CN102520130A

CN102520130A - Screening method of medicines for inducing formation of myocardial cells by using model organism zebrafish

Info

Publication number: CN102520130A
Application number: CN2011104030996A
Authority: CN
Inventors: 钟涛
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2011-12-07
Filing date: 2011-12-07
Publication date: 2012-06-27

Abstract

The invention, belonging to the technical field of medicament, particularly relates to a screening method of medicines for inducing the formation of myocardial cells by using a model organism zebrafish, comprising the following steps: first, letting 25-30 pairs of Tg (cmlc2: EGFP) transgenic zebrafish homozygotes mate to obtain embryons, then transferring the embryons into a culture plate, adding a proper amount of zebrafish embryons at the stage of 50% epiboly in every pore, using an embryon culture solution containing 1-30 mu M small molecular compounds in an incubator for culture; then carefully checking the embryon heart size, heart shape and the number of myocardial cells; in addition, checking the whole shape (such as dorsal-ventral body axis, anterior-posterior body axis, etc.) and whether the formation of other organs of the embryons is influenced; and finally preliminarily judging whether the small molecular compounds promote the formation of the myocardial cells without influencing the growth of the other organs according to the experiment results. The new medicines which are screened out by the method has strong specificity and low toxic and side effect.

Description

Induce the method for the medicine that the cardiac muscle cell forms with model organism zebra fish screening

Technical field

The invention belongs to medical technical field, be specifically related to a kind of high intension screening technique of drug candidate, relating in particular to the heart transgenic zebrafish is model, and screening promotes the method for the prodrug (micromolecular compound) that the cardiac muscle cell forms.

Background technology

Angiocardiopathy is the healthy common disease of harm humans, and its M & M has surpassed the malignant tumour position that ranks first place.Although modern medicine constantly makes progress; But the treatment to ischemic heart diseases such as myocardial infarction and heart failure still faces huge challenge; Development can excite the cardiac muscle cell to form in the myocardial infarction district and regenerate to increase the methods of treatment of number of myocardial cells, has huge medical prospect and economic benefit clinically.Find that new biological reagent that external source or endogenous adult cardiac stem cells be divided into the cardiac muscle cell and the small-molecule substance of can inducing has the treatment meaning for the injury repair of heart.Therefore set up a kind of effective screening and promote that the method for the micromolecular compound that the cardiac muscle cell forms is significant.

Zebra fish is a kind of extraordinary model organism, and the characteristic that the zebra fish whole body is transparent helps to observe the change of its phenotype, is the effective tool of studying medicine both at home and abroad.Foundation has the transgenic zebrafish strain of green or red fluorescence, is easy to observe the influence of different compounds to its phenotype.The long 3-4 of adult zebra fish centimetre, a mating can obtain 100-200 piece of embryo, lays eggs weekly once, and its nursing and the expense of keeping are lower, and the requisite space place is little, is suitable for indoor large-scale breeding.At present, zebra fish is mainly used in hereditary development, and screening can promote the method for the micromolecular compound that the cardiac muscle cell forms also not have relevant report as research object to utilize it.

Summary of the invention

The object of the invention provides a kind of easy to operate, and cost is low, and experimental result is reliably accurate, and rapid screening is induced the method for the micromolecular compound that the cardiac muscle cell forms.

The method of the micromolecular compound that the cardiac muscle cell forms is induced in screening provided by the invention, adopts the model organism transgenic zebrafish, and concrete steps are following:

1, to the micromolecular compound of the various different structures that from drug reservoir, take out, as cosolvent, is made into the storage liquid of 1mM with DMSO (dimethyl sulfoxide); When phenotypic screen, every kind of compound is diluted to 1-30 μ M with embryo medium.

2, make the heart transgenic zebrafish Tg (cmlc2:EGFP)Homozygote carries out mating, obtains the embryo, is cultured to the 50% outsourcing phase (after fertilization 5 hours); Then the embryo is transferred in the culture plate (like 96 orifice plates), an amount of embryo (like 3 pieces of embryos) is inserted in every hole, adds the embryo medium of different micromolecular compounds simultaneously, cultivates at 20-30 ℃ of Embryo Culture case; Wherein, DMSO is as the blank group; Embryonic development different phase (for example be cultured to 24 hours, 48 hours, different phase such as 72 hours), the embryo heart size carried out double check with cardiac shape, and pass through the transgenic zebrafish of nucleus mark Tg (cmlc2:DsRed-nuc)Carry out cardiac muscle cell's counting; At last experimental result is carried out preliminary judgement, analyze the influence that each micromolecular compound forms the cardiac muscle cell.

3, whether inspection embryo's configuration (such as back of the body abdomen axon and front and back axon etc.) and other organ (comprising brain, eyes, notochord and body segment etc.) are affected; Judge that through the observations analysis whether each micromolecular compound is to the toxic effect of other organ of embryo.

