CN101810866A - New method for screening anti-liver injury medicament by using model organism zebra fish - Google Patents
New method for screening anti-liver injury medicament by using model organism zebra fish Download PDFInfo
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Abstract
The invention discloses a new method for screening an anti-liver injury medicament by using a model organism zebra fish. The method comprises the following steps: continuously molding the zebra fish serving as a molding object for 3 days by using preferred carbon tetrachloride at the concentration of 1mmol/L to obtain the zebra fish of a liver injury model, and then evaluating the anti-liver injury activity of different receptor medicaments by using ALT and AST levels in the liver tissue of the zebra fish and the pathological change condition of the liver tissue of the zebra fish. Experimental results show that the new method for screening the anti-liver injury medicament by using the model organism zebra fish has high molding success rate, can simulate an in vivo environment to perform quick screening, and has the advantages of strong operability, accurate experimental results, little amount of required reacceptance medicament, strong repeatability, low cost, wide application range, capability of performing batch screening experiments and high work efficiency.
Description
Technical field
The present invention relates to a kind of screening technique of medicine, be specifically related to a kind of new method with model organism zebra fish rapid screening anti-liver injury medicament.
Background technology
The anti-liver injury medicament screening study often comprises (animal) and external (cell, molecule etc.) test in the body.The anti-liver injury test is normal in the body adopts animals such as mice, rat to carry out, and main liver injury model has acute liver damage model and chronic hepatic injury scale-model investigation.Wherein select the chmice acute liver injury model at present mostly for use for the rapid screening of anti-liver injury medicament, but because the required drug dose of experiment is big in the mice body, the experimental cost height, the rapid screening that is unfavorable for small amount of drug, especially the amount of extracting the monomeric compound that obtains from Chinese medicine is few sometimes, is restricted with mouse model screening anti-liver injury medicament.Methods such as cell, molecule are adopted in external anti-liver injury medicament screening study more, method is simple, condition is controlled, be applicable to the quick primary dcreening operation of liver injury medicament, but isolated cells, molecule etc. are difficult to the accurately physiological disposition of Simulation of Complex, false positive results often appears in process of the test.Therefore, set up a kind of can the simulation preferably in the body process, the research method of liver-protecting efficacy that again can quick and convenient prediction anti-liver injury medicament is significant.Brachydanio rerio is a kind of fabulous model organism, is to replace the good test model fish as object of study such as Rana nigromaculata, fruit bat, white mice.The expense that Brachydanio rerio is fed and keeps is more cheap, and the requisite space place is little, is easy to indoor large-scale breeding.Long 3～the 4cm of adult fish, a copulation can obtain 100～200 embryos, lays eggs weekly once, and medium scale like this fish jar is annual just can culture millions of of adult fishes, and cost is only for supporting the 0.1%-1% of mice.Brachydanio rerio is suitable with human gene's similarity and mice, and on protein level, the homology of its key position almost is 100%, so available Brachydanio rerio simulating human disease.At present, what people were successful sets up many human diseases models with Brachydanio rerio, as the disease of aspects such as nervous system, blood circulation, audition, vision, cancer.A kind of method with zebra opah research drug toxicity is disclosed as Chinese patent 200810019040.5, patent 200780051025.2 disclose a kind of organ that is undertaken by stem cell nutrition formation and and the renovation process of alcohol damaged organ, 200310108710.8 disclose diseases such as utilizing Brachydanio rerio gene therapy paraplegia, Brachydanio rerio becomes a kind of study of pharmacy instrument of hot topic at home and abroad, the research of Brachydanio rerio at present mainly is limited to the developmental genetics aspect, the application of Shang Weijian aspect anti-liver injury medicament research.
Summary of the invention
Goal of the invention: technical problem to be solved by this invention is to overcome the deficiencies in the prior art, provide a kind of easy to operate, cost is low, experimental result is accurate, required to be tried medication amount few, goes out the new method that anti-liver decreases medicine with the model organism zebra fish rapid screening.
