CN102023207B - Method for carrying out enzyme-linked immunoadsorption detection on integral zebra fish and application thereof - Google Patents
Method for carrying out enzyme-linked immunoadsorption detection on integral zebra fish and application thereof Download PDFInfo
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Abstract
The invention relates to a method for carrying out enzyme-linked immunoadsorption detection on an integral zebra fish and application of the method in drugs for identifying genotoxic compounds, screening anti-tumor drugs and repairing DNA damage. On one hand, the invention can be used for evaluating the genotoxicity of the compounds including environmental compounds, drugs, cosmetics, food additives and the like, and on the other hand, the invention can be used for screening new drugs, such as the anti-tumor drugs, antioxidants, anti-aging drugs, anti-neurodegenerative disease drugs and the like. The invention has very important guiding significance on diagnosing and treating certain diseases and novel drug research and development and compound toxicity screening. By applying the zebra fish to evaluate the compound toxicity and screen the novel drugs, the advantages of low cost, low use level of samples, automatic operation and the like can be achieved, and an in vivo physiological environment of a live animal can be provided, therefore, the goals of simple and convenient evaluating and screening works, rapidness, economy, high efficiency and high flux can be achieved.
Description
Technical field
The present invention relates to a kind of enzyme linked immunosorbent assay, more particularly, relate to a kind of method of carrying out the Enzyme-linked Immunosorbent Assay detection on integral zebra fish, and the application of the method in the medicine of sldh gene toxic chemical, antitumor medicine screening, DNA plerosis damage.
Background technology
Genotoxicity refers to direct or indirect DNA Damage.DNA is a kind of important biomacromolecule that has the hereditary information transfer function in cell, is the important research object of Biochemistry and Molecular Biology.DNA molecular consists of double-spiral structure by antiparallel two polynucleotide chains via basepairing rule, its entrained hereditary information is determined by kind, quantity and the ordering of the base on polynucleotide chain, base sequence is hidden in the inside of DNA double spiral deeply, article two, the double-spiral structure of polynucleotide chain composition has formed stable DNA molecular and (the Phillips DH that can correctly convey hereditary information, Arlt VM.Genotoxicity:damage to DNA and its consequences.EXS., 2009,99:87-110.).Yet extraneous various physics and chemistry and biotic factor all may cause DNA damage.(Ma Yuntong, the Qi Hao such as the form of these damages has single stranded DNA chain damage, and double-stranded DNA damage, the formation of pyrimidine dimer, DNA adduct, point mutation, DNA are crosslinked, Luo Mingzhi, Techniques of Detecting DNA Single-Strand Breakage in Cells. Xi'an school of Arts and Sciences journal, 2005,8 (3): 22-25.).
Along with the development of industrial or agricultural and the raising of living standards of the people, can cause the chemical substance of DNA damage more and more in daily life, as biological biochemistry medicine, industrial chemistry product, agriculture chemistry product (as agricultural chemicals, herbicide), pure natural (plant) product, food additives, nutrient and healthcare products, cosmetology product, three industrial wastes.If the DNA damage that various chemical substances cause occurs in reproduction cell, may affect the reproduction cell genetic stability, and may exert an influence to the offspring, for example cause miscarriage, stillborn foetus, deformity and some genetic disease.If DNA damage occurs in body cell, might cause disease to comprise cancer, threaten human health (Chai Guolin, Zhu Weiguo, the damage of DNA and reparation, Chinese Journal of Oncology, 2005,27 (10): 557-580.).Also the important content that the medicine non-clinical safety evaluation is estimated for genotoxicity research (Genotoxicity Study) simultaneously, especially mutagenesis and the carcinogenicity research of it and other toxicologic studies, genotoxicity research have close contacting, and are that before medicine enters clinical testing and listing, essential toxicity detects data.
Cancer is one of principal disease of serious harm human health, and capture cancer is the research topic of attracting attention in the world always.World Health Organization's investigation shows that the cancer patient increases year by year, China's cancer year number of the infected is in 1,200,000 left and right, die from the number of cancer every year up to more than 900,000 people, now become the second largest killer (Liu Feng, the Wei Xinting that are only second to cardiovascular disease, Wang Dongdong, Sun Dezhi, Li Lin is luxuriant, the present Research of cancer therapy drug, Liaocheng University's journal 2006,19:39-43.).Utilize determining presence of genotoxic compound can cause the character of cancer cell DNA damage, determining presence of genotoxic compound might be used for the treatment of cancer.
When cell was subject to the stimulation of DNA damage, genomic integrality faced threat, will produce a series of biological effects.When DNA damage can't be repaired, with cell death inducing.Apoptosis is the apoptosis of cytogene coding guiding under physiological condition, so be called again apoptosis.It guarantees balance between cell proliferation and cell death, is the part of normal physiological processes, is the cell oneself extinction process of being controlled by gene.Apoptosis embryonic development, hematopoiesis, immune maturation and keep normal structure and the cell number of organ constant with the growth balance, even human senility and disease all play an important role in occuring.Because there is defective in apoptosis, the cell death program exception is expressed, and perhaps apoptotic cell death program execution defectiveness and apoptosis are promoted, and be all relevant with disease incidence.Comprise tumour, nerve degenerative diseases.Thereby we can pass through screening-gene drug toxicity, regulating cell apoptosis.Equally, the screening DNA renovation agent is to disease prevention and cure and anti-ageing being of great immediate significance (Jiang Xue, Yang Hongli, Apoptosis and the effect in tumor development and treatment thereof, clinical assembling, 1997,12 (17): 774-776.).
Utilizing the method that detects the damage of DNA single chain is the effective ways (Chai Guolin of screening-gene toxic chemical, Zhu Weiguo, the damage of DNA and reparation, Chinese Journal of Oncology, 2005,27 (10): 557-580.), because the damage of DAN strand is the highest DNA damage type of occurrence frequency in mammalian cell.
The method of current discriminating DNA single chain damage is mainly single cell gel electrophoresis (comet) and micronucleus test, is also present topmost screening-gene toxic chemical method.
Single cell gel electrophoresis (single cell gel electrophoresis assay is called for short SCGE, has another name called Using Comet Assay, comet assay) is one and is widely used in the means that eukaryotic gene toxicity detects.This technology is pioneering in 1984 by Ostling, is improved respectively by Singh and Olive again, has formed present alkalescence and neutral cracking two class methods.Its ultimate principle is: under neutrality or alkali condition, be embedded in the lysis in agar gel, carry out electrophoresis after the DNA uncoiling, be without damage as cell, the DNA part is less, fragment is larger, and during electrophoresis, DNA rests in paralinin greatly because of its molecular mass, and fluorophore rounded after fluorescent dye is without conditions of streaking; If cell is impaired, the DNA part is more, the DNA uncoiling becomes strand under alkali condition, during electrophoresis because of DNA molecular weight I to enter in gel, anode stretches, be a bright head and afterbody under fluorescent microscope, the likeness in form comet is therefore claim again Using Comet Assay (Klaude M, Eriksson S, Nygren J, Ahnstrom G, The comet assay:mechanisms and technical considerations, Mutation Research, 1996,363 (2): 89 – 96.).Cell and DNA are impaired more serious, the part that produces is more and fragment is less, the DNA amount of moving during electrophoresis is also just larger, migration distance is longer, under fluorescent microscope, the long increase of observable tail, afterbody fluorescence intensity strengthen, and move the optical density of part and the DNA damage degree that migration length just can be measured individual cells by mensuration.Its operating process comprises: offset plate preparation, lysis, electrophoresis, neutralization, dyeing, microscopy, analysis (Li Hong, the progress of micronucleus test, Agriculture of Anhui science, 2009,37 (7): 2864-2866.).
