.35) %, (43.92 ± 3.17) %, (52.61 ± 2.01) %, (63.16 ± 2.27) %, (65.31 ± 1.83) % (see figure 6).Environmental toxicity compound treatment group to be measured is compared difference with the solvent control group have statistical significance (p＜0.05).

(5) qualitative analysis

Mitochondria normally can be observed bright green fluorescence in solvent control group and the blank group; Injury of mitochondria can be observed the green fluorescence deepening in the positive controls; Environmental toxicity compound treatment group green fluorescence to be measured deepening can judge that vinclozolin has caused injury of mitochondria.But by observing contrast fluorescence color lightness qualitative evaluation environmental toxicity compound.

Embodiment 3Target mitochondria screening antineoplastic

Mitochondria can mediate the apoptosis of tumour cell.Antineoplastic acts on mitochondria, causes mitochondrial permeability conversion hole open, and mitochondrial membrane potential descends or disappears, and respiratory chain is taken off the coupling connection then, and glutathione exhausts that ROS generation and cromoci and inducible factor discharge, and inducing apoptosis of tumour cell.Therefore, the mitochondrial toxicity compound might be used as potential anticarcinogen.

Present embodiment is undertaken by following step:

(1) zebra fish is chosen

The zebra fish of 4dpf is placed observation under the dissecting microscope, and the normotrophic zebra fish of picking moves in 96 orifice plates, every hole 1 tail.

(2) compound treatment

8 experimental group are set: 5 candidate's antineoplastic processed group, 1 injury of mitochondria positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, add 150 μ L concentration in 5 candidate's antineoplastic processed group respectively and be respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M candidate antineoplastic Teniposides ^[28]Solution; Adding 150 μ L concentration in the positive controls is the Etoposide solution of 50 μ M; Adding 150 μ L concentration are 0.1% DMSO in the solvent control group; Add 150 μ L breeding waters in the blank group.In 28 ℃ of constant incubators, cultivate 24h.

(3) dyeing is handled

The solvent control group is divided into two groups at random, and wherein one group is not dyeed, as a setting group; With other one group of group of solvents, candidate's antineoplastic processed group, positive controls, the dyeing of blank group, select for use JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

A, remove the liquid in the microwell plate, draw 150 μ L, 10 μ M JC-1 in microwell plate with liquid-transfering gun;

B, place 28 ℃ of constant incubators to cultivate 1h microwell plate;

Add 150 μ L breeding waters in D, the microwell plate, in 28 ℃ of constant incubators, cultivate 5min;

E, repeated experiments operation D) 2 time;

F, zebra fish is changed in the 96 new orifice plates, remove the liquid in the microwell plate, add 150 μ L breeding waters.

(4) quantitative test

Experimental data with

Expression is relatively adopted variance analysis between many groups, relatively adopts Student ' s t check to carry out statistical procedures between two groups.But by statistical procedures result quantitative screening antineoplastic.

Calculate according to above-mentioned formula, statistical procedures result shows that positive controls injury of mitochondria rate is (69.11 ± 1.68) %, and 5 candidate's antineoplastic processed group injury of mitochondria rates are respectively (20.22 ± 1.33) %, (38.55 ± 3.04) %, (52.19 ± 2.12) %, (66.42 ± 2.17) %, (67.31 ± 1.83) % (see figure 7).Candidate's antineoplastic processed group is compared difference with the solvent control group have statistical significance (p＜0.05).

(5) qualitative analysis

Remove the liquid in the microwell plate, add 150 μ L concentration and be the methane-sulforic acid anesthesia zebra fish of 0.64mM, be fixed on the concave-concave microslide with 3% methylcellulose glue after, place observation fluorescence color under the fluorescent microscope.Can qualitative screening antineoplastic by observing the lightness of contrast fluorescence color.

Mitochondria normally can be observed red fluorescence in solvent control group and the blank group; Injury of mitochondria can be observed green fluorescence in the positive controls; Candidate's antineoplastic processed group has produced green fluorescence, can judge that Teniposide has caused injury of mitochondria.