Technical scheme: in order to realize above purpose, the technical scheme that the present invention takes is:
A kind of new method with model organism zebra fish screening anti-liver injury medicament specifically may further comprise the steps:
(1) the adult fish of getting Brachydanio rerio is put in the container that fills water, be divided into normal control group, blank group immediately, be subjected to reagent thing group, every group of Brachydanio rerio quantity equates, blank group and be subjected to reagent thing group to add carbon tetrachloride, the molar concentration that makes carbon tetrachloride is 0.5～2mmol/L, and the normal control group adds the solvent of equivalent, continues modeling 3～5 days, promptly get the acute liver damage zebra fish model, standby;
(2) will be subjected to the reagent thing to be made into desired concn, join in the aqueous solution that is subjected to reagent thing group, normal control group and blank group add the distilled water of equivalent, after the administration 48 hours to 72 hours, each group Brachydanio rerio is taken out execution, get and respectively organize the Brachydanio rerio liver and be divided into 2 parts at random, portion is made 1% liver tissue homogenate, detects ALT in the homogenate, AST level and total protein content, and calculate the proteic ALT of every gram in the homogenate, the AST level, another part liver organization specimen is fixed through concentration 10% formalin solution, paraffin section, H-E dyeing, the pathological change situation of Brachydanio rerio hepatic tissue is respectively organized in observation under the light microscopic, carry out the pathological grading scoring, normally be 1 minute, slight steatosis is 2 minutes, and the moderate pathological changes is 3 minutes, the steatosis severe patient is 4 minutes, estimates and screen the anti-liver injury activity that each group is subjected to the reagent thing.
As preferred version, above-described new method with model organism zebra fish screening anti-liver injury medicament, when wherein step (1) was with the carbon tetrachloride modeling, the whole molar concentration of carbon tetrachloride was 1mmol/L.Because if the concentration of carbon tetrachloride is too high, may produce serious toxicity to Brachydanio rerio, cause the Brachydanio rerio mortality, thereby make modeling the failure of an experiment, if but the concentration of carbon tetrachloride is low excessively, can not produce damage to the liver of Brachydanio rerio, can not obtain satisfactory acute liver damage model equally, the present invention screens by the carbon tetrachloride concentration to variable concentrations, is evaluation index with the Brachydanio rerio hepatic injury, and drawing the required carbon tetrachloride concentration of best modeling is 1mmol/L.
As preferred version; above-described new method with model organism zebra fish screening anti-liver injury medicament; wherein the polarity of used carbon tetrachloride is smaller during step (1) modeling; can not well be dissolved in the aqueous solution; thereby influence the success of modeling; the present invention adds an amount of dimethyl sulfoxide in carbon tetrachloride be that DMSO is as cosolvent; the final concentration of control dimethyl sulfoxide in solution is 0～1%; normal activity to Brachydanio rerio does not have influence; and the DMSO of corresponding adding equivalent in the normal control group, to guarantee the science of experiment.
As preferred version, new method with model organism zebra fish screening anti-liver injury medicament of the present invention, wherein the described modeling time of step (1) is 3 days, because too short as the modeling time, the Brachydanio rerio liver fails to take place pathological changes, modeling can not meet the requirements, if but the modeling time oversize, Brachydanio rerio may occur the tolerance, even it is dead, also can cause the modeling failure, the present invention is by screening the different modeling time, and the Best Times of determining the carbon tetrachloride modeling is 3 days.
As preferred version, new method with model organism zebra fish screening anti-liver injury medicament of the present invention, wherein after 72 hours each group Brachydanio rerio is taken out execution in administration in the step (2), and step (2) adopts automatic clinical chemistry analyzer to detect ALT, the AST level of respectively organizing in the Brachydanio rerio hepatic homogenate, and it is active to be with the level of ALT and AST that index evaluation is decreased by the anti-liver of reagent thing respectively.
As preferred version, new method with model organism zebra fish screening anti-liver injury medicament of the present invention, the reagent thing that is subjected to of screening can be chemical drugs, Chinese medicine and extract thereof, Chinese medicine compound and extract thereof, natural drug, or their compositions, and compare with animal models such as mices, the new method of screening anti-liver injury medicament provided by the invention, the required amount of reagent thing that is subjected to just can experimentize between 10～100 μ g, and range of application is wider.
