CN111388684A - A method for evaluating the efficacy of drugs against Alzheimer's disease using transgenic zebrafish - Google Patents

Abstract

The invention discloses a method for evaluating the drug effect of a drug on Alzheimer's disease by using transgenic zebra fish, which is specifically characterized by constructing a transgenic zebra fish drug screening model with Alzheimer's disease, taking juvenile fish as a research object, culturing the juvenile fish in a solution with a specific drug concentration, detecting relevant biochemical indexes of Alzheimer's disease and researching brain pathological forms by analyzing behavior tracks of the juvenile fish, and preliminarily judging whether the drug has the effect of resisting Alzheimer's disease. In view of the multiple advantages of zebra fish as a model organism, the method can quickly and efficiently carry out preliminary evaluation on whether the medicine has the effect of resisting Alzheimer's disease, and further promote the development and marketing of the medicine.

Description

A method for evaluating the efficacy of drugs against Alzheimer's disease using transgenic zebrafish

technical field

The invention relates to the technical field of drug screening, in particular to a method for evaluating the efficacy of a drug against Alzheimer's disease by utilizing transgenic zebrafish.

Background technique

Alzheimer's disease (AD) is a severe neurodegenerative disease, mainly in the elderly over 65 years old, characterized by neurofibrillary tangles and senile plaque accumulation, accompanied by neuronal loss and cognitive decline. According to a report, the number of people diagnosed with AD worldwide will reach 130 million by 2050. Therefore, the development of effective AD drugs is imminent. Although AD has been extensively studied by scientists in the past few decades, the complete mechanism of AD remains elusive. Given the complexity of AD etiology, a good animal model is crucial for AD research.

The existing AD models are mainly large and mouse models, but they have the defects of poor pathological simulation, long modeling time, low repeatability and high price, which cannot fully meet the needs of AD research. The study found that the neuroanatomical and neurochemical pathways of the zebrafish (Danio Rerio) brain showed great similarities with the human brain, as well as similarities in physiological, emotional and social behavior patterns between them. establishment. Thus, the zebrafish is emerging as an increasingly successful model for human neurological disease research. At the same time, the current drug research and development cycle is long, the cost is high, and there is a lack of evaluation methods for rapid and preliminary evaluation of the anti-AD effect of drugs. Therefore, the establishment of a simple, efficient and reproducible high-throughput method for evaluating the anti-AD effect of drugs will greatly reduce the number of The investment and consumption of human and material resources in the early stage shorten the research and development cycle and further promote the research and development of anti-AD drugs.

Chinese patent document CN108935231A (application number CN201810724850.4) discloses a method and application for the establishment of a zebrafish model of senile dementia. The zebrafish are placed in water culture medium and glucose culture medium for alternating culture, and each culture medium is cultured for 22~ 26 hours for a total of 50 to 60 days, and then the obtained zebrafish were alternately cultured in a glucose medium and an Okadaic acid medium, and each medium was cultured for 22 to 26 hours for a total of 6 to 10 days. In this invention, the total cultivation and modeling time is as long as 2 months, and the transfer operation needs to be performed every day at the same time, which is very inconvenient, and the excessively long modeling time increases the influence of external factors and increases the consumption of manpower and material resources.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides a method for evaluating the anti-Alzheimer's disease efficacy of a drug using transgenic zebrafish, which can quickly and efficiently evaluate whether the drug has an anti-Alzheimer's disease effect.