Another kind method is micronucleus test, and it is take animals and plants as material, and the micronuclear rates that utilizes cell biology method to observe its appearance represents that material is subjected to a kind of method that detects the gene poisonous substance of genetic damage degree.Micronucleus (micronucleus, MN) refers to that the tenuigenin that is arranged in biological cell is independent of main core, and diameter is less than main core 1/ 20～1/ 3, the circle or the oval-shaped small core that separate with main core fully.It can be whole chromosome, can be also chromosome fragment, and dyeability is consistent with main core, and wherein the part micronucleus has the DNA replication dna ability.Micronucleus is because extraneous damage factor makes the chromosome fracture, and cell enters when dividing next time, and chromosome can not enter daughter cell with mitosis, and causes chromosome diminution or fracture, thereby forms one or several small nuts.Micronuclear rates refers to biomaterial after the cell biology method film-making, and the shared ratio of micronucleus cell in the middle of 1000 cells of microscopically observation can be also to calculate with the mean value that contains the micronucleus number in the middle of each cell.Concrete operation step comprises: smear, fixing, dyeing, mounting, observation and counting, analysis (Wang Lan, Liu Xinrong, the application and development of single cell gel electrophoresis technique, Chinese Journal of Health Laboratory Technology, 2008,12 (18): 2829-2831.).
These two kinds of methods can detect genotoxicity on external level, any eukaryotic can be used for the outer-gene toxicity research, and the most frequently used cell is human peripheral lymphocyte, mouse peripheral blood lymphocyte and erythrocyte.But in vitro test because cell is not in vivo in environment, the absorption, distribution, metabolism, the discharge process that there is no medicine, the result of test can not reflect the truth in the medicine body, and in these tests, false positive results is very general, micronucleus test particularly, the method is simple, but confidence level is lower.Micronucleus detects cell is made picture, dyes erythrocytic micronucleus number with more than 1000 of microscopic countings after fixing dyeing, and the technical work amount is very big, and artifical influence factor is many, and the micronucleus erroneous judgement easily occurs, and operating process is complicated, poor repeatability.
These two tests also can be differentiated the compound genotoxicity by in vivo studies, the most frequently used animal model is rodent, in most of bodies, experiment is directly processed animal used as test by medicine, then get tissue or the blood of animal, make single cell suspension, then carrying out single cell gel electrophoresis or micronucleus test.Such in vivo studies is actually the way that in vivo studies (compound treatment) combines with in vitro test (analysis of zooblast genotoxicity), and common experimental period is long, complicated operation, expensive, poor specificity, and cannot carry out high flux screening, false positive results is high.
The method of the genotoxicity of zebra fish detection compound then occurs utilizing, mainly contained following several method:
1) experiment in body: by the compound treatment zebra fish, juvenile fish or adult fish, then with the zebra fish juvenile fish, the embryo pulverizes or gets tissue or the blood of Adult Zebrafish, make single cell suspension, prepare by offset plate, lysis, electrophoresis, neutralization, dyeing, microscopy, analyze, utilize comet to differentiate genotoxicity (the Jarvis RB of compound, Knowles JF, DNA damage in zebrafish larvae induced by exposure to low-dose rate γ-radiation:detection by the alkaline comet assay, Mutation Research, 2003, 541:63 – 69.).Or zebra fish juvenile fish, embryo are pulverized or get tissue or the blood of Adult Zebrafish, after fixing, Giemsa staining, probability (the Oliveira R that occurs micronucleus in observation of cell, Domingues I, Effects of triclosan on zebrafish early-life stages and adults. Environ. Sci. Pollut. Res., 2009,16:679 – 688.).But these two kinds of method complicated operations easily produce false positive results, can not carry out high flux screening.
2) experiment in vitro: the liver cell of zebra fish is carried out former culture, utilize testing compound to process cell a period of time, by comet sldh gene toxicity (Sun LW, Qu MM, Li YQ, et al, Toxic Effects of Aminophenols on Aquatic Life Using the Zebrafish Embryo Test and the Comet Assay. Bull. Environ. Contam. Toxicol., 2004, 73:628 – 634.), although the method test period is short, can carry out high flux screening, but complicated operation, false positive results is many, the more important thing is that cell is in the external absorption that there is no medicine, distribute, metabolism, discharge process, the result of test can not reflect compound truth in vivo.
Compare with comet with single cell gel electrophoresis, said method has the advantages such as cost is low, dosage is few, operation is simpler, but still have still comparatively complicated, poor specificity of length experimental period, operation, and cannot carry out high flux screening, false positive results is high.
Summary of the invention
The objective of the invention is to solve problem set forth above, provide that a kind of cost is low, dosage is few, operation is simple, experimental period is short, can carry out high flux screening, predictability is strong, susceptibility is high, false positive rate is low a kind ofly carries out the method that Enzyme-linked Immunosorbent Assay detects on integral zebra fish, and the application of the method in sldh gene toxic chemical and antitumor medicine screening, DNA plerosis damage medicine.
Technical scheme of the present invention is such:
A kind of method of carrying out the Enzyme-linked Immunosorbent Assay detection on integral zebra fish, step is as follows:
1) fixing:
Integral zebra fish is fixing in containing the microwell plate of immobile liquid, and the time is 15-17 hour;
2) aquation:
2.1) zebra fish was placed 5-10 minute under 20-25 ℃ of room temperature;
2.2) aquation in the methyl alcohol of variable concentrations-contain 0.1% Tween-20 phosphate buffer (PBST) solution, step is as follows:
1. under 20-25 ℃ of room temperature, aquation in 75% methyl alcohol of 10-20 ml/25% contains 0.1% Tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
2. under 20-25 ℃ of room temperature, aquation in 50% methyl alcohol of 10-20ml/50% contains 0.1% Tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
3. under 20-25 ℃ of room temperature, aquation in 25% methyl alcohol of 10-20 ml/75% contains 0.1%tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
4. under 20-25 ℃ of room temperature, contain aquation in 0.1% tween-20 phosphate buffer (PBST) solution at 10-20ml, duration is 5-10 minute;
3) increase permeability:
3.1) adding Proteinase K, temperature was bathed 10-15 minute under 35-37 ℃;
3.2) clean 2 times each 5-10 minute with containing 0.1% Tween-20 phosphate buffer (PBST) solution;
4) under 20-25 ℃ of room temperature, processed 1-1.5 hour with blocking-up (blocking) endogenous peroxydase solution (containing 5% sheep blood serum, 1% bovine serum albumin(BSA), 0.1% Tween-20 phosphate buffer of 1% dimethyl sulfoxide (DMSO));
5) zebra fish and anti-single stranded DNA damage antibody temperature are bathed, temperature 4-6 ℃, 15-17 hour warm bath time;
6) clean zebra fish with containing 0.1% Tween-20 phosphate buffer (PBST) solution, at least 4 times, each 10-15 minute;
7) add good horseradish peroxidase (HRP) mark two of dilution anti-, hatched 2-3 hour under 20-25 ℃ of room temperature;
8) with phosphate buffer (PBS, NaCl 8g, KCL 0.2g, Na
2HPO
4(12H
2O) 1.44g, KH
2PO
40.24g adding distil water is to 1000ml, PH7.0) solution washes 3 times, and each 10-15 minute;
9) zebra fish is transferred in another hole microwell plate, each micropore is put into 1 tail zebra fish;
10) add dyeing substrate chemical illuminating reagent horseradish peroxidase substrate (PS-atto), hatched 20-30 minute;
11) utilize microplate reader to read chemical light (chemiluminescence) intensity level of each micropore.
As preferably, described microwell plate can be 6 hole microwell plates or 12 hole microwell plates or 24 hole microwell plates or 48 hole microwell plates or 96 hole microwell plates or 384 hole microwell plates.
As preferably, described immobile liquid can be 4% paraformaldehyde, Deng Shi immobile liquid (Dent ' s fixation, methyl alcohol/dimethyl sulfoxide (DMSO)=4:1), biological tissue fixing agent (Dietrich ' s solution), cloth iS-One immobile liquid (Bouin ' s fixtion, picric acid saturated aqueous solution 75 ml, 10% formalin solution 25 ml, glacial acetic acid 5 ml).