Different with mammals such as mice, rat, dogs, the volume of Brachydanio rerio is less, oral or the drug administration by injection mode is very difficult, therefore, the present invention is dissolved in carbon tetrachloride solution in the water that Brachydanio rerio lives in the step (1), thereby Brachydanio rerio can be caused hepatic injury by the autonomous successive carbon tetrachloride that absorbs from solution; Brachydanio rerio adult fish is about 3～4cm, its body inner blood amount denier, be difficult to realize blood sampling, so detect the feasibility existing problems of serum biochemistry index observing liver function, experimental studies have found that, the liver organization of Brachydanio rerio proportion in its body is relatively large, liver is got in dissection, and to carry out the relative operability of detection of histopathologic examination and liver organization mesophytization index stronger, and the pathology of livers of direct observation Brachydanio rerio and biochemical indicator are more obvious, objective than other index, and can repeat experiment under the same conditions.Therefore, the present invention estimates the liver function of Brachydanio rerio by ALT (ALT), aspartic acid aminotransferase (AST) level in the mensuration Brachydanio rerio liver tissue homogenate, and further estimate the degree of its liver organization pathological changes by the liver organization pathological examination, the method is workable.
Beneficial effect: the new method with model organism zebra fish screening anti-liver injury medicament provided by the invention is compared with prior art and is had the following advantages:
Preparation method of screening the new method of anti-liver injury medicament with model organism zebra fish provided by the invention, modeling success rate height, can carry out rapid screening by simulated in vivo environment, workable, experimental result is accurate, it is especially required that to be tried medication amount few, repeatable strong, cost is low, has wide range of applications, and can carry out screening experiment, high efficiency in batch.
The specific embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, process conditions and result thereof only are used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1 acute liver damage modeling screening experiment
1, the selection of the used carbon tetrachloride concentration of Brachydanio rerio acute liver damage model
Get 60 of Brachydanio rerio adult fishes; place the bottle that contains 200mL solution respectively; 2 every bottle; be divided into the normal control group at random; the carbon tetrachloride group of variable concentrations; every group contains 10 Brachydanio rerio adult fishes; the carbon tetrachloride group is given the carbon tetrachloride with variable concentrations respectively; because carbon tetrachloride polarity is low; therefore add an amount of DMSO hydrotropy, the concentration of DMSO is lower than 1% in the whole solution of each experimental group, and the normal activity of Brachydanio rerio is not had influence; and corresponding adding contains the pure water of equivalent DMSO in the normal control group, to guarantee the science of experiment.Different time points dynamic observes the active situation of Brachydanio rerio behind the adding carbon tetrachloride, writes down death toll in its 24 hours, calculates mortality rate.Concrete experimental result is as shown in table 1.
Table 1 variable concentrations carbon tetrachloride is to Brachydanio rerio hepatic injury situation
Show that by table 1 experimental result when carbon tetrachloride concentration surpassed 2mmol/L, typical poisoning symptom appearred in the part Brachydanio rerio, acutely move about, whipping and twisting, the very fast death of part fish, when carbon tetrachloride concentration surpassed 5mmol/L, all Brachydanio rerio were all dead in 1 hour.And when carbon tetrachloride concentration was lower than 1mmol/L, then active state of Brachydanio rerio and normal group were as good as.Therefore, Brachydanio rerio carbon tetrachloride maximum concentration suitable and easily tolerance is 1～2mmol/L, and as optimum selection, the present invention selects the carbon tetrachloride solution of 1mmol/L concentration for use when causing the Brachydanio rerio liver injury model with carbon tetrachloride.
2, the examination of Brachydanio rerio acute liver damage model modeling time
Get 100 of Brachydanio rerio adult fishes, place the bottle that contains 200mL solution respectively, 2 every bottle, be divided into 5 groups at random, every group contains 20.Wherein 1 group is the normal control group, other 4 groups is the carbon tetrachloride group, the carbon tetrachloride group adds the carbon tetrachloride with the DMSO hydrotropy, the final concentration that makes carbon tetrachloride is that 1mmol/L carries out the modeling experiment, the normal control group adds the pure water that contains equivalent DMSO, adding behind the carbon tetrachloride different time points dynamically gets the Brachydanio rerio hepatic tissue and carries out every index and detect, observe liver injury model and have or not formation, every group of 20 livers that fish is got are divided into 2 parts at random, every part is the liver of 10 fishes, portion is made 1% liver tissue homogenate, detect ALT in the homogenate with automatic clinical chemistry analyzer, the AST level, another part liver organization specimen is fixed paraffin section through conventional 10% formalin solution, H-E dyeing, the pathological change situation of Brachydanio rerio hepatic tissue is respectively organized in observation under the light microscopic.According to ALT, AST level and hepatic pathology histological examination evaluation of result Brachydanio rerio hepatic injury degree in the liver tissue homogenate.Concrete experimental result is as shown in table 2.