The technical scheme of the present invention is as follows:

A method for evaluating the efficacy of a drug against Alzheimer's disease using transgenic zebrafish, comprising the following steps:

(1) Construction of transgenic zebrafish

First, construct a target vector containing Alzheimer's disease-related gene sequences, and then obtain a transgenic zebrafish model fluorescently labeled with the gene by microinjection of zebrafish embryos. The positive embryos (showing green fluorescence) are cultured to adult fish, which is F Generation ₀ transgenic zebrafish;

(2) Culture of transgenic zebrafish embryos

After breeding the transgenic zebrafish, collect fertilized fish eggs, remove dead eggs and impurities, place them in a petri dish, add embryo culture medium and then put them into a constant temperature incubator for cultivation to obtain transgenic zebrafish juveniles;

(3) Grouping and administration

Zebrafish larvae were selected, and wild-type control group (WT), transgenic group and drug treatment groups with different concentrations were set. Among them, wild-type control group was maintained with wild-type zebrafish larvae in circulating water of zebrafish system, and transgenic group was treated with transgenic The zebrafish larvae were maintained in the circulating water of the zebrafish system, and the transgenic zebrafish larvae were exposed to different concentrations of the drug solution in the treatment groups with different concentrations of the drug. group, the vehicle group was exposed to the corresponding concentration of the vehicle;

(4) Behavior Trajectory Analysis

After the drug acts, use the zebrafish behavioral trajectory analysis system to set the experimental condition parameters, observe and analyze the behavioral trajectories of each group of zebrafish, and use the speed-related variables for quantitative analysis;

(5) Brain pathological study

After the behavioral trajectory analysis, multiple samples were selected from each group, quickly dissected on ice after anesthesia, and the fish brains were isolated for ultrastructural pathological analysis;

(6) Biochemical index detection

The fish brain obtained with reference to the method described in step (5) is prepared into tissue homogenate, and molecular biology methods are used to measure the changes of Alzheimer's disease-related indicators;

(7) Data statistics and analysis processing

GraphPad Prism 5 software was used for statistics and data visualization, and one-way analysis of variance was used for comparison between groups.

Preferably according to the present invention, the steps include but are not limited to the construction of transgenic zebrafish, the cultivation of transgenic zebrafish embryos, the grouping and administration of transgenic zebrafish larvae, behavioral trajectory analysis, brain pathological morphological research, biochemical index detection and One or more steps in the statistical and analytical processing of data.

Preferably according to the present invention, the Alzheimer's disease-related genes in the step (1) are obtained from GeneCards (https://www.genecards.org/), and the Alzheimer's disease-related scores are greater than 100.

Preferably according to the present invention, the Alzheimer's disease-related genes in the step (1) include but are not limited to APP (correlation score=160.64), PSEN1 (correlation score=146.86), APOE (correlation score=133.71) ), one or more of MAPT (relevance score = 110.73) and PSEN2 (relevance score = 109.16).

Preferably according to the present invention, the transgenic zebrafish in the step (2) is homozygous, and the preparation method is as follows _: breeding the female F0 generation transgenic zebrafish obtained in the step ( ₁ ) with a wild-type male zebrafish to obtain F1 Generation, through caudal fin sequencing, screening out heterozygotes, selfing to obtain F ₂ generation, the self-obtained progeny through caudal fin sequencing, screening homozygous transgenic zebrafish.

Preferably according to the present invention, the method for breeding zebrafish in the step (2) is as follows: in the evening according to female:male=1:1 or 2:1, the female zebrafish is placed in a special tank for fish breeding, and the middle Separate with a baffle, and remove the baffle the next morning.

Preferably according to the present invention, the diameter of the culture dish in the step (2) is 80-90mm, and the density is controlled at 10-50 eggs/dish.

Preferably according to the present invention, the embryo culture liquid in the described step (2) is the system circulating water containing 0.5mg/L methylene blue, and the liquid is changed every day to keep the water clean.

According to a preferred embodiment of the present invention, the conditions for culturing in the constant temperature incubator are: culturing in a constant temperature incubator at 28.0-30.0°C to 2-7 dpf (days post fertilization, days after fertilization); the constant temperature incubator has no photoperiod regulation.

Preferably according to the present invention, the drug solvent in the step (3) includes, but is not limited to, dimethyl sulfoxide (DMSO) and zebrafish system circulating water, which is specifically selected according to the physicochemical properties of the drug.

