CN101968484B

CN101968484B - Method for screening mitochondria targeted compounds by using zebra fish

Info

Publication number: CN101968484B
Application number: CN 201010296696
Authority: CN
Inventors: 彭恩泽
Original assignee: Hangzhou Hunter Biotechnology Inc
Current assignee: Nanjing Xinhuan Detection Technology Co., Ltd.
Priority date: 2010-09-29
Filing date: 2010-09-29
Publication date: 2013-06-12
Anticipated expiration: 2030-09-29
Also published as: CN101968484A

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 the zebra fish screening mitochondria targeted compounds

Technical field

The present invention relates to compound (chemicals) detection field, be specifically related to a kind of method that simple, economy, high flux realize utilizing the zebra fish screening mitochondria targeted compounds.

Background technology

Mitochondria is important organelle in eukaryotic, participates in the multiple important physiology and chemistry processes such as tricarboxylic acid cycle, fatty acid metabolism, oxidative phosphorylation in cell.Mitochondria plays important pivotal role in Apoptosis, in mitochondria, a series of metabolic process and Apoptosis are closely related, as the excessive generation of oxygen radical, the release of cromoci, the abnormal opening of mitochondrial permeability transition pore etc.Mitochondrial membrane has the different kinds of ions passage can mediate ion transport, and the adjusting of ion channel may affect the even function of cell of mitochondria.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 apoptotic pathways main process 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.These albumen are the non-mitochondria components that are positioned at cell under normal circumstances, in case cell is subject to inducing of inducer of apoptosis, they just to the mitochondria transposition, regulate 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 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 exists in tenuigenin, 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 Bcl-2 gene family.Albumen such as Bcl-2, Bax, the Bcl-xl etc. of a lot of Bcl-2 family are positioned on mitochondrial membrane.

Nerve degenerative diseases (Degenerative disease of the central nervous system, ND) be one group take the primary neuronal degeneration as the basis the chronic progressive external the nervous system disease.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, discharges high-caliber active oxygen (reactive oxygen species, ROS), thereby the active cell apoptosis reaches the damage to the karyogene group ^[7-9]Antineoplastic acts on mitochondria, can cause opening of mitochondria permeability transition pore, and mitochondrial membrane potential descends or disappears, and respiratory chain is taken off and is coupled then, and glutathione is exhausted, ROS generation and cromoci and inducible factor release, and inducing apoptosis of tumour cell.

Old and feeble evolution and mitochondrial function are extremely closely related ^[10]Free radical theory is one of representative causes of senescence.Mitochondria as the center of cellular respiration and oxidation, is also the important place that produces active oxygen ^[11]Generation and the elimination of ROS is in mobile equilibrium in 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 pass through peroxidation attack line Mitochondria Membrane, causes the mitochondrial membrane permeability changes, and mitochondrial membrane potential descends or disappears, final cell death inducing.

Research finds that the action target spot of many medicine or poisonous substances is positioned at mitochondrial membrane or mitochondrial inner membrane compound, its pharmacology or toxicological effect mainly realize by regulating mitochondrial respiration-chain function, metabolic enzyme activity, membrane permeability, and 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 the detection to mitochondrial permeability transition pore, the detection of membrane phospholipid, the detection of film potential and the damage check of mitochondrial DNA.At present the detection method of injury of mitochondria is mainly to be based upon on cellular level or molecular level basis.The detection method of apoptosis process Mitochondria damage mainly contains following several ^[15]:

(1) Western blot: by different Western blots, measure the albumen distributions such as cromoci, AIF in different cell components.The method treatment capacity is relatively low, and needs the mitochondrial separation of subcellular structure.The method experimentation complicated operation is difficult to avoid non-specific impact, easily produces 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.The method experimental implementation is complicated, easily causes cellular damage, and can not reflect medicine at the absorption,distribution,metabolism,excretion of 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, although the method can be applicable to living cells, and dynamically observe, 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, successively with primary antibodie and two anti-reactions, corresponding two resist and carry out fluorescence labeling, fluorescence microscope.Can utilize active caspase antibody and the carrying out of checking than staining examine apoptotic process.But the method sample throughput is lower, and the dynamics of different intermembranous protein delivery processes is different, easily produces false negative result.