Table 2 carbon tetrachloride act on the Brachydanio rerio different time to the influence of its liver function (
N=10)
Compare t-check, * P＜0.05, * * P＜0.01 with the normal control group.
Show by table 2 experimental result, when the 1mmol/L carbon tetrachloride acted on Brachydanio rerio in the time of 1 day, influence to its liver function is not remarkable, when acting on 3 days, its liver function occurs obviously unusual, ALT, AST level significantly are lower than the normal control group in the liver tissue homogenate, and tangible steatosis appears in the visible hepatocyte of histopathologic examination, show that its liver function obviously sustains damage.When the 1mmol/L carbon tetrachloride acted on Brachydanio rerio in the time of 5 days, its liver function still occurs obviously unusual, and is similar during to 3 days, and when effect in the time of 7 days, the hepatic injury degree alleviates on the contrary to some extent, and this may be that toleration appears in its body, and alleviates liver dysfunction.This shows that when causing Brachydanio rerio acute liver damage model with the 1mmol/L carbon tetrachloride, its modeling time was advisable with 3-5 days.
The present invention screens definite best modeling method by concentration and modeling time to carbon tetrachloride, can give security for the screening of anti-liver damage pharmaceutically active.
Embodiment 2 Brachydanio rerio acute liver damage models are used for the rapid screening of anti-liver injury medicament
Experimental technique: get 160 of Brachydanio rerio, place the wide mouthed bottle that fills 200mL solution respectively, be divided into 8 groups at random, every group contains 20, be respectively the normal control group, carbon tetrachloride mould shape matched group, the high and low concentration group of carbon tetrachloride modeling+compound glycyrrhizin, the high and low concentration group of carbon tetrachloride modeling+radix cynanchi bungei total sterioside and the high and low concentration group of carbon tetrachloride modeling+Indian Herba Swertiae bimaculatae extract.The normal control group is given the distilled water with equivalent, and other administration component is the high-concentration and low-concentration group.Wherein modeling method is the carbon tetrachloride modeling 3 days of 1mmol/l for adopting concentration, obtains the Brachydanio rerio of liver injury model.Normal control group and carbon tetrachloride mould shape matched group are given and the equivalent normal saline, with behind the administration 72h each group Brachydanio rerio take out is put to death, every group of 20 livers that fish is got are divided into 2 parts at random, every part is the liver of 10 fishes, portion is made 1% liver tissue homogenate, detect ALT in the homogenate with automatic clinical chemistry analyzer, AST level and total protein content, and the proteic ALT of every gram in the calculating homogenate, the AST level, another part liver organization specimen is fixed through conventional 10% formalin solution, paraffin section, H-E dyeing, the pathological change situation of Brachydanio rerio hepatic tissue is respectively organized in observation under the light microscopic, carries out the pathological grading scoring, it normally is 1 minute, slight steatosis is 2 minutes, and the moderate pathological changes is 3 minutes, and the steatosis severe patient is 4 minutes.Concrete experimental result is as shown in table 3.
Table 3 different pharmaceutical to the protective effect of Brachydanio rerio hepatic injury due to the carbon tetrachloride (
N=10)
Compare t-check, * P＜0.05, * * P＜0.01 with model control group.