As preferably, described dyeing substrate can also be chromogenic substrate or fluorogenic substrate.
A kind of method of quantitative detection determining presence of genotoxic compound or antitumor medicine screening, step is as follows:
Get Adult Zebrafish kind fish, the pairing mating gathers zebra fish, and the sucking-off deposit is put into temperature and hatched at the incubator of 20-30 ℃;
Step 4, quantitative test:
DNA damage rate computing formula is,
As preferably, in described step 1, after the zebra fish feritilization of ovum 6-8 hour and 24-26 hour, take out dead embryo.
A kind of method of screening the DNA plerosis damage medicine, step is as follows:
Get Adult Zebrafish kind fish, the pairing mating gathers zebra fish, and the sucking-off deposit is put into temperature and hatched at the incubator of 20-30 ℃;
Step 4, quantitative test:
DNA damage rate computing formula is,
As preferably, in described step 1, after the zebra fish feritilization of ovum 6-8 hour and 24-26 hour, take out dead embryo.
Beneficial effect of the present invention is as follows:
1. the method for the sldh gene toxicity on integral level---can be at zebra fish individuality, organ, organize the genotoxicity of detection compound on level, this is that in the past method is not available.Method in the past is all to differentiate the genotoxicity of compound on the individual cells level, thereby can not estimate genotoxicity on tissue, organ (as eyes) level, also lost the chance of estimating other potential target organs (as heart, brain, liver, kidney, intestines, gonad, skin etc.) genotoxicity simultaneously.
2. 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.
3. high flux---the zebra fish volume is little, and zebra fish children fish length only has 1-4 mm, can be in 6 of a standard, 12, analyzing in 24,48,96 or 384 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.And rodent body length is in the 10-30cm left and right.
4. experimental period is short---can complete in 2～3 days; And mouse often needs the time of several weeks to several months, and primate 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.
5. experimental expenses is low---expend greater than 10 dollars every every day take the screening experiment of primate as the experiment carrier, expend greater than 1 dollar every every day take the screening experiment of rodent 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.
6. compound amount is few---and the consumption of required test compounds is only several mg, and traditional screening experiment take rodent as representative needs the compound more than several g.
7. predictability is high---and zebra fish is vertebra class animal, similar to the function height to the mammalian biological structure, 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, and the comparability of zebra fish sldh gene toxicity test result is compared with mammal and can be reached more than 80%.
8. 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.
9. administering mode is simple and safe---zebra fish can
Nutrient solution in one week of survival, thereby compound is directly added in the zebra fish nutrient solution, facilitate drug delivery and accelerate analytic process.Administering mode in rodent is mainly administration by gavage.But the method inefficiency easily causes the alimentary canal damage or is strayed into air flue lethal.
10. good reproducibility, specificity is high---and utilize the genotoxicity specificity of antigen-antibody reaction detection compound more than 95%.Through many experiments, experimental result is stable.
11. susceptibility is good---the present invention utilizes antigen-antibody reaction, and reaction signal is amplified step by step.
12. application prospect is good---the present invention carries out the application of the method sldh gene toxic chemical of Enzyme-linked Immunosorbent Assay detection on integral zebra fish, will greatly improve the breakneck acceleration of determining presence of genotoxic compound.The evaluation of the genotoxicity of the discriminating of determining presence of genotoxic compound in environment, new drug and Development of Novel antineoplastic and DNA damage repair medicine are significant.
Description of drawings
Fig. 1 is flow chart of steps of the present invention;
Fig. 2 is zebra fish different developmental phases DNA damage rate comparison diagram;
Fig. 3 is that staurosporine is processed different time DNA damage rate comparison diagram;
Fig. 4 is DNA damage rate comparison diagram after cisplatin treated, and wherein 50 μ M staurosporine processed group are as positive control;
Fig. 5 is DNA damage rate comparison diagram after benzo (a) pyrene is processed, and wherein 50 μ M staurosporine processed group are as positive control;
Fig. 6 is DNA damage rate comparison diagram after carboplatin is processed, and wherein 50 μ M staurosporine processed group are as positive control;
Fig. 7 is 1,7-bis(3,4-dimethoxyphenyl)-1,6-heptadiene-3,5-dione DNA damage repair rate comparison diagram, wherein 50 μ M staurosporines+DNA damage renovation agent selenomethionine (seleno-L-methionine) is organized as positive control, and positive controls DNA damage repair rate is (29.8 ± 0.80) %;
Fig. 8 is DNA damage rate comparison diagram after cisplatin treated, and wherein 50 μ M staurosporine processed group are as positive control;
* in Fig. 2,3,4,5,6,8 represents to test processed group and compares with control group and have significant difference (P＜0.05).
Embodiment
Below in conjunction with accompanying drawing, embodiments of the invention are further elaborated:
The method of carrying out the Enzyme-linked Immunosorbent Assay detection on integral zebra fish shown in Figure 1, step is as follows:
1) fixing:
Integral zebra fish is fixing in containing the microwell plate of immobile liquid, and the time is 15-17 hour;
2) aquation:
2.1) zebra fish was placed 5 minutes under 20-25 ℃ of room temperature;
2.2) aquation in the methyl alcohol of variable concentrations-contain 0.1% Tween-20 phosphate buffer (PBST) solution, step is as follows:
1. under 20-25 ℃ of room temperature, aquation in 75% methyl alcohol of 10-20ml/25% contains 0.1% Tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
2. under 20-25 ℃ of room temperature, aquation in 50% methyl alcohol of 10-20ml/50% contains 0.1% Tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
3. under 20-25 ℃ of room temperature, aquation in 25% methyl alcohol of 10-20ml/75% contains 0.1%tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
4. under 20-25 ℃ of room temperature, contain aquation in 0.1% tween-20 phosphate buffer (PBST) solution at 10-20ml, duration is 5-10 minute;
3) increase permeability:
3.1) adding Proteinase K, temperature was bathed 10-15 minute under 35-37 ℃;
3.2) clean 2 times each 5-10 minute with containing 0.1% Tween-20 phosphate buffer (PBST) solution;
4) under 20-25 ℃ of room temperature, processed 1-1.5 hour with blocking-up (blocking) endogenous peroxydase solution (containing 5% sheep blood serum, 1% bovine serum albumin(BSA), 0.1% Tween-20 phosphate buffer of 1% dimethyl sulfoxide (DMSO));
5) zebra fish and anti-single stranded DNA damage antibody temperature are bathed, temperature 4-6 ℃, 15-17 hour warm bath time;
6) clean zebra fish, at least 4 times, 10-15 minute with containing 0.1% Tween-20 phosphate buffer (PBST) solution;
7) add good horseradish peroxidase (HRP) mark two of dilution anti-, hatched 2 hours under 20-25 ℃ of room temperature;
8) with phosphate buffer (PBS, NaCl 8g, KCL 0.2g, Na
2HPO
4(12H
2O) 1.44g, KH
2PO
40.24g adding distil water is to 1000ml, PH7.0) solution washes 3 times, and each 10-15 minute;
9) zebra fish is transferred in another hole microwell plate, each micropore is put into 1 tail zebra fish;
10) add dyeing substrate chemical illuminating reagent horseradish peroxidase substrate (PS-atto), hatched 20-30 minute;
11) utilize microplate reader to read chemical light (chemiluminescence) intensity level of each micropore.
Described microwell plate can be 6 hole microwell plates or 12 hole microwell plates or 24 hole microwell plates or 48 hole microwell plates or 96 hole microwell plates or 384 hole microwell plates.
Described immobile liquid can be 4% paraformaldehyde, Deng Shi immobile liquid (Dent ' s fixation, methyl alcohol/dimethyl sulfoxide (DMSO)=4:1), biological tissue fixing agent (Dietrich ' s solution), cloth iS-One immobile liquid (Bouin ' s fixtion, picric acid saturated aqueous solution 75 ml, 10% formalin solution 25 ml, glacial acetic acid 5 ml); Described dyeing substrate can also be chromogenic substrate or fluorogenic substrate.