(5) electronics/immunoelectron microscope: the ultrastructure of observable apoptotic cell, thus direct observation line plastochondria outer membrane rupture and local inner membrance hernia go out.The method sample throughput is very low, is difficult to carry out quantitative test, and the saturatingization phenomenon that occurs can not detect outer film integrality and do not destroy the time.

(6) cell fluorescence is measured: utilize fluorescent activation cell sorting system (fluorescence activated cell sorting, FACS) to measure.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 mitochondrial membrane space, presents hyperfluorescenceCeng Yongminggaoyingguang, and spills from adventitia as cromoci, 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 processing procedure.

But no matter above-mentioned is that 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 that cell is cultivated is taked in the detection of cellular level, although the method is effective, required expense is lower, cell in vitro has lacked the metabolic cycles conversion of biological integral and the loop distribution in body, can not really reflect whole biologically active;

(2) detection of molecular level needs mitochondria is separated, and has lacked equally medicine absorption, distribution, metabolism and discharge process in vivo, 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 impact, 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 is complicated, and 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 the research of zebra fish detection injury of mitochondria also seldom.Zhang YZ etc. ^[18]Utilize Laser Scanning Confocal Microscope to detect mitochondria behavior in the zebra fish Process of oogenesis in conjunction with the JC-1 fluorescence colour, this research female zebra fish ovary of at first growing up exsomatizes, make early stage egg mother cell suspension after compound treatment, then be placed on observational study under Laser Scanning Confocal Microscope through the mitochondria fluorescent dyeing.This research is the mitochondria behavior of observing in the zebra fish Process of oogenesis, 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 mitochondria targeted compounds.Publication number is China's invention of CN1866025A, discloses a kind of via zebra fish embryo model and detects the method that anti-new vessels generates protein factor or short new vessels generation protein factor activity, but should invention and non-targeted mitochondria screening compounds.

JC-1(iodate tetrachloro is for tetraethylbenzene imidazoles carbocyanine) be a kind of carbon cyanogen compound cationoid fluorescent dye, can enter in living cells and the mitochondrial inner membrane specific binding.JC-1 is good voltage-dependent fluorescent dye, is considered at present the specific fluorescent dye of the most responsive the most reliable mitochondrial membrane potential.Its principle is: the NM film potential is higher, and JC-1 is gathered in 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 be gathered in mitochondrial matrix, and this moment, JC-1 was monomer, and during exciting light 488nm, maximum emission wavelength is 530nm, can produce green fluorescence ^[16-17]Therefore, can represent by detecting red green fluorescence intensity the variation of mitochondrial membrane potential.In addition, the present invention also can adopt mitochondria fluorescent dye DASPEI(2 in living cells, 4-dimethylamino styryl-N-ethylpyridine iodide) as coloring agent, it can produce green fluorescence at exciting light 488nm during utilizing emitted light 515nm.

Detection method after mitochondria dyes through JC-1 is mainly to adopt flow cytometer, fluorescence microplate reader or laser confocal microscope etc. ^[19-22]Instrument is analyzed and researched.But adopt at present the research of these methods still to be in the experiment in vitro stage, and be based upon on the cellular level basis.Although these methods are effective, but experimental implementation is complicated, experimental expenses is higher, be difficult to avoid non-specific impact, 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 body, can not really reflect whole biologically active.