Experimental result by above table 3 shows, compare with the normal control group, ALT, AST level have reduction (P＜0.01) extremely significantly in its liver tissue homogenate of Brachydanio rerio of carbon tetrachloride mould shape matched group, the liver function that shows Brachydanio rerio has been subjected to obvious damage, this and the result of histopathologic examination are in full accord, and the liver injury model that shows the Brachydanio rerio of causing with carbon tetrachloride is successful.And after giving different medicine 72h behind the carbon tetrachloride model, then can obviously alleviate the hepatic injury degree of the Brachydanio rerio that carbon tetrachloride causes, raising significantly, (P＜0.05-0.01), the visible hepatic injury degree of pathologic finding also obviously alleviates (P＜0.05-0.01) for ALT in the liver tissue homogenate, AST level.And experimental result shows that also compound glycyrrhizin has than radix cynanchi bungei total sterioside and the better anti-liver harm usefulness of Indian Herba Swertiae bimaculatae extract, better liver-protecting efficacy is promptly arranged, this shows that the liver injury model that method provided by the invention is set up is successful, and can be used for the interior rapid screening of body of anti-liver injury medicament.
Brachydanio rerio is suitable with human gene's similarity and mice, and on protein level, the homology of its key position almost is 100%, so the present invention selects for use Brachydanio rerio as hepatic injury modeling object, preferred by to modeling method, physiological disposition that can Simulation of Complex, and experimental result shows by this model and can quick and conveniently filter out the medicine with anti-liver injury.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (7)
1. the new method with model organism zebra fish screening anti-liver injury medicament is characterized in that, may further comprise the steps:
(1) the adult fish of getting Brachydanio rerio is put in the container that fills water, be divided into normal control group, blank group immediately, be subjected to reagent thing group, every group of Brachydanio rerio quantity equates, blank group and be subjected to reagent thing group to add carbon tetrachloride, the molar concentration that makes carbon tetrachloride is 0.5～2mmol/L, and the normal control group adds the solvent of equivalent, continues modeling 3～5 days, promptly get the acute liver damage zebra fish model, standby;
(2) will be subjected to the reagent thing to be made into desired concn, join in the aqueous solution that is subjected to reagent thing group, normal control group and blank group add the distilled water of equivalent, after the administration 48 hours to 72 hours, each group Brachydanio rerio is taken out execution, get and respectively organize the Brachydanio rerio liver and be divided into 2 parts at random, portion is made 1% liver tissue homogenate, detects ALT in the homogenate, AST level and total protein content, and calculate the proteic ALT of every gram in the homogenate, the AST level, another part liver organization specimen is fixed through concentration 10% formalin solution, paraffin section, H-E dyeing, the pathological change situation of Brachydanio rerio hepatic tissue is respectively organized in observation under the light microscopic, carry out the pathological grading scoring, normally be 1 minute, slight steatosis is 2 minutes, and the moderate pathological changes is 3 minutes, the steatosis severe patient is 4 minutes, estimates and screen the anti-liver injury activity that each group is subjected to the reagent thing.
2. the new method with model organism zebra fish screening anti-liver injury medicament according to claim 1 is characterized in that when step (1) was used the carbon tetrachloride modeling, the whole molar concentration of carbon tetrachloride was 1mmol/L.
3. the new method with model organism zebra fish screening anti-liver injury medicament according to claim 1, it is characterized in that, the used carbon tetrachloride of step (1) adds the dimethyl sulfoxide hydrotropy, the final concentration of dimethyl sulfoxide in solution is 0～1%, and the solvent that the normal control group adds is the dimethyl sulfoxide of equivalent.
4. the new method with model organism zebra fish screening anti-liver injury medicament according to claim 1 is characterized in that the described modeling time of step (1) is 3 days.
5. the new method with model organism zebra fish screening anti-liver injury medicament according to claim 1 is characterized in that, after 72 hours each group Brachydanio rerio is taken out execution in administration in the step (2) and gets hepatic tissue.
6. the new method with model organism zebra fish screening anti-liver injury medicament according to claim 1 is characterized in that step (2) adopts automatic clinical chemistry analyzer to detect ALT, the AST level of respectively organizing in the Brachydanio rerio hepatic homogenate.
7. according to each described new method of claim 1 to 6, it is characterized in that the described reagent thing that is subjected to of step (2) is chemical drugs, Chinese medicine, Chinese medicine compound, natural drug or their compositions with model organism zebra fish screening anti-liver injury medicament.
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| CN103430890B (en) * | 2013-09-05 | 2019-04-16 | 南京新环检测科技有限公司 | The method for building up of ROS detection model and its application in a kind of zebra fish body |
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