Embodiment 4Target mitochondria screening antioxidant

The oxidative stress reaction can cause multiple and old and feeble relevant human degenerative disease, and mitochondria is a cell within a cell device of being responsible for energetic supersession, is to cause the free radical of oxidative stress and the main source of active oxygen in most of cell.Old and feeble evolution and mitochondrial function are closely related unusually.Free radical theory is one of representative causes of senescence.Along with the increase of organismic age, antioxidation activity successively decreases, and the oxygen radical generation increases and accumulates.Too much oxygen radical can be attacked mitochondrial membrane by peroxidation, causes the mitochondrial membrane permeability changes, and mitochondrial membrane potential descends or disappears, final cell death inducing.Therefore, antioxidant can be used as potential antiaging agent.

Present embodiment is undertaken by following step:

(1) zebra fish is chosen

(2) compound treatment

9 experimental group are set: 5 testing compound combined treatment groups, 1 injury of mitochondria model group, 1 chondriosome protective positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, add 150 μ L deoxycholic acids (oxidative stress derivant) in 5 testing compound combined treatment groups respectively ^[29](the deoxycholic acid final concentration is all 0.5mM to the mixed solution of+candidate antioxidant in 5 groups, candidate's antioxidant Dipyridamole ^[30]Concentration be respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M); Add 150 μ L 0.5mM deoxycholic acids in the injury of mitochondria model group; Add 150 μ L deoxycholic acid+glutathione (antioxidant) in the positive controls ^[31]Mixed solution (in the mixed solution: the concentration of deoxycholic acid is that the concentration of 0.5mM, glutathione is 25 μ M); Adding 150 μ L concentration are 0.1% DMSO in the solvent control group; The breeding water that adds 150 μ L in the blank group.In 28 ℃ of constant incubators, cultivate 24h.

(3) dyeing is handled

The solvent control group is divided into two groups at random, and wherein one group is not dyeed, as a setting group; With testing compound combined treatment group, injury of mitochondria model group, chondriosome protective positive controls, blank group and another group group of solvents dyeing, select for use JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

B, place 28 ℃ of constant incubators to cultivate 1h microwell plate;

E, repeated experiments operation D) 2 time;

F, zebra fish is changed in the 96 new orifice plates, every hole 1 tail removes the liquid in the microwell plate, adds 150 μ L breeding waters.

(4) quantitative test

Microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyser.Exciting light is set to 488nm, gathers radiative fluorescence intensity at the 530nm place, and test repeats 3 times and gets its mean value.Injury of mitochondria protection ratio computing formula is:

。

Experimental data with

Expression is relatively adopted variance analysis between many groups, relatively adopts Student ' s t check to carry out statistical procedures between two groups.But according to statistical procedures result quantitative screening antioxidant.

Calculate according to above-mentioned formula; statistical procedures result shows that positive controls injury of mitochondria protection ratio is (68.80 ± 0.80) %, and 5 testing compound combined treatment group injury of mitochondria protection ratios are respectively (23.16 ± 1.33) %, (35.32 ± 3.04) %, (54.17 ± 2.12) %, (67.14 ± 1.62) %, (68.31 ± 1.83) % (see figure 8).Testing compound combined treatment group is compared difference with the injury of mitochondria model group have statistical significance (p＜0.05).

(5) qualitative analysis

Remove the liquid in the microwell plate, add 150 μ L concentration and be the methane-sulforic acid anesthesia zebra fish of 0.64mM, be fixed on the concave-concave microslide with 3% methylcellulose glue after, place observation fluorescence color under the fluorescent microscope.Solvent control group and blank group are red fluorescence, and the injury of mitochondria model group can be observed green fluorescence.Green fluorescence lightness in testing compound combined treatment group and the injury of mitochondria model group is compared can qualitative screening antioxidant, the observations of the testing compound combined treatment group of present embodiment is: the green fluorescence lightness is dark than the injury of mitochondria model group, and the red fluorescence lightness is bright than the injury of mitochondria model group.