Summary of the invention

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

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

Summary of the invention

A kind of method of utilizing the zebra fish screening mitochondria targeted compounds mainly comprises the following steps:

(1) zebra fish is chosen

Choose the zebra fish of the normal development of after fertilization 2-7 days, put into microwell plate,

(2) compound treatment

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

B scheme: remove the breeding water in 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 respectively corresponding testing compound+injury of mitochondria derivant according to the specification of microwell plate, injury of mitochondria derivant solution, the mixed solution of injury of mitochondria derivant+mitochondrion protecting agent, solvent, breeding water, then microwell plate was in 28 ℃ of lower constant temperature culture 6-72 h(hours), wherein: testing compound solution concentration is 0.1-1000 μ M,

(3) JC-1 or DASPEI dyeing is processed

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

B, microwell plate is placed in 28 ℃ of constant temperature cultivated 0.5-1 h(hour);

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

Specification according to microwell plate in D, 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 new microwell plate, removes the liquid in 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 to detect mitochondria targeted compounds.The principle of JC-1 is: the NM film potential is higher, and JC-1 is gathered in 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 be gathered in mitochondrial matrix, and this moment, JC-1 was monomer, and during exciting light 488nm, maximum emission wavelength is 530nm, can produce green fluorescence.Therefore, can represent by detecting red green fluorescence intensity the variation of mitochondrial membrane potential.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 utilizing emitted light 515nm.

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

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

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

As preferably, according to the method for utilizing the zebra fish screening mitochondria targeted compounds of the present invention, wherein: described microwell plate is selected the microwell plate in 6-96 hole.The reason of selecting these specification microwell plates is that research is found, zebra fish of the present invention is suitable to be analyzed in 6,12,24,48 or 96 orifice plates of a standard.

As preferably, according to the method for utilizing the zebra fish screening mitochondria targeted compounds 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) operated as follows:

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

。

As preferably, according to the method for utilizing the zebra fish screening mitochondria targeted compounds 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) operated as follows:

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

。

As preferably, according to the method for utilizing the zebra fish screening mitochondria targeted compounds of the present invention, wherein: described step (4) fluorescent microscope qualitative analysis operates as follows:

Remove the liquid in microwell plate, adding concentration is 0.64mM methane-sulforic acid anesthesia zebra fish, after being fixed on the concave-concave microslide with 3% methylcellulose glue, is placed in fluorescence microscopy Microscopic observation fluorescence color.The JC-1 principle is: under fluorescent microscope, JC-1 forms polymer in NM, produces red fluorescence; Exist with monomeric form in the lesion wire plastochondria, produce green fluorescence.The DASPEI principle is: under fluorescent microscope, produce bright green fluorescence in NM, in the lesion wire plastochondria, green fluorescence compared with normal mitochondria is dimmed or disappear.

As preferably, according to the method for utilizing the zebra fish screening mitochondria targeted compounds 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 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 excretion 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 after fertilization 24h, the main former base of histoorgan of zebra fish forms, can be

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

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

The official system;

5) embryo has the yolk that nutrition can be provided, and does not need feeding in the first week, the interaction of compound and food component in the time of can avoiding compound treatment;

6) embryo is very little, and young fish length only has 1-4 mm, can be in 6,12,24,48 or 96 of a standard

Analyze in 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), staurosporine (staurosporine) and Etoposide (etoposide) are 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, determine the zebra fish optimization process stage

(1) zebra fish is chosen

The zebra fish of 2dpf, 3dpf, 4dpf, 5dpf, 6dpf, 7dpf is placed under dissecting microscope observes, the normotrophic zebra fish of picking moves into respectively in six 48 orifice plates, every hole 10 tails.(annotate: the dpf=day post fertilization in the present invention, Chinese refers to zebra fish after fertilization number of days, refers to 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 microwell plate; Water temperature is 28 ℃; PH is 7.2-7.6; Total hardness is 200-250mg/L, and is lower same), add 1mL 50 μ M staurosporines in injury of mitochondria derivant processed group; Adding 1mL concentration in the solvent control group is 0.1% DMSO; Add the 1mL breeding water in the blank group, cultivate in 28 ℃ of constant incubators.