A kind of method of quantitative sldh gene toxic chemical or antitumor medicine screening, step is as follows:
Get Adult Zebrafish kind fish, the pairing mating gathers zebra fish, and the sucking-off deposit is put into temperature and hatched at the incubator of 20-30 ℃;
1) fixing:
Integral zebra fish is fixing in containing the microwell plate of immobile liquid, and the time is 15-17 hour;
2) aquation:
2.1) zebra fish was placed 5-10 minute under 20-25 ℃ of room temperature;
2.2) aquation in the methyl alcohol of variable concentrations-contain 0.1% Tween-20 phosphate buffer (PBST) solution, step is as follows:
1. under 20-25 ℃ of room temperature, aquation in 75% methyl alcohol of 10-20ml/25% contains 0.1% Tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
2. under 20-25 ℃ of room temperature, aquation in 50% methyl alcohol of 10-20ml/50% contains 0.1% Tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
3. under 20-25 ℃ of room temperature, aquation in 25% methyl alcohol of 10-20 ml/75% contains 0.1%tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
4. under 20-25 ℃ of room temperature, contain aquation in 0.1% tween-20 phosphate buffer (PBST) solution at 10-20ml, duration is 5-10 minute;
3) increase permeability:
3.1) adding Proteinase K, temperature was bathed 10-15 minute under 35-37 ℃;
3.2) clean 2 times each 5-10 minute with containing 0.1% Tween-20 phosphate buffer (PBST) solution;
4) under 20-25 ℃ of room temperature, processed 1-1.5 hour with blocking-up (blocking) endogenous peroxydase solution (containing 5% sheep blood serum, 1% bovine serum albumin(BSA), 0.1% Tween-20 phosphate buffer of 1% dimethyl sulfoxide (DMSO));
5) zebra fish and anti-single stranded DNA damage antibody temperature are bathed, temperature 4-6 ℃, 15-17 hour warm bath time;
6) clean zebra fish with containing 0.1% Tween-20 phosphate buffer (PBST) solution, at least 4 times, each 10-15 minute;
7) add good horseradish peroxidase (HRP) mark two of dilution anti-, hatched 2-3 hour under 20-25 ℃ of room temperature, be labeled as the compound treatment group; Separately get 0.1% dimethyl sulfoxide (DMSO) (DMSO) solvent and press the zebra fish of step 2 processing as negative control group, this control group is divided into two groups, one group adds good horseradish peroxidase (HRP) mark two of dilution anti-, hatches 2-3 hour under 20-25 ℃ of room temperature, is labeled as group of solvents; Another group does not add good horseradish peroxidase (HRP) mark two of dilution anti-, hatches under room temperature 2-3 hour, is labeled as the background group;
8) with phosphate buffer (PBS, NaCl 8g, KCL 0.2g, Na
2HPO
4(12H
2O) 1.44g, KH
2PO
40.24g adding distil water is to 1000ml, PH7.0) solution washes 3 times, each 10-15 minute, zebra fish translated in microwell plate, 1, every hole zebra fish;
9) zebra fish is transferred in another hole microwell plate, each micropore is put into 1 tail zebra fish;
10) add dyeing substrate chemical illuminating reagent horseradish peroxidase substrate (PS-atto), hatched 20-30 minute;
11) utilize microplate reader to read chemical light (chemiluminescence) intensity level of each micropore;
Step 4, quantitative test:
Microwell plate is placed under microplate reader detects chemiluminescence intensity, DNA damage rate computing formula is:
1) if the processing of compound is single concentration, compare with T check and control group so, confirm whether to have significant difference;
2) if whether a plurality of concentration group exists significant difference with the mean intensity of each concentration of variance analysis (ANOVA) assessing compound.
In described step 1, dead embryo is taken out in 6-8 hour and 24-26 hour after the zebra fish feritilization of ovum.
Described microwell plate can be 6 hole microwell plates or 12 hole microwell plates or 24 hole microwell plates or 48 hole microwell plates or 96 hole microwell plates or 384 hole microwell plates; Described immobile liquid can be 4% paraformaldehyde, Deng Shi immobile liquid (Dent ' s fixation, methyl alcohol/dimethyl sulfoxide (DMSO)=4:1), biological tissue fixing agent (Dietrich ' s solution), cloth iS-One immobile liquid (Bouin ' s fixtion, picric acid saturated aqueous solution 75 ml, 10% formalin solution 25 ml, glacial acetic acid 5 ml); Described dyeing substrate can also be chromogenic substrate or fluorogenic substrate.
A kind of method of screening the DNA plerosis damage medicine, step is as follows:
Get Adult Zebrafish kind fish, the pairing mating gathers zebra fish, and the sucking-off deposit is put into temperature and hatched at the incubator of 20-30 ℃;
Step 3: integral zebra fish is carried out Enzyme-linked Immunosorbent Assay detect:
1) fixing:
Integral zebra fish is fixing in containing the microwell plate of immobile liquid, and the time is 15-17 hour;
2) aquation:
2.1) zebra fish is taken out, placed 5-10 minute under 20-25 ℃ of room temperature;
2.2) aquation in the methyl alcohol of variable concentrations-contain 0.1% Tween-20 phosphate buffer (PBST) solution, step is as follows:
1. under 20-25 ℃ of room temperature, aquation in 75% methyl alcohol of 10-20 ml/25% contains 0.1% Tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
2. under 20-25 ℃ of room temperature, aquation in 50% methyl alcohol of 10-20ml/50% contains 0.1% Tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
3. under 20-25 ℃ of room temperature, aquation in 25% methyl alcohol of 10-20ml/75% contains 0.1%tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
4. under 20-25 ℃ of room temperature, contain aquation in 0.1% tween-20 phosphate buffer (PBST) solution at 10-20ml, duration is 5-10 minute;
3) increase permeability:
3.1) adding Proteinase K, temperature was bathed 10-15 minute under 35-37 ℃;
3.2) clean twice, each 5-10 minute with containing 0.1% Tween-20 phosphate buffer (PBST) solution;
4) under 20-25 ℃ of room temperature, processed 1-1.5 hour with blocking-up (blocking) endogenous peroxydase solution (containing 5% sheep blood serum, 1% bovine serum albumin(BSA), 0.1% Tween-20 phosphate buffer of 1% dimethyl sulfoxide (DMSO));
5) zebra fish and anti-single stranded DNA damage antibody temperature are bathed, temperature 4-6 ℃, 15-17 hour warm bath time;
6) clean the embryo with containing 0.1% Tween-20 phosphate buffer (PBST) solution, at least 4 times, each 10-15 minute;
7) add good horseradish peroxidase (HRP) mark two of dilution anti-, hatched under room temperature 2-3 hour, be labeled as the drug combination group; Separately get 50 μ M staurosporines (staurosporine) and add zebra fish that the DNA damage renovation agent processes by step 2 as a control group, this control group is divided into two groups, one group adds good horseradish peroxidase (HRP) mark two of dilution anti-, hatched 2-3 hour under 20-25 ℃ of room temperature, be labeled as model group; Another group does not add good horseradish peroxidase (HRP) mark two of dilution anti-, hatches under room temperature 2-3 hour, is labeled as the background group;
8) with phosphate buffer (PBS, NaCl 8g, KCL 0.2g, Na
2HPO
4(12H
2O) 1.44g, KH
2PO
40.24g adding distil water is to 1000ml, PH7.0) solution washes 3 times, each 10-15 minute, zebra fish translated in microwell plate, one, every hole zebra fish;
9) zebra fish is transferred in another hole microwell plate, each micropore is put into 1 tail zebra fish;
10) add dyeing substrate chemical illuminating reagent horseradish peroxidase substrate (PS-atto), hatched 20-30 minute;
11) utilize microplate reader to read chemical light (chemiluminescence) intensity level of each micropore;
Step 4, quantitative test:
Microwell plate is placed under microplate reader detects chemiluminescence intensity, DNA damage repair rate computing formula is:
1) if the processing of compound is single concentration, compare with T check and control group so, confirm whether to have significant difference;
2) if whether a plurality of concentration group exists significant difference with the mean intensity of each concentration of variance analysis (ANOVA) assessing compound.