Embodiment 5The target mitochondria screens anti-nerve degenerative diseases medicine

Nerve degenerative diseases is one group of chronic progressive external the nervous system disease based on the primary neuronal degeneration.Such disease mainly comprises Alzheimer disease, Parkinson's, Huntington chorea, dissimilar spinocerebellar ataxia, ALS and spinal cord muscular atrophy etc.One of pathomechanism that nerve retrograde affection is common is the apoptosis of neurocyte, the medicine of the apoptosis process screening control nerve retrograde affection that therefore can mediate by the mitochondria of block nerves cell degenerative change.

With the screening antiparkinsonism drug is example, and present embodiment is undertaken by following step:

(1) zebra fish is chosen

(2) compound treatment

9 experimental group are set: 5 testing compound combined treatment groups, 1 injury of mitochondria model group, 1 chondriosome protective positive controls, 1 solvent control group, 1 blank group.Remove the breeding water in the microwell plate, add 150 μ L 1-methyl-4-phenyl-1,2 in 5 testing compound combined treatment groups respectively, 3,6-tetrahydropyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyritine, MPTP) (Parkinson's derivant) ^[32]+ candidate antiparkinsonism drug tanshin polyphenolic acid B ^[33]Mixed solution (in 5 mixed solutions: the concentration of MPTP is all 10 μ g/mL, and the concentration of candidate's antiparkinsonism drug tanshin polyphenolic acid B is respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M); Add 150 μ L, 10 μ g/mL MPTP in the injury of mitochondria model group; MPTP+the selegiline (L-deprenyl) (Parkinson's therapeutic agent) that adds 150 μ L in the positive controls ^[32]Mixed solution (in the mixed solution: the MPTP final concentration is 10 μ g/mL, and selegiline concentration is 4 μ g/mL); Adding 150 μ L concentration are 0.1% DMSO in the solvent control group; Add 150 μ L breeding waters in the blank group.In 28 ℃ of constant incubators, cultivate 24h.

(3) dyeing is handled

The solvent control group is divided into two groups at random, and wherein one group is not dyeed, as a setting group; With testing compound combined treatment group, injury of mitochondria model group, positive controls, blank group and another group group of solvents dyeing, select for use JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

B, place 28 ℃ of constant incubators to cultivate 1h microwell plate;

E, repeated experiments operation D) 2 time;

(4) quantitative test

。

Experimental data with

Expression is relatively adopted variance analysis between many groups, relatively adopts Student ' s t check to carry out statistical procedures between two groups.But according to the anti-nerve degenerative diseases medicine of statistical procedures result quantitative screening.

Calculate according to above-mentioned formula; statistical procedures result shows that positive controls injury of mitochondria protection ratio is (68.80 ± 0.80) %, and 5 testing compound combined treatment group injury of mitochondria protection ratios are respectively (32.15 ± 1.58) %, (48.41 ± 2.89) %, (58.93 ± 3.51) %, (66.43 ± 2.31) %, (67.65 ± 2.13) % (see figure 9).Testing compound combined treatment group is compared difference with the injury of mitochondria model group have statistical significance (p＜0.05).

(5) qualitative analysis

Remove liquid in the microwell plate, add 150 μ L concentration and be the methane-sulforic acid anesthesia zebra fish of 0.64mM, be fixed on the concave-concave microslide with 3% methylcellulose glue after, place observation fluorescence color under the fluorescent microscope.Solvent control group and blank group are red fluorescence, and the injury of mitochondria model group can be observed green fluorescence.Green fluorescence lightness in testing compound combined treatment group and the injury of mitochondria model group is compared, can the anti-nerve degenerative diseases medicine of qualitative screening.The observations of present embodiment testing compound combined treatment group is: the green fluorescence lightness is dark than the injury of mitochondria model group, and the red fluorescence lightness is bright than the injury of mitochondria model group.

By above preferred embodiment as seen: live body zebra fish fluorescence colour provided by the invention can be easy, quick, economical, efficient, high flux the ground accurately environmental toxicity and the screening new drug of evaluating chemical thing.Method step provided by the invention is simple, and is with low cost, the accuracy height, have good stability and reliability, the live body zebra fish can really be reflected the whole biologically active 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, the foregoing description is modified and/or flexible or to adopt the replacement scheme that is equal to be obvious, the essence that all can not break away from 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 solution of the present invention and understanding.