Preferably according to the present invention, the drug treatment method in the step (3) is as follows: using a 1/10,000 analytical balance to precisely weigh the quantitative drug, select a suitable solvent according to the drug properties to dissolve, and dilute to the desired concentration, 0.22 μm Filter through the filter head and store at -20°C for later use.

Preferably according to the present invention, the medicines in the step (3) include but are not limited to chemical raw materials, chemical medicine-related preparations, traditional Chinese medicine extracts, single traditional Chinese medicines, traditional Chinese medicine preparations, and traditional Chinese medicine compounds.

Preferably according to the present invention, the administration period in the step (3) is 3dpf-7dpf, including but not limited to 3 days, 4 days or 5 days.

Preferably according to the present invention, the drug concentration in the step (3) is set to 2-3 gradient doses, and the specific determination method is as follows: placing the transgenic zebrafish larvae in a reaction vessel, such as a 96-well plate, Expose them to drugs with gradient concentrations, observe the morphology of juvenile fish under a microscope, determine whether there is toxic effect, determine the safe concentration range, and further determine 2-3 gradient doses.

Preferably according to the present invention, the administration requirements in the step (3) are: the reaction vessel includes but not limited to 96-well plate, 48-well plate, 24-well plate, 12-well plate and 6-well plate, and the administration volume is 200 μL -4mL, change the same concentration and volume of drug solution every day to maintain stable drug concentration.

Preferably according to the present invention, the experimental condition parameters in the step (4) are: the reaction vessel includes but is not limited to 96-well plate, 48-well plate, 24-well plate, 12-well plate and 6-well plate, and the temperature is kept at 28.0- At 30.0 °C, the behavioral tracking duration was 1-24 h, and each hour included 3 light/dark cycles (10 min of light and 10 min of darkness).

The zebrafish behavioral trajectory analysis system in the step (4) adopts the Danio Vision zebrafish behavioral trajectory analysis system, namely the DanioVision zebrafish behavioral trajectory tracking system.

Preferably according to the present invention, the motion-related variables in the step (4) include but are not limited to the moving distance within 1 h and the average motion speed within 1 h.

Preferably according to the present invention, the ultrastructural analysis in the step (5) includes, but is not limited to, transmission electron microscopy and other methods.

In step (5), the multiple samples are 3-10 samples.

Preferably according to the present invention, the Alzheimer's disease-related indicators in the step (6) include but are not limited to acetylcholinesterase (ChE), acetylcholinesterase (ChAT), malondialdehyde (MDA) and superoxide. One or more of dismutase (SOD).

Preferably according to the present invention, the molecular biology method in the step (6) includes but is not limited to Western Blot, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry One or more of (IHC) and immunofluorescence (IF).

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

The invention constructs a transgenic Alzheimer's disease zebrafish drug screening model, takes juvenile fish as the research object, puts them in a solution with a specific drug concentration and culture, and detects the Alzheimer's disease-related biochemistry by analyzing the behavioral trajectory of the juvenile fish. Indicators and study of brain pathological morphology, to preliminarily determine whether the drug has anti-Alzheimer's disease effect. In view of the multiple advantages of zebrafish as a model organism, this method can quickly and efficiently conduct a preliminary evaluation of whether a drug has anti-Alzheimer's disease effect, thereby promoting drug development and marketing.