In described step 1, dead embryo is taken out in 6-8 hour and 24-26 hour after the zebra fish feritilization of ovum.
Described microwell plate can be 6 hole microwell plates or 12 hole microwell plates or 24 hole microwell plates or 48 hole microwell plates or 96 hole microwell plates or 384 hole microwell plates; Described immobile liquid can be 4% paraformaldehyde, Deng Shi immobile liquid (Dent ' s fixation, methyl alcohol/dimethyl sulfoxide (DMSO)=4:1), biological tissue fixing agent (Dietrich ' s solution), cloth iS-One immobile liquid (Bouin ' s fixtion, picric acid saturated aqueous solution 75 ml, 10% formalin solution 25 ml, glacial acetic acid 5 ml); Described dyeing substrate can also be chromogenic substrate or fluorogenic substrate.
The mensuration of best stage of development of zebra fish during embodiment 1 compound treatment
Embryo Collection
Get 5 pairs of zebra fishs early morning, independent assortment is divided into 5 groups of mating hatched blastocysts, gathers 1200 zebra fishs, and the sucking-off deposit is put into artemia hatching solution, hatches in 28 ℃ of incubators.Middle in time to take out white embryo (in heaven) bad to prevent the water qualitative change, obtains nutriment due to zebra fish during this period by yolk, therefore do not need other nutriment.
Drug treating
Zebra fish is divided into 7 experimental group, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, the 7 days experimental group that are respectively the zebra fish after fertilization.Each experimental group is divided into again three groups, blank group, 0.1% dimethyl sulfoxide (DMSO) (DMSO) negative control group and experiment processed group (50 μ M staurosporines (staurosporine), 0.1% dimethyl sulfoxide (DMSO) (DMSO) dissolving), began to process a period of time in 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days at the zebra fish after fertilization respectively.
The anti-single stranded DNA damage of zebra fish antibody bulk dyeing
At first the embryo being placed in 4 ℃ of 4% paraformaldehydes processed 15-17 hour, generally be no more than 24 hours, utilize Proteinase K temperature under 37 ℃ to bathe, increase the permeability of zebra fish cell membrane, nuclear membrane, then carry out the zebra fish bulk dyeing, the anti-single stranded DNA damage of mouse antibody is as primary antibodie.After primary antibodie reaction, negative control group is divided into two groups, wherein one group of sheep anti-mouse antibody (two is anti-) with horseradish peroxidase (HRP) mark reacts (group of solvents), and another group is not reacted (background group) with the sheep anti-mouse antibody (two is anti-) of HRP mark.Then add chemiluminescence agent horseradish peroxidase substrate (PS-atto) as substrate, utilize multi-functional microwell plate analyser to read the chemical light of each micropore (chemiluminescence) intensity level.
Measure genotoxicity
Microwell plate is placed under multi-functional microwell plate analyser detects chemiluminescence intensity.To test repetition and get its mean value 3 times.DNA damage rate computing formula is:
Experimental data with
Expression is relatively adopted variance analysis between many groups, relatively adopts the T check to carry out statistical procedures between two groups.
The comparative statistics result, the p value is minimum, that the DNA damage rate is the highest experimental group is as the live body zebra fish medicine optimization process stage.as shown in Figure 2, the statistical procedures result shows: after fertilization 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, the zebrafish dna damage ratio of 7 days is respectively (6.3 ± 1.37) %, (27.4 ± 1.17) %, (25.3 ± 1.67) %, (17.7 ± 1.18) %, (15.3 ± 1.74) %, (12.3 ± 1.48) %, (11.9 ± 1.34) %, result shows that zebra fish is 2, compared significant difference (p＜0.05) during 3dpf with control group, and the DNA damage rate of zebra fish is the highest when 2dpf, therefore 2dpf is zebra fish the best stage of development during compound treatment in this experiment.
The mensuration of embodiment 2 compound treatment Best Times length
Embryo Collection
Get 5 pairs of zebra fishs early morning, independent assortment is divided into 5 groups of mating hatched blastocysts, gathers 1200 zebra fishs, and the sucking-off deposit is put into artemia hatching solution, hatches in 28 ℃ of incubators.Middle in time to take out white embryo (in heaven) bad to prevent the water qualitative change, obtains nutriment due to zebra fish during this period by yolk, therefore do not need other nutriment.
Drug treating
Zebra fish is divided into five experimental group, and every group comprises blank group, processed group (50 μ M staurosporines (staurosporine), 0.1% dimethyl sulfoxide (DMSO) (DMSO) dissolving) and negative control group (0.1% dimethyl sulfoxide (DMSO) (DMSO)).Began to process experimental group in 2 days at the zebra fish after fertilization, the processing time was respectively 6 hours, 12 hours, 24 hours, and 48 hours, 72 hours.
The anti-single stranded DNA damage of zebra fish antibody bulk dyeing
At first the embryo being placed in 4 ℃ of 4% paraformaldehydes processed 15-17 hour, generally be no more than 24 hours, utilize Proteinase K temperature under 37 ℃ to bathe, increase the permeability of zebra fish cell membrane, nuclear membrane, then carry out the zebra fish bulk dyeing, the anti-single stranded DNA damage of mouse antibody is as primary antibodie.After primary antibodie reaction, negative control group is divided into two groups, wherein one group of sheep anti-mouse antibody (two is anti-) with horseradish peroxidase (HRP) mark reacts (group of solvents), and another group is not reacted (background group) with the sheep anti-mouse antibody (two is anti-) of HRP mark.Then add chemiluminescence agent horseradish peroxidase substrate (PS-atto) as substrate, utilize multi-functional microwell plate analyser to read the chemical light of each micropore (chemiluminescence) intensity level.
Measure genotoxicity
Microwell plate is placed under multi-functional microwell plate analyser detects chemiluminescence intensity.To test repetition and get its mean value 3 times.DNA damage rate computing formula is:
Experimental data with
Expression is relatively adopted variance analysis between many groups, relatively adopts the T check to carry out statistical procedures between two groups.
The comparative statistics result, the p value is minimum, that the DNA damage rate is the highest experimental group is as live body zebra fish medicine optimization process time span.as shown in Figure 3, the statistical procedures result shows: processed zebra fish 6 hours with staurosporine, 12 hours, 24 hours, 48 hours, 72 hours, the zebrafish dna damage ratio is respectively (4.3 ± 1.37) %, (12.4 ± 1.17) %, (28.4 ± 1.67) %, (30.7 ± 1.18) %, (31.3 ± 1.14) %, result shows that zebra fish processed zebra fish 24 hours at staurosporine, 48 hours, compared significant difference (p＜0.05) in 72 hours with control group, staurosporine process zebra fish in the time of 24 hours the DNA damage rate of zebra fish significant difference appears the earliest, therefore select SPT 24h as the compound optimization process time.The different compound optimization process times are not identical yet, need particular compound to make a concrete analysis of, and the present invention tests according to the optimization process time span of DNA damage derivant.
The detection of embodiment 3 medicine genotoxicities
Embryo Collection
Get 10 pairs of zebra fishs early morning, independent assortment is divided into 10 groups of mating hatched blastocysts, gathers 1200 zebra fishs, and the sucking-off deposit is put into artemia hatching solution, hatches in 28 ℃ of incubators.Middle in time to take out white embryo (in heaven) bad to prevent the water qualitative change, obtains nutriment due to zebra fish during this period by yolk, therefore do not need other nutriment.