According to above-mentioned two screening criterias, can obtain the drug molecule that promotes that the cardiac muscle cell forms, these drug molecules then do not have toxic action or toxicity less to embryo and other organ;

Provided by the inventionly promote the new method and the prior art of the micromolecular compound that the cardiac muscle cell forms that following advantage is relatively arranged: workable with model organism zebra fish screening; The experiment accuracy is high; The success ratio of compound primary dcreening operation is high, in vivo observes simultaneously, and the compound tissue specificity is strong; But the toxic and side effect of Direct observation compound improves the efficient of drug candidate screening.

Description of drawings

Fig. 1 is the micromolecular compound screening process synoptic diagram that Screening and Identification influences heart development in the body.

Fig. 2 is the influence that myocardium micromolecular compound forms heart.Wherein, A is the influence of myocardium micromolecular compound to cardiac muscle cell's quantity; B cardiac muscle micromolecular compound forms the synoptic diagram of development impact to the different development stage cardiac muscle cell; C is the influence of myocardium micromolecular compound to the different development stage number of myocardial cells.

Embodiment

According to following embodiment, can better understand the present invention.Yet, those skilled in the art will readily understand, implement the source of described concrete compound, concentration, selection of zebra fish and result thereof are only applicable to explain the present invention, and the present invention that should also can not limit in the claims to be described in detail.

1. select suitable compound storehouse or synthetic new compound

The source of this instantiation compound; From U.S. Univ Vanderbilt (Vanderbilt University) chemical-biological the high-flux medicaments sifting storehouse obtain the compound of 4000 different structures, every kind of micromolecular compound all is mixed with the storage liquid of 1mM with DMSO.

2. select to observe the relevant transgenic zebrafish strain of purpose

This instance purpose is to observe the micromolecular compound that promotes that the cardiac muscle cell forms, so select tape has the zebra fish of myocardium marker gene Tg (cmlc2:EGFP)Strain (international zebra fish resource center can buy) and Tg (cmlc2:DsRed-nuc)Strain (international zebra fish resource center can buy)

3. the optium concentration compound is selected in the selection of different compound concentrations

Selected concentration is 10 μ M in this instance compound library, therefore before the compound treatment embryo, earlier every kind of compound is become 10 μ M for 100 times with the embryo medium dilution.

4. the selection of fish-egg

To use the transgenic zebrafish of cardiac marker Tg (cmlc2:EGFP)Homozygote carries out mating and obtains the embryo, is cultured to the 50% outsourcing phase (after fertilization 5 hours).

5. the processing procedure of compound

The embryo of (after fertilization 5 hours) is transferred in 96 orifice plates with 50% outsourcing phase, inserts 3 pieces of embryos in every hole, adds the embryo medium of different micromolecular compounds simultaneously, cultivates at 28.5 ℃ of Embryo Culture casees, and wherein DMSO is as the blank group.Detailed process such as synoptic diagram 1.

6. observe

The embryo grew respectively to 24 hours, and 48 hours, cardiac shape and cardiac contractility carried out double check to the embryo heart size in 72 hours, and passed through the transgenic zebrafish of nucleus mark Tg (cmlc2:DsRed-nuc)Carry out cardiac muscle cell's counting (Fig. 2).In addition, gone back scrutiny embryo's configuration (such as the back of the body abdomen axon and front and back axon etc.) and other organ comprises brain, eyes, and whether notochord and body segment etc. is affected.Come the whether specific cardiac muscle cell's of inducing the formation of preliminary judgement micromolecular compound through these observationss at last, and whether these micromolecular compounds are to embryo and the toxic effect of other organ.

The above only is an instance of the present invention; Should be understood 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 improvement and retouching; For example compound is to the influence of other internal organs phenotype etc., and these improvement and retouching also should be regarded as protection category of the present invention.

Claims

1. induce the method for the medicine that the cardiac muscle cell forms with model organism zebra fish screening for one kind, it is characterized in that concrete steps are:

(1), to the micromolecular compound of the various different structures that from drug reservoir, take out, as cosolvent, be made into the storage liquid of 1mM with dimethyl sulfoxide; When phenotypic screen, every kind of compound is diluted to 1-30 μ M with embryo medium;

(2), make the heart transgenic zebrafish Tg (cmlc2:EGFP)Homozygote carries out mating, obtains the embryo, is cultured to for 50% outsourcing phase; Then the embryo is transferred in the culture plate, an amount of embryo is inserted in every hole, adds the embryo medium of different micromolecular compounds simultaneously, cultivates at 20-30 ℃ of Embryo Culture case; Wherein, dimethyl sulfoxide is as the blank group; In the different phase of embryonic development, embryo heart size and cardiac shape are carried out double check, and pass through the transgenic zebrafish of nucleus mark Tg (cmlc2:DsRed-nuc)Carry out cardiac muscle cell's counting; At last experimental result is carried out preliminary judgement, analyze the influence that each micromolecular compound forms the cardiac muscle cell;

(3), inspection embryo's configuration, and other organ comprises brain, eyes, whether notochord and body segment etc. is affected; Judge that through the observations analysis whether each micromolecular compound is to the toxic effect of other organ of embryo.