Description of drawings

Fig. 1 is the flow chart of screening technology;

Among them, 1. Construction of transgenic zebrafish; 2. Culture of transgenic zebrafish embryos; 3. Grouping and drug administration; 4. Behavioral trajectory analysis; Analytical processing;

Fig. 2 shows the swimming trajectory within 30s of the wild-type control group (WT) and the APPsw transgenic group (A) and the swimming heat map within 30s (B);

Figure 3 is an electron microscope ultrastructural image of the brain structure of the wild-type control group (WT) (A, C) and the APPsw transgenic group (B, D);

Fig. 4 is the moving distance of embodiment 1, 2, 3 and 4 in 1h to this figure;

Fig. 5 is the average motion speed comparison chart of embodiment 1, 2, 3 and 4 in 1h;

Fig. 6 is embodiment 1,2,3 and 4 acetylcholinesterase (AChE) activity comparison chart;

Fig. 7 is embodiment 1, 2, 3 and 4 acetylcholinesterase (ChAT) activity comparison chart;

Fig. 8 is embodiment 1,2,3 and 4 malondialdehyde (MDA) content comparison chart;

Figure 9 is a comparison chart of the superoxide dismutase (SOD) activity of Examples 1, 2, 3 and 4.

Detailed ways

In order to illustrate the technical solutions and outstanding beneficial effects of the present invention, the following examples are now given for further description. It should be understood that the specific embodiments listed here are only used to explain the present invention, but not to limit the present invention.

Example 1:

1.1 Construction of transgenic zebrafish

A target vector containing the APP gene Swedish mutant (APPsw) sequence was constructed, and then a transgenic zebrafish model fluorescently labeled with the gene was obtained by microinjection of zebrafish embryos. Positive embryos (showing green fluorescence) were cultured to adult fish, which was the F ₀ generation.

1.2 Culture of transgenic zebrafish embryos

The F ₀ generation female transgenic zebrafish was bred with wild-type male zebrafish to obtain the F ₁ generation, and the heterozygotes were screened by caudal fin sequencing, and the F ₂ generation was obtained by selfing. Zygote. In the evening, the homozygous zebrafish were placed in a special fish tank according to female: male = 1:1, separated by a baffle in the middle, and the baffle was removed the next morning. Collect fertilized fish eggs, remove dead eggs and impurities, place them in 80mm diameter petri dishes, control the density at 25 eggs/dish, circulate water with a system containing 0.5mg/L methylene blue, change the liquid every day, and keep the water clean, at 28.5 The cells were cultured to 6 dpf (days post fertilization, days after fertilization) in a constant temperature incubator without photoperiod.

1.3 Behavior Trajectory Analysis

At 7dpf, 12 zebrafish were randomly selected from each of the wild-type control group (WT) and the APPsw transgenic group and placed in a 96-well plate. The Danio Vision zebrafish behavioral trajectory analysis system was used to set the experimental condition parameters to observe and analyze the zebrafish in each group. Behavioral trajectories were quantitatively analyzed using two parameters, the moving distance within 1 hour and the average movement speed within 1 hour.

1.4 Brain pathological morphology research

After the behavioral trajectory analysis, 3 samples were selected from each group, quickly dissected on ice after anesthesia, isolated fish brains, photographed under electron microscope, and ultrastructural pathological analysis.

1.5 Biochemical index detection

The zebrafish brains of the WT group and the APPsw transgenic group were prepared into tissue homogenates, and acetylcholinesterase (AChE), acetylcholinesterase (ChAT), malondialdehyde (MDA) and superoxide dismutase ( SOD) content or activity.

1.6 Data Statistics and Analysis Processing

GraphPad Prism 5 software was used for statistics and data visualization, and one-way analysis of variance was used for comparison between groups.

1.7 Experimental results

Using the zebrafish behavioral trajectory tracking system to count the swimming trajectories of zebrafish within 30s, the swimming trajectories of zebrafish in the APPsw transgenic group were significantly less than those in the WT group (Fig. 2A). The heat map of zebrafish within 30s was counted. The more red, the longer the zebrafish stayed in this area. It can be seen that the APPsw group spent significantly more time in the same area than the WT group (Fig. 2B). At the same time, the moving distance (P<0.001) within 1 h (Fig. 4) and the average movement speed (P < 0.001) within 1 h (Fig. 5) of zebrafish in the APPsw transgenic group were significantly decreased.