(3) dyeing is processed

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 JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

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

B, microwell plate is placed in 28 ℃ of constant incubators cultivates 1h;

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

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

E, repeated experiments operation D) 2 times;

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

(4) quantitative test

Microwell plate is placed in fluorescence intensity under multi-functional microwell plate analyser.Exciting light is set to 488nm, and in the radiative fluorescence intensity of 590nm place's collection, 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.The 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 rear reduction, 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 microwell plate, adding 350 μ L concentration is the methane-sulforic acid anesthesia zebra fish of 0.64mM, after being fixed on the concave-concave microslide with 3% methylcellulose glue, is placed in fluorescence microscopy Microscopic observation fluorescence color.Under fluorescent microscope, JC-1 forms polymer in control group, produces red fluorescence; Exist with monomeric form in the mitochondria damaging cells, produce green fluorescence.Along with the increase at zebra fish age, injury of mitochondria derivant processed group green fluorescence first brightens rear dimmed, and 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, deterministic compound optimization process time span

(1) zebra fish is chosen

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

(2) compound treatment

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

(3) dyeing is processed

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 JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

B, microwell plate is placed in 28 ℃ of constant incubators cultivates 1h;

E, repeated experiments operation D) 2 times;

(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.The statistical procedures result shows: the zebra fish injury of mitochondria rate that the injury of mitochondria derivant is processed 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, injury of mitochondria derivant processed group is processed the injury of mitochondria rate of 12h, 24h, 48h, 72h higher than the solvent control group, difference has statistical significance (p＜0.05), and injury of mitochondria derivant processed group is processed no difference of science of statistics between 24h, 48h, 72h, therefore selects SPT 24h as the compound optimization process time.The different compound optimization process times are not identical yet, need the particular compound concrete analysis, and the present invention tests according to the optimization process time span of injury of mitochondria derivant.

(5) qualitative analysis

Remove the liquid in microwell plate, adding 350 μ L concentration is the methane-sulforic acid anesthesia zebra fish of 0.64mM, after being fixed on the concave-concave microslide with 3% methylcellulose glue, is placed in fluorescence microscopy Microscopic observation fluorescence color.Under fluorescent microscope, JC-1 forms polymer in control group, produces red fluorescence; Exist with monomeric form in the mitochondria damaging cells, produce green fluorescence.Along with the prolongation in processing time, injury of mitochondria derivant processed group green fluorescence brightens gradually, processes the 24h green fluorescence intensity and reaches maximum, and 48h and 72h have no obvious enhancing.Therefore, qualitative definite 24h is 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 mitochondria targeted compounds of screening damage

(1) zebra fish is chosen

Get 4～5 couples of zebra fish parents mating, according to Westerfield ^[25]The method hatched blastocyst.The zebra fish that will be in the optimization process stage (being 4dpf) is placed under dissecting microscope to be observed, 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 microwell plate, add respectively certain volume (deciding according to the microwell plate specification) concentration to be respectively the testing compound solution of 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M in 5 testing compound processed group; Add isochor plastochondria wound inducement agent 50 μ M staurosporines (staurosporine) or 50 μ M Etoposides (etoposide) in positive controls; Add equal-volume solvent (being generally 0.1% DMSO) in the solvent control group; Add the equal-volume breeding water in the blank group.Cultivate 24h according to the optimization process time span in 28 ℃ of constant incubators.

(3) dyeing is processed

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

B, microwell plate is placed in 28 ℃ of constant incubators cultivates 1h;

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

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

F, zebra fish is changed in 48 or 96 new orifice plates, every hole 1 tail removes the liquid in 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 in fluorescence intensity under multi-functional microwell plate analyser.Exciting light is set to 488nm, at 590nm place (JC-1 is coloring agent) or 515nm place (DASPEI is coloring agent) gather radiative fluorescence intensity, 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 utilizing emitted 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, 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 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, after being fixed on the concave-concave microslide with 3% methylcellulose glue, is placed in fluorescence microscopy Microscopic observation fluorescence color.

JC-1 is as coloring agent: solvent control group and blank group Mitochondria normally can be observed red fluorescence; The damage of positive controls Mitochondria can be observed green fluorescence; 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: solvent control group and blank group Mitochondria normally can be observed bright green fluorescence; It is dimmed that the damage of positive controls Mitochondria can be observed green fluorescence; Whether compound treatment group green fluorescence is dimmed judges that can this compound cause injury of mitochondria by observing.But contrast fluorescence color lightness qualitative evaluation toxicity of compound by observation.