List of references:

[1] Wang Yunliang, Shao Fenghua, Li Ping, etc. mitochondriopathy progress [J]. Chinese practical sacred disease magazine, 2007,10(8): 137-140.

[2]?Li?C.Q.,?Wogan?G.?N..?Nitric?oxide?as?a?modulator?of?apoptosis[J].?Cancer?letters,?2005,?226(1):1-15.

[3] Liu Weili, Qi Wencheng. the progress that mitochondria acts in Apoptosis [J]. the medical science summary, 2007,13(8): 578-580.

[4] Chen Ru. Parkinson's progress [J]. Chinese rehabilitation theory and practice, 2007,13(7): 637-639.

[5] Cheng Yinxia, Du Guanhua. the drug research progress [J] of control nerve degenerative diseases. the Chinese Medicine guide, 2006,8(5): 320-322.

[6] Xin Ying. mitochondria and nerve retrograde affection [J]. the medical science summary, 2008,14(4): 520-521.

[7]?Hayakawa?M.,?Ogawa?T.,?Sugiyama,?et?al.?Massive?conversion?of?gunaosine?to?8-hydroxy-gunaosine?in?mouse?liver?mitochondrial?DNA?by?administration?of?azidot?hymidine?[J].?Biochemical?and?Biophysical?Research?Communication,?1991,?176(1):?87-93.

[8]?Bandy?B.,?Davison?A.J..?Mitochondrial?mutations?may?increase?oxidative?stress:?implications?for?cracinogenesis?and?aging[J].?Free?Radical?Biology?＆?Medicine,?1990,?8(6):?523-539.

[9]?Anthony?S.,?Don,?Philip?J.,?et?al.?Mitochondria?as?cancer?drug?targets[J].?Trends?in?Molecular?Medicine,?2004,?10(8):?372-378.

[10] Chen Zhirong. the progress of antiaging agent [J]. Strait Pharmaceutical Journal, 2008,20(1): 8-11.

[11] Sun Xiaosheng, willow. the progress of anti-ageing mechanism and medicine [J]. Traditional Chinese Medicine University Of Guangzhou's journal, 2009,26(6): 593-597.

[12]?Anders?M.W.,?Robotham?J.L.,?Sheu?S.S..?Mitochondria:?new?drug?targets?for?oxidative?stress-induced?diseases[J].?Expert?Opinion?Drug?Metabolism?＆Toxicology,?2006,?2(1):?71-79.

[13]?Armstrong?J.S..?Mitochondrial?medicine:?pharmacological?targeting?of?mitochondria?in?disease[J].?British?Journal?of?Pharmacology,?2007,?151(8):?1154-1165.

[14]?Wallace?K.B.,?Starkov?A.A..?Mitochondrial?Targets?of?Drug?Toxicity[J].?Annual?Review?of?Pharmacology?and?Toxicology,?2000,?40:?353-388.

[15] peace Ran, Dong Qiang. the detection method commonly used [J] of saturatingization of mitochondrial membrane in the apoptotic process. Chinese cerebrovascular disease magazine, 2009,3(1): 33-37.

[16]?Li?C.Q.,Trudel?L.J.,?Wogan?G.N..Nitric?oxide-induced?genotoxicity,?mitochondrial?damage,?and?apoptosis?in?human?lymphoblastoid?cells?expressing?wild-type?and?mutant?p53[J].?PANS,?2002,?99(16):?10364-10369.

[17]?Li?C.Q.,Trudel?L.J.,?Wogan?G.N..?Genotoxicity,?mitochondrial?damage,?and?apoptosis?in?human?lymphoblastoid?cells?exposed?to?peroxynitrite?generated?from?SIN-1[J].?Chemical?Research?in?Toxicology,?2002,?15(4):527-35.

[18]?Zhang?Y.Z.,?Ouyang?Y.C.,?Hou?Y.,?et?al.?Mitochondrial?behavior?during?oogenesis?in?zebrafish:?A?confocal?microscopy?analysis[J].?Development,?Growth＆Differentiation,?2008,?50(3):?189-201.