The ultrastructure of the zebrafish brain was observed by transmission electron microscopy. The zebrafish neurons in the WT group had a round nucleus with a clear nuclear membrane and abundant organelles such as mitochondria and endoplasmic reticulum in the cytoplasm (Fig. 3A, C). The nuclei of zebrafish neurons in the APPsw transgenic group were irregular in shape, and some neurons had disintegration and destruction of the cell contents, the endoplasmic reticulum was swollen and deformed, and there were many vacuoles.

Biochemical index detection showed that compared with the WT group, the zebrafish AchE activity in the APPsw transgenic group was significantly increased (P<0.001) (Fig. 6), the ChAT activity was significantly decreased (P<0.001) (Fig. 7), and the MDA content significantly increased (P<0.05) (Figure 8), and SOD activity was significantly decreased (P<0.001) (Figure 9).

In conclusion, the APPsw transgenic zebrafish model was successfully constructed.

Example 2:

2.1 Construction of transgenic zebrafish

A target vector containing the APP gene Swedish mutant (APPsw) sequence was constructed, and then a transgenic zebrafish model fluorescently labeled with the gene was obtained by microinjection of zebrafish embryos. Positive embryos (showing green fluorescence) were cultured to adult fish, which was the F ₀ generation.

2.2 Culture of transgenic zebrafish embryos

The F ₀ generation female transgenic zebrafish was bred with wild-type male zebrafish to obtain the F ₁ generation, the heterozygotes were screened by caudal fin sequencing, and the F ₂ generation was obtained by selfing. Zygote. In the evening, the homozygous zebrafish were placed in a special fish tank according to female: male = 1:1, separated by a baffle in the middle, and the baffle was removed the next morning. Collect fertilized fish eggs, remove dead eggs and impurities, place them in 80mm diameter petri dishes, control the density at 25 eggs/dish, use the system circulating water containing 0.5mg/L methylene blue, change the liquid every day, keep the water clean, at 28.0 The cells were cultured to 2 dpf (days post fertilization, days after fertilization) in a constant temperature incubator without photoperiod.

2.3 Grouping and Administration

Select 2dpf larval zebrafish, set up wild-type control group (WT), APPsw transgenic group and Huperzine A (HUPA) group, with 30 in each group in a 6-well plate. The quantitative Huperzine A was accurately weighed with a 1/10,000 analytical balance, 200nM and 800nM solutions were prepared with the circulating water of the zebrafish system, and filtered with a 0.22 μm filter. Wild-type control (WT) and APPsw transgenic groups were maintained in zebrafish systemic circulating water, and Huperzine A (HUPA) groups were exposed to 200 nM and 800 nM drug solutions.

2.4 Behavior Trajectory Analysis

The duration of drug action was 3dpf-6dpf, and half of the drug solution was changed every day. At 7dpf, 12 zebrafish were randomly selected from Huperzine A at 200nM and 800nM concentrations and placed in a 96-well plate. The Danio Vision zebrafish behavioral trajectory analysis system was used to set Experiment condition parameters, observe and analyze the behavioral trajectories of zebrafish in each group, and use two parameters, the moving distance within 1 hour and the average movement speed within 1 hour, for quantitative analysis.

2.5 Data statistics and analysis processing

GraphPad Prism 5 software was used for statistics and data visualization, and one-way analysis of variance was used for comparison between groups.

2.6 Experimental results

Compared with the APPsw transgenic group, Huperzine A at a concentration of 200nM and Huperzine A at a concentration of 800nM can significantly increase the moving distance within 1h (P<0.05) of zebrafish (Fig. 4) and the average movement within 1h. speed (P<0.001) (Fig. 5), proving that Huperzine A at this concentration indeed has the effect of improving Alzheimer's disease.

Example 3:

3.1 Construction of transgenic zebrafish

A target vector containing the APP gene Swedish mutant (APPsw) sequence was constructed, and then a transgenic zebrafish model fluorescently labeled with the gene was obtained by microinjection of zebrafish embryos. Positive embryos (showing green fluorescence) were cultured to adult fish, which was the F ₀ generation.