3.2 the mitochondrial mitochondria targeted compounds of screening protection

(1) zebra fish is chosen

The zebra fish that will be in the optimization process stage is placed under dissecting microscope to be observed, 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 microwell plate, add respectively the testing compound solution of the injury of mitochondria derivant of certain volume (deciding according to the microwell plate specification)+0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M in 5 testing compound combined treatment groups; Add the injury of mitochondria derivant in the injury of mitochondria model group; Add injury of mitochondria derivant+mitochondrion protecting agent in positive controls; Adding equal-volume concentration in the solvent control group is 0.1% DMSO; Add isopyknic breeding water in the blank group.Cultivate 24h according to the optimization process time span in 28 ℃ of constant incubators.

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

First: after adding injury of mitochondria derivant 6～24h, then add final concentration to be respectively the testing compound solution of 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M; (being applicable to screen curative)

Second: after at first adding final concentration to be respectively testing compound solution 6～24h of 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M, then add the injury of mitochondria derivant.(being applicable to screen preventive medicine)

(3) dyeing is processed

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 3.1 mitochondrial target mitochondria compounds of screening damage.

(4) quantitative test

Microwell plate is placed in fluorescence intensity under multi-functional microwell plate analyser.Exciting light is set to 488nm, gathers radiative fluorescence intensity at 530nm place (JC-1 is coloring agent), 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 utilizing emitted 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; 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 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, after being fixed on the concave-concave microslide with 3% methylcellulose glue, then is placed in fluorescence microscopy Microscopic observation fluorescence color.Solvent control group and blank group Mitochondria normally can be observed red fluorescence; The damage of injury of mitochondria model group Mitochondria can be observed green fluorescence; Green fluorescence lightness in testing compound processed group and injury of mitochondria model group is compared, if the testing compound processed group is dimmed or disappear than injury of mitochondria model group Green fluorescence, can judge that this compound can save injury of mitochondria.

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

The present invention has the 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 excretion 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 through Britain expert's predictive genes, and 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 the mammal such as muroid, zebrafish embryo is transparent, a plurality of organs of observation analysis simultaneously, and experimental period is short, and sample size is large, and the credible result degree is high, and required expense is low ^[24]Specifically, the present invention has following advantage:

(1) in vivo-experiment material is the live body zebra fish, and as a kind of vertebrate, its screening model belongs to the body inner model, can truly reflect medicine absorption,distribution,metabolism,excretion in vivo, really reflects the whole biologically active of medicine, and predictability is good.

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

(3) economy-required expense is low, expend greater than 10 dollars every every day take the screening experiment of monkey as the experiment carrier, expend greater than 1 dollar every every day take the screening experiment of mouse as the experiment carrier, and expend less than 0.01 dollar every every day take the screening experiment of zebra fish as the experiment carrier.

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

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

(6) fast-experimental period is short, can complete in 2～3 days; 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 completed 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 needed respectively 21 days and 9 months can complete embryonic development.

(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, the comparability of zebra fish screening experiment result (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 high-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 Apoptosis cascade reaction process, in case mitochondrial membrane potential drastic change, Apoptosis is with irreversible.Therefore, the present invention utilize JC-1 or DASPRI fluorescence colour detection line mitochondrial membrane potential susceptibility very high.

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

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

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 processed.

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 present invention 1 variable concentrations adriamycin.

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

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

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

Fig. 9 is the injury of mitochondria protection ratio change curve of the embodiment of the present 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, any pro forma accommodation and/or change that the present invention is made all will fall into protection domain of the present invention.

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.If without specializing, the method that embodiment adopts is this area current techique.