[19]?Widlansky?M.E.,?Wang?J.L.,?Shenouda?S.M.,?et?al.?Altered?mitochondrial?membrane?potential,?mass,?and?morphology?in?the?mononuclear?cells?of?humans?with?type?2?diabetes[J].?Translational?Research,?2010,?156(1):?15-25.

[20]?Wu?S.Y.,?Li?Y.Y.,?Xu?Y.,?et?al.?Macrophage?apoptosis?associated?with?Salmonella?enterica?serovar?Typhi?plasmid[J].?Indian?Journal?of?Experimential?Biology,?2010,?48(8):?773-777.

[21]?Zhao?X.J.,?Huang?Y.H.,?Yu?Y.C.,?et?al.?GnRH?antagonist?cetrorelix?inhibits?mitochondria-?dependent?apoptosis?triggered?by?chemotherapy?in?granulosa?cells?of?rats[J].?Gyneologic?Oncology,?2010,?118(1):?69-75.

[22]?Cheng?W.Y.,?Tong?H.Y.,?Miller?E.W.,?et?al.?An?integrated?imaging?approach?to?the?study?of?oxidative?stress?generation?by?mitochondrial?dysfunction?in?living?cells[J].?Environmental?Health?Perspectives,?2010,?118(7):?902-908.

[23] Yin Wu, Zou Suqi, Wang Guanghui, etc. the application [J] of model animal zebra fish in the nervous system disease research. life science, 2008,20(5): 773-777.

[24]?McGrath?P.,?Li?C.Q..?Zebrafish:?a?predictive?model?for?assessing?drug-induced?toxicity[J].?Drug?Discover?Today,?2008,?13(9):?394-401.

[25]?Westerfield?M..?The?Zebrafish?Book:?A?Guide?for?the?Laboratory?Use?of?Zebrafish.?Eugene,?Oregon:?The?University?of?Oregon?Press,?1993.

[26] Liu Zhe, Song Xiaodong, Hui Rutai, etc. the prevention of adriamycin cardiac toxic [J]. Chinese molecular cardiology magazine, 2007,7(6): 379-380.

[27] Li Xiuxiu, Yang Xin army, Yan Hongtao. the toxicity [J] that vinclozolin is grown zebrafish embryo. environment and occupational medicine, 2008,25(4): 378-380.

[28] Xu Rong, Hua Yong, Zhong Ping, etc. Teniposide and lomustine are induced brain glioblastoma cell mechanism of apoptosis [J]. Fudan Journal, and 2009,36(6): 692-706.

[29]?Payne?C.M.,?Weber?C.,?Crowley-Skillicorn,?et?al.?Deoxycholate?induces?mitochondrial?oxidative?stress?and?activates?NF-κB?through?multiple?mechanisms?in?HCT-116?colon?epithelial?cells[J].?Carcinogenesis,?2007,?28(1):?215-222.

[30]?Kusmic?C.,?Picano?E.,?Busceti?C.L.,?et?al.?The?antioxidant?drug?dipyridamole?spares?the?vitamin?E?and?thiols?in?red?blood?cells?after?oxidative?stress[J].?Cardiovascular?Research,?2000,?47:?510-514.

[31]?Tomas?R.,?Zuzana?C.,?Vladimira?M.,?et?al.?Glutathione?and?glutathione?assays[J].?Acta?Medical(Hradec?Kralove)?Suppl,?2005,?48(1):?15-20.

[32]?McKinley?E.T.,?Baranowski?T.C.,?Blavo?D.O..?Neuroprotection?of?MPTP-induced?toxicity?in?zebrafish?dopaminergic?neurons[J].?Molecular?Brain?Research,?2005,?141(2):?128-137.

[33] Cheng Jingli, and Ji Ling, Geng Hong, etc. Nimodipine is to the research [J] of nerve cell apoptosis influence after the cerebral ischemia. the contemporary Chinese doctor, and 2010,48(6): 1-2.