3.2 Culture of transgenic zebrafish embryos

The F ₀ generation female transgenic zebrafish was bred with wild-type male zebrafish to obtain the F ₁ generation, the heterozygotes were screened by caudal fin sequencing, and the F ₂ generation was obtained by selfing. Zygote. In the evening, the homozygous zebrafish were placed in a special fish tank according to female: male = 1:1, separated by a baffle in the middle, and the baffle was removed the next morning. Collect fertilized fish eggs, remove dead eggs and impurities, place them in 80mm diameter petri dishes, control the density at 40 eggs/dish, use the system circulating water containing 0.5mg/L methylene blue, change the liquid every day, keep the water clean, at 28.0 The cells were cultured to 2 dpf (days post fertilization, days after fertilization) in a constant temperature incubator without photoperiod.

3.3 Grouping and Administration

Select 2dpf larval zebrafish, set up wild-type control group (WT), APPsw transgenic group and donepezil hydrochloride (DPZ) group, with 30 in each group in a 6-well plate. The quantitative donepezil hydrochloride was accurately weighed with a 1/10,000 analytical balance, and the solutions of 2 μM, 4 μM and 8 μM were prepared with the circulating water of the zebrafish system, and filtered with a 0.22 μm filter head. The wild-type control group (WT) and the APPsw transgenic group were maintained in zebrafish systemic circulating water, and the donepezil hydrochloride group was exposed to 2 μM, 4 μM and 8 μM of the drug solution, respectively.

3.4 Behavior Trajectory Analysis

The duration of drug action is 3dpf-6dpf, and half of the drug solution is changed every day. At 7dpf, 12 zebrafish are randomly selected from the concentration of donepezil hydrochloride at 2 μM and 4 μM and placed in a 96-well plate. The Danio Vision zebrafish behavioral trajectory analysis system is used to set the experimental conditions. Parameters, observe and analyze the behavioral trajectories of zebrafish in each group, and use two parameters, the moving distance within 1 h and the average movement speed within 1 h, for quantitative analysis.

3.5 Biochemical index detection

The zebrafish brain at a concentration of 8 μM of donepezil hydrochloride was prepared into tissue homogenate, and acetylcholinesterase (AChE), acetylcholinesterase (ChAT), malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by ELISA. ) content or activity.

3.6 Data Statistics and Analysis Processing

GraphPad Prism 5 software was used for statistics and data visualization, and one-way analysis of variance was used for comparison between groups.

3.7 Experimental results

Compared with the APPsw transgenic group, donepezil hydrochloride at a concentration of 2 μM and donepezil hydrochloride at a concentration of 4 μM could significantly increase the zebrafish moving distance within 1 h (P<0.05) (Fig. 4) and the average movement speed within 1 h (P<0.05). 0.001) (Fig. 5), donepezil hydrochloride at a concentration of 8 μM can significantly reduce the activity of AchE (P<0.001) (Fig. 6) and increase the activity of ChAT (P<0.01) (Fig. 7), while reducing the content of MDA and increasing The effect trend of high SOD activity (Figures 8 and 9) proves that donepezil hydrochloride at this concentration does have the effect of improving Alzheimer's disease.

Example 4:

4.1 Construction of transgenic zebrafish

A target vector containing the APP gene Swedish mutant (APPsw) sequence was constructed, and then a transgenic zebrafish model fluorescently labeled with the gene was obtained by microinjection of zebrafish embryos. Positive embryos (showing green fluorescence) were cultured to adult fish, which was the F ₀ generation.