Key instrument and reagent explanation:

Breeding water in 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

The present embodiment carries out in the steps below:

(1) zebra fish is chosen

The zebra fish of 4dpf is placed under dissecting microscope observes, 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 microwell plate, add respectively 1mL concentration to be respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M adriamycins in 5 drug treating groups to be measured ^[26]Solution; Adding 1mL concentration in positive controls is the Etoposide solution of 50 μ M; Adding 1mL concentration in the solvent control group is 0.1% DMSO; Add the 1mL breeding water in the blank group.Cultivate 24h in 28 ℃ of constant incubators.

(3) dyeing is processed

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 JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

B, microwell plate is placed in 28 ℃ of constant incubators cultivates 1h;

E, repeated experiments operation D) 2 times;

(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, the 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 microwell plate, adding 350 μ L concentration is the methane-sulforic acid anesthesia zebra fish of 0.64mM, after being fixed on the concave-concave microslide with 3% methylcellulose glue, is placed in fluorescence microscopy Microscopic observation fluorescence color.

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

Embodiment 2The target mitochondria is estimated the environmental toxicity compound

The present embodiment carries out in the steps below:

(1) zebra fish is chosen

The zebra fish of 4dpf is placed under dissecting microscope observes, 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 microwell plate, add respectively 1mL concentration to be respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M environmental toxicity compound vinclozolins in 5 environmental toxicity compound treatment groups to be measured ^[27]Solution; Adding 1mL concentration in positive controls is the staurosporine solution of 50 μ M; Adding 1mL concentration in the solvent control group is 0.1% DMSO; Add the 1mL breeding water in the blank group.Cultivate 24h in 28 ℃ of constant incubators.

(3) dyeing is processed

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 DASPEI as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

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

B, microwell plate is placed in 28 ℃ of constant incubators cultivates 0.5h;

E, repeated experiments operation D) 3 times;

(4) quantitative test

Microwell plate is placed in fluorescence intensity under multi-functional microwell plate analyser.Exciting light is set to 488nm, and in the radiative fluorescence intensity of 515nm place's collection, 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, the 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

Solvent control group and blank group Mitochondria normally can be observed bright green fluorescence; It is dimmed that the damage of positive controls Mitochondria can be observed green fluorescence; Environmental toxicity compound treatment group green fluorescence to be measured is dimmed, can judge that vinclozolin has caused injury of mitochondria.But contrast fluorescence color lightness qualitative evaluation environmental toxicity compound by observation.

Embodiment 3Target mitochondria antitumor medicine screening

Mitochondria can mediate the apoptosis of tumour cell.Antineoplastic acts on mitochondria, causes opening of mitochondria permeability transition pore, and mitochondrial membrane potential descends or disappears, and respiratory chain is taken off and is coupled then, and glutathione is exhausted, ROS generation and cromoci and inducible factor release, and inducing apoptosis of tumour cell.Therefore, the mitochondrial toxicity compound might be used as potential anticarcinogen.

The present embodiment carries out in the steps below:

(1) zebra fish is chosen

The zebra fish of 4dpf is placed under dissecting microscope observes, 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 microwell plate, add respectively 150 μ L concentration to be respectively 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M candidate antineoplastic Teniposides in 5 candidate's antineoplastic processed group ^[28]Solution; Adding 150 μ L concentration in positive controls is the Etoposide solution of 50 μ M; Adding 150 μ L concentration in the solvent control group is 0.1% DMSO; Add 150 μ L breeding waters in the blank group.Cultivate 24h in 28 ℃ of constant incubators.

(3) dyeing is processed

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 JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

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

B, microwell plate is placed in 28 ℃ of constant incubators cultivates 1h;

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

E, repeated experiments operation D) 2 times;

F, zebra fish is changed in 96 new orifice plates, remove the liquid in 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, the 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 microwell plate, adding 150 μ L concentration is the methane-sulforic acid anesthesia zebra fish of 0.64mM, after being fixed on the concave-concave microslide with 3% methylcellulose glue, is placed in fluorescence microscopy Microscopic observation fluorescence color.Contrasting the fluorescence color lightness by observation can qualitative antitumor medicine screening.