Claims

1. method of utilizing zebra fish to screen Mitochondrially targeted compound mainly may further comprise the steps:

(1) zebra fish is chosen

(2) compound treatment

A scheme: remove the breeding water in the microwell plate, then according to testing compound processed group, injury of mitochondria positive controls, solvent control group, blank group, specification according to microwell plate adds corresponding testing compound solution, injury of mitochondria derivant solution, solvent, breeding water respectively, microwell plate was in 28 ℃ of following constant temperature culture 6-72 hours then, wherein: testing compound solution concentration is 0.1-1000 μ M, perhaps

B scheme: remove the breeding water in the microwell plate; then according to testing compound combined treatment group; the injury of mitochondria model group; the chondriosome protective positive controls; the solvent control group; the blank group; the mixed solution that adds corresponding testing compound+injury of mitochondria derivant according to the specification of microwell plate respectively; injury of mitochondria derivant solution; the mixed solution of injury of mitochondria derivant+mitochondrion protecting agent; solvent; breeding water; microwell plate was in 28 ℃ of following constant temperature culture 6-72 hours then; wherein: testing compound solution concentration is 0.1-1000 μ M

(3) JC-1 or DASPEI dyeing is handled

B, place 28 ℃ of constant temperature to cultivate 0.5-1 hour microwell plate;

C, remove the liquid in the microwell plate, clean with breeding water;

Specification according to microwell plate in D, the microwell plate adds breeding water, cultivates 5 minutes in 28 ℃ of constant temperature;

E, repeated experiments operation D) 2-3 time;

2. the method for utilizing zebra fish to screen Mitochondrially targeted compound according to claim 1 is characterized in that: described step (1) zebra fish is chosen the zebra fish of fertilization after 4 days; Microwell plate was in 28 ℃ of following constant temperature culture 24 hours in described step (2) compound treatment.

3. the method for utilizing zebra fish to screen Mitochondrially targeted compound according to claim 1 and 2 is characterized in that: the whole dyeing course that described step (3) dyeing is handled needs the lucifuge operation.

4. the method for utilizing zebra fish to screen Mitochondrially targeted compound according to claim 1 and 2 is characterized in that: described step (1) zebra fish is chosen the employing dissecting microscope.

5. the method for utilizing zebra fish to screen Mitochondrially targeted compound according to claim 1 and 2 is characterized in that: described microwell plate is selected the microwell plate in 6-96 hole for use.

6. the method for utilizing zebra fish to screen Mitochondrially targeted compound according to claim 1 and 2, it is characterized in that: after described step (2) adopted the A scheme to carry out compound treatment, the quantitative test of the multi-functional microwell plate analyser of step (4) was operated as follows:

Microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyser, and exciting light is set to 488nm, at the 590nm place or 515nm gather radiative fluorescence intensity, calculate the injury of mitochondria rate by following formula:

Figure 2010102966969100001DEST_PATH_IMAGE002

。

7. the method for utilizing zebra fish to screen Mitochondrially targeted compound according to claim 1 and 2, it is characterized in that: after described step (2) adopted the B scheme to carry out compound treatment, the quantitative test of the multi-functional microwell plate analyser of step (4) was operated as follows:

Microwell plate is placed fluorescence intensity under the multi-functional microwell plate analyser, and exciting light is set to 488nm, gathers radiative fluorescence intensity at the 530nm place, calculates the injury of mitochondria protection ratio by following formula:

Figure 2010102966969100001DEST_PATH_IMAGE004

。

8. the method for utilizing zebra fish to screen Mitochondrially targeted compound according to claim 1 and 2 is characterized in that: described step (4) fluorescent microscope qualitative analysis is operated as follows:

After changing zebra fish over to new microwell plate, remove the breeding water in the microwell plate, adding concentration according to the microwell plate specification is the methane-sulforic acid of 0.64mM, be fixed on the concave-concave microslide with 3% methylcellulose glue after, place observation fluorescence color under the fluorescent microscope.

9. the method for utilizing zebra fish to screen Mitochondrially targeted compound according to claim 1 and 2 is characterized in that: the solvent in described step (2) compound treatment is that volumetric concentration is 0.1% dimethyl sulfoxide (DMSO).