Drug treating
Zebra fish is divided into eight experimental group, is respectively 0.1,1,10,100,1000 μ M genotoxicity medicine cis-platinums (cisplatin), then be dissolved in artemia hatching solution by above-mentioned concentration; Blank group is for being left intact, and 0.1% dimethyl sulfoxide (DMSO) (DMSO) negative control group can cause 50 μ M staurosporine (staurosporine) positive controls of genotoxicity.Begin to process at 2dpf, the processing time is 24 hours.
The anti-single stranded DNA damage of zebra fish antibody bulk dyeing
At first the embryo being placed in 4 ℃ of 4% paraformaldehydes processed 15-17 hour, generally be no more than 24 hours, utilize Proteinase K temperature under 37 ℃ to bathe, increase the permeability of zebra fish cell membrane, nuclear membrane, then carry out the zebra fish bulk dyeing, the anti-single stranded DNA damage of mouse antibody is as primary antibodie.After primary antibodie reaction, negative control group is divided into two groups, wherein one group of sheep anti-mouse antibody (two is anti-) with horseradish peroxidase (HRP) mark reacts (group of solvents), and another group is not reacted (background group) with the sheep anti-mouse antibody (two is anti-) of HRP mark.Then add chemiluminescence agent horseradish peroxidase substrate (PS-atto) as substrate, utilize multi-functional microwell plate analyser to read the chemical light of each micropore (chemiluminescence) intensity level.
Measure genotoxicity
Microwell plate is placed under multi-functional microwell plate analyser detects chemiluminescence intensity.To test repetition and get its mean value 3 times.DNA damage rate computing formula is:
Experimental data with
Expression is relatively adopted variance analysis between many groups, relatively adopts the T check to carry out statistical procedures between two groups.But the genotoxicity by statistical procedures result quantitative evaluation medicine to be measured.As shown in Figure 4, when estimating genotoxicity medicine cis-platinum (cisplatin), statistical procedures gained positive controls DNA damage rate as a result is (28.4 ± 1.67) %, and drug treating group DNA damage rate is respectively (6.4 ± 1.69) %, (19.5 ± 1.07) %, (25.5 ± 1.01) %, (36.9 ± 51.36) %, (40.5 ± 1.57) %.Result shows that the drug treating zebra fish is compared with control group in 100,1000 μ M cis-platinum concentration groups and has significant difference (p＜0.05), and higher than staurosporine positive controls (28.4 ± 1.67) %, so we can draw this medicine and have genotoxicity medicine in the DNA damage rate of these two concentration.
The detection of embodiment 4 environment determining presence of genotoxic compound
Embryo Collection
Get 10 pairs of zebra fishs early morning, independent assortment is divided into 10 groups of mating hatched blastocysts, gathers 1200 zebra fishs, and the sucking-off deposit is put into artemia hatching solution, hatches in 28 ℃ of incubators.Middle in time to take out white embryo (in heaven) bad to prevent the water qualitative change, obtains nutriment due to zebra fish during this period by yolk, therefore do not need other nutriment.
Drug treating
Environment determining presence of genotoxic compound benzo to be measured (a) pyrene (benzo (a) pyrene) is divided into 5 different processed group, is respectively 0.1,1,10,100,1000 μ M, then be dissolved in artemia hatching solution by above-mentioned concentration; Blank group is for being left intact, and 0.1% dimethyl sulfoxide (DMSO) (DMSO) negative control group can cause 50 μ M staurosporine (staurosporine) positive controls of genotoxicity.Begin to process at 2dpf, the processing time is 24 hours.
The anti-single stranded DNA damage of zebra fish antibody bulk dyeing
At first the embryo being placed in 4 ℃ of 4% paraformaldehydes processed 15-17 hour, generally be no more than 24 hours, utilize Proteinase K temperature under 37 ℃ to bathe, increase the permeability of zebra fish cell membrane, nuclear membrane, then carry out the zebra fish bulk dyeing, the anti-single stranded DNA damage of mouse antibody is as primary antibodie.After primary antibodie reaction, negative control group is divided into two groups, wherein one group of sheep anti-mouse antibody (two is anti-) with horseradish peroxidase (HRP) mark reacts (group of solvents), and another group is not reacted (background group) with the sheep anti-mouse antibody (two is anti-) of HRP mark.Then add chemiluminescence agent horseradish peroxidase substrate (PS-atto) as substrate, utilize multi-functional microwell plate analyser to read the chemical light of each micropore (chemiluminescence) intensity level.
Measure genotoxicity
Microwell plate is placed under multi-functional microwell plate analyser detects chemiluminescence intensity.To test repetition and get its mean value 3 times.DNA damage rate computing formula is:
Experimental data with
Expression is relatively adopted variance analysis between many groups, relatively adopts the T check to carry out statistical procedures between two groups.But the genotoxicity by compound in statistical procedures result quantitative evaluation environment to be measured.As shown in Figure 5, when estimating benzo (a) pyrene, statistical procedures gained positive controls DNA damage rate as a result is (28.4 ± 1.67) %, and drug treating group DNA damage rate is respectively (3.4 ± 1.69) %, (10.5 ± 1.07) %, (19.5 ± 1.01) %, (26.9 ± 1.36) %, (31.5 ± 1.57) %.Result shows, the benzo of 1000 μ M (a) pyrene processed group is compared with negative control group and is had significant difference (p＜0.05), can draw us tests environmental compound used and has genotoxicity, can be to the DNA injury, thus proof environmental compound benzo (a) pyrene has the potential ability that causes human diseases such as cancer.
Embryo Collection
Get 10 pairs of zebra fishs early morning, independent assortment is divided into 10 groups of mating hatched blastocysts, gathers 1200 zebra fishs, and the sucking-off deposit is put into artemia hatching solution, hatches in 28 ℃ of incubators.Middle in time to take out white embryo (in heaven) bad to prevent the water qualitative change, obtains nutriment due to zebra fish during this period by yolk, therefore do not need other nutriment.
Drug treating
Carboplatin (carboplatin) is divided into 5 different processed group, is respectively 0.1,1,10,100,1000 μ M, then be dissolved in artemia hatching solution by above-mentioned concentration; Blank group is for being left intact, and 0.1% dimethyl sulfoxide (DMSO) (DMSO) negative control group can cause 50 μ M staurosporine (staurosporine) positive controls of genotoxicity.Begin to process at 2dpf, the processing time is 24 hours.
The anti-single stranded DNA damage of zebra fish antibody bulk dyeing
At first the embryo being placed in 4 ℃ of 4% paraformaldehydes processed 15-17 hour, generally be no more than 24 hours, utilize Proteinase K temperature under 37 ℃ to bathe, increase the permeability of zebra fish cell membrane, nuclear membrane, then carry out the zebra fish bulk dyeing, the anti-single stranded DNA damage of mouse antibody is as primary antibodie.After primary antibodie reaction, negative control group is divided into two groups, wherein one group of sheep anti-mouse antibody (two is anti-) with horseradish peroxidase (HRP) mark reacts (group of solvents), and another group is not reacted (background group) with the sheep anti-mouse antibody (two is anti-) of HRP mark.Then add chemiluminescence agent horseradish peroxidase substrate (PS-atto) as substrate, utilize multi-functional microwell plate analyser to read the chemical light of each micropore (chemiluminescence) intensity level.
Measure genotoxicity
Microwell plate is placed under multi-functional microwell plate analyser detects chemiluminescence intensity.To test repetition and get its mean value 3 times.DNA damage rate computing formula is:
Experimental data with
Expression is relatively adopted variance analysis between many groups, relatively adopts the T check to carry out statistical procedures between two groups.But the genotoxicity medicine that is used for oncotherapy by statistical procedures result quantitative evaluation screening.As shown in Figure 6 when estimating carboplatin (carboplatin), statistical procedures gained positive controls DNA damage rate as a result is (28.4 ± 1.67) %, and drug treating group DNA damage rate is respectively (5.4 ± 1.69) %, (17.5 ± 1.07) %, (28.5 ± 1.01) %, (35.9 ± 1.36) %, (43.5 ± 1.57) %.Result shows this medicine 10,100, and the genotoxicity that 1000 μ M cause is compared with control group and had significant difference (p＜0.05).Can draw thus this medicine and have genotoxicity, and the apoptosis of induced tumor cell (death) thus.