Solvent control group and blank group Mitochondria normally can be observed red fluorescence; The damage of positive controls Mitochondria can be observed green fluorescence; 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 mankind's degenerative disease, and mitochondria is the 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 cells.Old and feeble evolution and mitochondrial function are extremely closely related.Free radical theory is one of representative causes of senescence.Along with the increase of organismic age, antioxidation activity successively decreases, and oxygen radical produces and increases and accumulate.Too much oxygen radical can pass through peroxidation attack line Mitochondria Membrane, 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.

The present embodiment carries out in the steps below:

(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 microwell plate, add respectively 150 μ L deoxycholic acids (oxidative stress derivant) in 5 testing compound combined treatment groups ^[29](in 5 groups, the deoxycholic acid final concentration is all 0.5mM to the mixed solution of+candidate antioxidant, 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 positive controls ^[31]Mixed solution (in mixed solution: the concentration of deoxycholic acid is that the concentration of 0.5mM, glutathione is 25 μ M); Adding 150 μ L concentration in the solvent control group is 0.1% DMSO; Add the breeding water of 150 μ L in the blank group.Cultivate 24h in 28 ℃ of constant incubators.

(3) dyeing is processed

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 JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

B, microwell plate is placed in 28 ℃ of constant incubators cultivates 1h;

E, repeated experiments operation D) 2 times;

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

(4) quantitative test

Microwell plate is placed in fluorescence intensity under multi-functional microwell plate analyser.Exciting light is set to 488nm, and in the radiative fluorescence intensity of 530nm place's collection, 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; the 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 microwell plate, adding 150 μ L concentration is the methane-sulforic acid anesthesia zebra fish of 0.64mM, after being fixed on the concave-concave microslide with 3% methylcellulose glue, is placed in fluorescence microscopy Microscopic observation fluorescence color.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 injury of mitochondria model group is compared get final product qualitative screening antioxidant, the observations of the testing compound combined treatment group of the 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 anti-nerve degenerative diseases medicine of target mitochondria screening

Nerve degenerative diseases be one group take the primary neuronal degeneration as the basis the chronic progressive external the nervous system disease.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.

Take the screening antiparkinsonism drug as example, the present embodiment carries out in the steps below:

(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 microwell plate, add respectively 150 μ L 1-methyl 4-phenyls-1,2 in 5 testing compound combined treatment groups, 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) (treatment of Parkinson disease agent) that adds 150 μ L in positive controls ^[32]Mixed solution (in mixed solution: the MPTP final concentration is 10 μ g/mL, and selegiline concentration is 4 μ g/mL); Adding 150 μ L concentration in the solvent control group is 0.1% DMSO; Add 150 μ L breeding waters in the blank group.Cultivate 24h in 28 ℃ of constant incubators.

(3) dyeing is processed

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 JC-1 as coloring agent, whole dyeing course needs the lucifuge operation, and step is as follows:

B, microwell plate is placed in 28 ℃ of constant incubators cultivates 1h;

E, repeated experiments operation D) 2 times;

(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 nerve degenerative diseases medicine anti-according to statistical procedures result quantitative screening.

Calculate according to above-mentioned formula; the 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 microwell plate, adding 150 μ L concentration is the methane-sulforic acid anesthesia zebra fish of 0.64mM, after being fixed on the concave-concave microslide with 3% methylcellulose glue, is placed in fluorescence microscopy Microscopic observation fluorescence color.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 injury of mitochondria model group is compared, can the anti-nerve degenerative diseases medicine of qualitative screening.The observations of the 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, environmental toxicity and the screening new drug of high flux ground accurate evaluation chemicals.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 zebra fish can really be reflected the whole biologically active of medicine, comprises absorption, distribution, metabolism, the excretion of medicine, can realize high flux screening.

Although the inventor has done comparatively detailed elaboration and has enumerated technical scheme of the present invention, be to be understood that, for one of this area those skilled in the art, above-described embodiment is modified and/or flexible or to adopt the replacement scheme that is equal to be obvious, all can not break away from the essence of spirit of the present invention, the term that occurs in the present invention is used for elaboration and the understanding to technical solution of the present invention, can not be construed as limiting the invention.

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]. Medical review, 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 Research progress of drugs [J] of control nerve degenerative diseases. the Chinese Medicine guide, 2006,8(5): 320-322.

[6] Xin Ying. mitochondria and nerve retrograde affection [J]. Medical review, 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 mechanism of resisting senility 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 apoptotic process Mitochondria film. Chinese Journal of Cerebrovascular Diseases, 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 doxorubicin cardiotoxicity [J]. Chinese molecular cardiology magazine, 2007,7(6): 379-380.

[27] Li Xiuxiu, Yang Xin army, Yan Hongtao. the toxicity [J] of vinclozolin to Zebrafish Embryo. environment and occupational medicine, 2008,25(4): 378-380.

[28] Xu Rong, Hua Yong, Zhong Ping, etc. Teniposide and lomustine are induced the mechanism [J] of brain glioblastoma cell apoptosis. 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. the research [J] of Nimodipine on the nerve cell apoptosis impact after cerebral ischemia/reperfusion injury of rats. the contemporary Chinese doctor, 2010,48(6): 1-2.

Claims

1. method of utilizing the zebra fish screening mitochondria targeted compounds mainly comprises the following steps:

(1) zebra fish is chosen

(2) compound treatment

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

B scheme: remove the breeding water in 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 respectively corresponding testing compound+injury of mitochondria derivant according to the specification of microwell plate, injury of mitochondria derivant solution, the mixed solution of injury of mitochondria derivant+mitochondrion protecting agent, solvent, breeding water, then microwell plate was in 28 ℃ of lower constant temperature culture 6-72 hours, wherein: testing compound solution concentration is 0.1-1000 μ M

Described solvent is that volumetric concentration is 0.1% dimethyl sulfoxide (DMSO),

(3) JC-1 or DASPEI dyeing is processed

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 processings of dyeing of other groups, whole dyeing course needs lucifuge to operate, step is as follows:

B, microwell plate is placed in 28 ℃ of constant temperature cultivated 0.5-1 hour;

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

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

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

(4) the fluorescence microplate reader quantitative test is or/and the fluorescent microscope qualitative analysis.

2. the method for utilizing the zebra fish screening mitochondria targeted compounds according to claim 1 is characterized in that: described step (1) zebra fish is chosen the zebra fish of fertilization after 4 days; In described step (2) compound treatment, microwell plate was in 28 ℃ of lower constant temperature culture 24 hours.

3. the method for utilizing the zebra fish screening mitochondria targeted compounds according to claim 1 and 2, it is characterized in that: described step (1) zebra fish is chosen the employing dissecting microscope.

4. the method for utilizing the zebra fish screening mitochondria targeted compounds according to claim 1 and 2, it is characterized in that: described microwell plate is selected the microwell plate in 6-96 hole.

5. the method for utilizing the zebra fish screening mitochondria targeted compounds according to claim 1 and 2 is characterized in that: after described step (2) adopted the A scheme to carry out compound treatment, step (4) fluorescence microplate reader quantitative test operated as follows:

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

Figure 2010102966969100001DEST_PATH_IMAGE001

。

6. the method for utilizing the zebra fish screening mitochondria targeted compounds according to claim 1 and 2 is characterized in that: after described step (2) adopted the B scheme to carry out compound treatment, step (4) fluorescence microplate reader quantitative test operated as follows:

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

。

7. the method for utilizing the zebra fish screening mitochondria targeted compounds according to claim 1 and 2, it is characterized in that: described step (4) fluorescent microscope qualitative analysis operates as follows:

After changing zebra fish over to new microwell plate, remove the breeding water in microwell plate, adding concentration according to the microwell plate specification is the methane-sulforic acid of 0.64mM, after fixing with 3% methylcellulose glue, is placed in fluorescence microscopy Microscopic observation fluorescence color.