The screening of embodiment 6 DNA plerosis damage medicines
Embryo Collection
Get 10 pairs of zebra fishs early morning, independent assortment is divided into 10 groups of mating hatched blastocysts, gathers 1200 zebra fishs, and the sucking-off deposit is put into artemia hatching solution, hatches in 28 ℃ of incubators.Middle in time to take out white embryo (in heaven) bad to prevent the water qualitative change, obtains nutriment due to zebra fish during this period by yolk, therefore do not need other nutriment.
Drug treating
9 experimental group are set: 5 drug combination processed group, 1 DNA damage derivant model group, 1 positive controls, 1 solvent control group, 1 blank group.Remove artemia hatching solution in microwell plate, testing compound 1,7-bis(3,4-dimethoxyphenyl)-1,6-heptadiene-3,5-dione (the dimethoxy Curcumin that adds 50 μ M staurosporines+0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M in the drug combination processed group, 1,7-bis (3,4-dimethoxyphenyl)-1,6-heptadiene-3,5-dione); Add 50 μ M staurosporines in DNA damage derivant model group; Adding the equal-volume final concentration in positive controls is 50 μ M staurosporines+DNA damage renovation agent selenomethionine (seleno-L-methionine) (Smith ML, Kumar MA., Seleno-L-Methionine Modulation of Nucleotide Excision DNA Repair Relevant to Cancer Prevention and Chemotherapy. Mol Cell Pharmacol, 2009,1(4): 218-221); Add equal-volume 0.1% dimethyl sulfoxide (DMSO) (DMSO) in the solvent control group.Cultivate 24h in 28 ℃ of constant incubators.
In addition, the drug combination processed group also can adopt following dual mode to process:
First: after adding certain volume (deciding according to the microwell plate specification) final concentration to be 50 μ M staurosporines or 6-24h, then add testing compound selenomethionine (seleno-L-methionine) solution of 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M;
Second: after at first adding testing compound selenomethionine (seleno-L-methionine) the solution 6-24h of 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M, then to add final concentration be 50 μ M staurosporines.
The anti-single stranded DNA damage of zebra fish antibody bulk dyeing
At first the embryo being placed in 4 ℃ of 4% paraformaldehydes processed 15-17 hour, generally be no more than 24 hours, utilize Proteinase K temperature under 37 ℃ to bathe, increase the permeability of zebra fish cell membrane, nuclear membrane, then carry out the zebra fish bulk dyeing, the anti-single stranded DNA damage of mouse antibody is as primary antibodie.After primary antibodie reaction, negative control group is divided into two groups, wherein one group of sheep anti-mouse antibody (two is anti-) with horseradish peroxidase (HRP) mark reacts (group of solvents), and another group is not reacted (background group) with the sheep anti-mouse antibody (two is anti-) of HRP mark.Then add chemiluminescence agent horseradish peroxidase substrate (PS-atto) as substrate, utilize multi-functional microwell plate analyser to read the chemical light of each micropore (chemiluminescence) intensity level.
Measure genotoxicity
Microwell plate is placed under multi-functional microwell plate analyser detects chemiluminescence intensity.To test repetition and get its mean value 3 times.DNA damage rate computing formula is:
Experimental data with
Expression is relatively adopted variance analysis between many groups, relatively adopts the T check to carry out statistical procedures between two groups.But by statistical procedures result quantitative screening can DNA plerosis the medicine of damage.For example: as shown in Figure 7; at screening DNA injury repair agent 1,7-bis(3,4-dimethoxyphenyl)-1,6-heptadiene-3,5-dione (dimethoxy Curcumin; 1; 7-bis (3; 4-dimethoxyphenyl)-1; 6-heptadiene-3; 5-dione) time; statistical procedures gained positive controls DNA damage repair rate as a result is (29.8 ± 0.80) %, and drug combination processed group injury of mitochondria protection ratio is respectively (6.3 ± 1.12) %, (13.4 ± 1.32) %, (20.6 ± 1.31) %, (30.4 ± 1.34) %, (31.1 ± 2.33) %.The drug combination processed group is compared difference with the solvent control group have statistical significance (p＜0.05).Thereby prove that this compound can damage by DNA plerosis, await further research and development and use.
The quantitative detection compound genotoxicity method of embodiment 7
Embryo Collection
Get 10 pairs of zebra fishs early morning, independent assortment is divided into 10 groups of mating hatched blastocysts, gathers 1200 zebra fishs, and the sucking-off deposit is put into artemia hatching solution, hatches in 28 ℃ of incubators.Middle in time to take out white embryo (in heaven) bad to prevent the water qualitative change, obtains nutriment due to zebra fish during this period by yolk, therefore do not need other nutriment.
Drug treating
Testing sample cis-platinum (cisplatin) is divided into 5 different processed group, be respectively 0.1,1,10,100,1000 μ M, blank group is for being left intact, 0.1% dimethyl sulfoxide (DMSO) (DMSO) negative control group can cause 50 μ M staurosporine (staurosporine) positive controls of genotoxicity.Zebra fish is put in 6 hole microwell plates, also can begin to process at 2dpf in 6 hole microwell plates or 12 hole microwell plates or 24 hole microwell plates or 48 hole microwell plates or 96 hole microwell plates or 384 hole microwell plates, the processing time is 24 hours.
The anti-single stranded DNA damage of zebra fish antibody bulk dyeing
At first the embryo being placed in 4 ℃ of 4% paraformaldehydes processed 15-17 hour, generally be no more than 24 hours, utilize Proteinase K temperature under 37 ℃ to bathe, to increase the permeability of zebra fish cell membrane, nuclear membrane, then carry out the zebra fish bulk dyeing, the anti-single stranded DNA damage of mouse antibody is as primary antibodie.After primary antibodie reaction, negative control group is divided into two groups, wherein one group of sheep anti-mouse antibody (two is anti-) with horseradish peroxidase (HRP) mark reacts (group of solvents), and another group is not reacted (background group) with the sheep anti-mouse antibody (two is anti-) of HRP mark.Then add tetramethyl benzidine (TMB) or horseradish peroxidase fluorogenic substrate (QuantaBlu) as chromogenic substrate, utilize multi-functional microwell plate analyser to read the optical density of each micropore (OD) value.
Quantitative test
Microwell plate is placed in the absorbance of fluorescence intensity under multi-functional microwell plate analyser or visible light.To test repetition and get its mean value 3 times.DNA damage rate computing formula is:
Experimental data with
Expression is relatively adopted variance analysis between many groups, relatively adopts the T check to carry out statistical procedures between two groups.But the genotoxicity by statistical procedures result quantitative evaluation testing compound.As shown in Figure 8, when estimating cis-platinum (cisplatin) genotoxicity, statistical procedures gained positive controls DNA damage rate as a result is (28.4 ± 1.67) %, and drug treating group DNA damage rate is respectively (6.4 ± 0.69) %, (19.5 ± 1.07) %, (21.5 ± 1.01) %, (33.9 ± 1.36) %, (40.3 ± 1.57) %.In figure, result shows that the drug treating zebra fish is compared with control group in 100,1000 μ M concentration groups and has significant difference (p＜0.05), and higher than staurosporine positive controls (28.4 ± 1.67) %, so we can draw cis-platinum (cisplatin) and have genotoxicity medicine in the DNA damage rate of these two concentration.
Drug screening is important link in discovery, developing drugs process, and screening model plays key effect in the drug screening process.Use zebra fish assessing compound toxicity and screening new drug, both had cost less, amount of samples is few, operation can realize the advantages such as robotization, the body physiological environment of living animal can be provided again, therefore can realize estimating and easy, quick, economic, efficient, the high-throughout purpose of screening operation.The present invention relates to a kind of method of utilizing genotoxicity and the screening new drug of integral zebra fish euzymelinked immunosorbent assay (ELISA) assessing compound.This invention can be used for the genotoxicity of assessing compound on the one hand, comprise environmental compound, medicine, cosmetics, food additives etc. can be used for screening new drug on the other hand, as medicines such as screening antineoplastic, antioxidant, anti-ageing, anti-nerve degenerative diseases.The present invention is for diagnosis and treat some disease, has very important directive significance for new drug development and toxicity of compound screening.
Contrast on effect between the present invention and prior art scheme is as follows:
The anti-single stranded DNA damage antibody that adopts in the present embodiment is available from Cell Technology company; Chemical illuminating reagent horseradish peroxidase substrate (PS-atto) is available from Lumigen company.Staurosporine (staurosporine) is available from U.S. Sigma company.QuantaBlu is available from U.S. Thermo company.1,7-bis(3,4-dimethoxyphenyl)-1,6-heptadiene-3,5-dione (Curcumin) is available from U.S. Cayman company.Other reagent are provided by the prosperous Science and Technology Ltd. of Beijing ancient cooking vessel state.
The multi-functional microwell plate analyser that adopts in the present embodiment, the Mithras LB940 that produces for Berthold Technologies company.
Above-described is only the preferred embodiment of the present invention; should be pointed out that the those of ordinary skill in the art, under the prerequisite that does not break away from core technology feature of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. one kind is carried out the method that Enzyme-linked Immunosorbent Assay detects on integral zebra fish, it is characterized in that, step is as follows:
1) fixing:
Integral zebra fish is fixing in containing the microwell plate of immobile liquid, and the time is 15-17 hour;
2) aquation:
2.1) zebra fish was placed 5-10 minute under 20-25 ℃ of room temperature;
2.2) aquation in the methyl alcohol of variable concentrations-contain 0.1% Tween-20 phosphate buffer (PBST) solution, step is as follows:
1. under 20-25 ℃ of room temperature, aquation in 75% methyl alcohol of 10-20ml/25% contains 0.1% Tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
2. under 20-25 ℃ of room temperature, aquation in 50% methyl alcohol of 10-20ml/50% contains 0.1% Tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
3. under 20-25 ℃ of room temperature, aquation in 25% methyl alcohol of 10-20ml/75% contains 0.1%tween-20 phosphate buffer (PBST) solution, duration is 5-10 minute;
4. under 20-25 ℃ of room temperature, contain aquation in 0.1% tween-20 phosphate buffer (PBST) solution at 10-20 ml, duration is 5-10 minute;
3) increase permeability:
3.1) adding Proteinase K, temperature was bathed 10-15 minute under 35-37 ℃;
3.2) clean 2 times each 5-10 minute with containing 0.1% Tween-20 phosphate buffer (PBST) solution;
4) under 20-25 ℃ of room temperature, with blocking-up endogenous peroxydase solution-treated 1-1.5 hour, described blocking-up endogenous peroxydase solution was to contain 5% sheep blood serum, 1% bovine serum albumin(BSA), 0.1% Tween-20 phosphate buffer of 1% dimethyl sulfoxide (DMSO);
5) zebra fish and anti-single stranded DNA damage antibody temperature are bathed, temperature 4-6 ℃, 15-17 hour warm bath time;
6) clean zebra fish with containing 0.1% Tween-20 phosphate buffer (PBST) solution, at least 4 times, each 10-15 minute;
7) add good horseradish peroxidase (HRP) mark two of dilution anti-, hatched 2-3 hour under 20-25 ℃ of room temperature;
8) wash 3 times with the phosphoric acid buffer liquor, each 10-15 minute, described phosphate buffer was NaCl 8g, KCL 0.2g, Na
2HPO
4(12H
2O) 1.44g, KH
2PO
40.24g adding distil water is to 1000ml, the solution of PH7.0;
9) zebra fish is transferred in another hole microwell plate, each micropore is put into 1 tail zebra fish;
10) add dyeing substrate chemical illuminating reagent horseradish peroxidase substrate PS-atto, hatched 20-30 minute;
11) utilize microplate reader to read chemical light (chemiluminescence) intensity level of each micropore.
2. method according to claim 1, is characterized in that, described microwell plate is 6 hole microwell plates or 12 hole microwell plates or 24 hole microwell plates or 48 hole microwell plates or 96 hole microwell plates or 384 hole microwell plates.
3. method according to claim 2, it is characterized in that, described immobile liquid is 4% paraformaldehyde, Deng Shi immobile liquid, biological tissue fixing agent Dietrich ' s solution, cloth iS-One immobile liquid, wherein, described Deng Shi immobile liquid is Dent ' s fixation, the solution of methyl alcohol/dimethyl sulfoxide (DMSO)=4:1, described cloth iS-One immobile liquid is Bouin ' s fixtion, picric acid saturated aqueous solution 75 ml, 10% formalin solution 25 ml, the solution of glacial acetic acid 5 ml.
4. method according to claim 3, is characterized in that, described dyeing substrate is chromogenic substrate or fluorogenic substrate.
5. a method that quantitatively detects determining presence of genotoxic compound or antitumor medicine screening, is characterized in that, step is as follows:
Step 1, Embryo Collection:
Get Adult Zebrafish kind fish, the pairing mating gathers zebra fish, and the sucking-off deposit is put into temperature and hatched at the incubator of 20-30 ℃;
Step 2 is placed in microwell plate with the zebra fish of after fertilization 6-72 hour, carries out testing compound and process in the water temperature of 20-30 ℃, and the processing time is 24-72 hour;
Step 3 is carried out Enzyme-linked Immunosorbent Assay as claimed in claim 1 to integral zebra fish and is detected;
Step 4, quantitative test:
DNA damage rate computing formula is,
6. the method for quantitative detection determining presence of genotoxic compound according to claim 5, is characterized in that, in described step 1, dead embryo is taken out in 6-8 hour and 24-26 hour after the zebra fish feritilization of ovum.
7. a method of screening the DNA plerosis damage medicine, is characterized in that, step is as follows:
Step 1, Embryo Collection:
Get Adult Zebrafish kind fish, the pairing mating gathers zebra fish, and the sucking-off deposit is put into temperature and hatched at the incubator of 20-30 ℃;
Step 2 is placed in microwell plate with an after fertilization 6-72 hour zebra fish, carries out 50 μ M staurosporines (staurosporine) and add drug treating to be measured in the water temperature of 20-30 ℃, and the processing time is 24-72 hour;
Step 3 is carried out Enzyme-linked Immunosorbent Assay as claimed in claim 1 to integral zebra fish and is detected;
Step 4, quantitative test:
DNA damage rate computing formula is,
8. the method for screening DNA plerosis damage medicine according to claim 7, is characterized in that, in described step 1, dead embryo is taken out in 6-8 hour and 24-26 hour after the zebra fish feritilization of ovum.
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| CN103063830B (en) * | 2012-12-21 | 2016-04-13 | 杭州茂天赛科技有限公司 | A kind of preparation method of ELISA pre-coated elisa plate |
| US20170356895A1 (en) * | 2016-06-08 | 2017-12-14 | Vitargent (International) Biotechnology Limited | Toxicant assays for consumable products |
| CN111766354B (en) * | 2020-03-22 | 2022-04-26 | 杭州环特生物科技股份有限公司 | Method for evaluating safety of meat product by using zebra fish |
| CN112198143B (en) * | 2020-09-04 | 2023-06-27 | 南京新环检测科技有限公司 | Method for evaluating photoaging resistance and glycosylation efficacy of cosmetics and health-care foods |
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