4.2 Culture of transgenic zebrafish embryos

The F ₀ generation female transgenic zebrafish was bred with wild-type male zebrafish to obtain the F ₁ generation, and the heterozygotes were screened by caudal fin sequencing, and the F ₂ generation was obtained by selfing. Zygote. In the evening, the homozygous zebrafish were placed in a special fish tank according to female: male = 2:1, separated by a baffle in the middle, and the baffle was removed the next morning. Collect fertilized fish eggs, remove dead eggs and impurities, place them in a 90mm diameter petri dish, control the density at 30 eggs/dish, use a system of circulating water containing 0.5mg/L methylene blue, change the liquid every day, keep the water clean, at 28.5 The cells were cultured to 2 dpf (days post fertilization, days after fertilization) in a constant temperature incubator without photoperiod.

4.3 Grouping and Administration

2dpf larval zebrafish were selected, and the wild-type control group (WT), the APPsw transgenic group and the memantine hydrochloride (Mem) group were set, with 30 in each group in a 6-well plate. A 1/10,000 analytical balance was used to accurately weigh the quantitative memantine hydrochloride, and the 50 μM and 100 μM solutions were prepared with the circulating water of the zebrafish system, and filtered with a 0.22 μm filter head. Wild-type control (WT) and APPsw transgenic groups were maintained in zebrafish systemic circulating water, and memantine hydrochloride groups were exposed to 50 μM and 100 μM drug solutions.

4.4 Behavior Trajectory Analysis

The duration of drug action was 3dpf-6dpf, and half of the drug solution was changed every day. At 7dpf, 12 zebrafish were randomly selected from the concentrations of memantine hydrochloride 50 μM and 100 μM and placed in a 96-well plate, and the Danio Vision zebrafish behavioral trajectory analysis system was used. , set the experimental condition parameters, observe and analyze the behavioral trajectories of zebrafish in each group, and use two parameters, the moving distance within 1 hour and the average movement speed within 1 hour, for quantitative analysis.

4.5 Biochemical index detection

The zebrafish brain at the concentration of memantine hydrochloride 50μM was prepared into tissue homogenate, and acetylcholinesterase (AChE), acetylcholinesterase (ChAT), malondialdehyde (MDA) and superoxide were determined by ELISA The content or activity of dismutase (SOD).

4.6 Data Statistics and Analysis Processing

GraphPad Prism 5 software was used for statistics and data visualization, and one-way analysis of variance was used for comparison between groups.

4.7 Experimental results

Compared with the APPsw transgenic group, both memantine hydrochloride at 50 μM concentration and memantine hydrochloride at 100 μM concentration could significantly increase the moving distance of zebrafish within 1 h (P<0.05) (Fig. 4) and 1 h Memantine hydrochloride at a concentration of 50 μM significantly decreased the viability of AchE (P<0.001) (Figure 6) and increased the viability of ChAT (P<0.01) within the mean speed of movement (P<0.01) (Fig. 5). (Fig. 7), and at the same time, there was a trend of reducing the content of MDA and increasing the activity of SOD (Fig. 8, 9), which proved that memantine hydrochloride at this concentration indeed had the effect of improving Alzheimer's disease.

Claims (10)

1. A method for evaluating the effect of a drug on Alzheimer's disease by using transgenic zebra fish is characterized by comprising the following steps:

(1) construction of transgenic Zebra fish

Firstly, constructing a target vector containing an Alzheimer disease related gene sequence, obtaining a transgenic zebra fish model of the gene by fluorescence labeling through zebra fish embryo microinjection, and culturing a positive embryo to a mature fish, namely F₀Transgenic zebrafish are bred;

(2) culture of transgenic zebra fish embryos

After breeding the transgenic zebra fish, collecting fertilized fish eggs, removing dead eggs and impurities, placing the fertilized fish eggs in a culture dish, adding an embryo culture solution, and placing the culture dish in a constant-temperature incubator for culture to obtain transgenic zebra fish juvenile fish;

(3) grouping and administration of drugs

Setting a wild type control group, a transgenic group and a treatment group with different concentrations of a medicament, wherein the wild type control group adopts wild type zebra fish juvenile fish to be maintained in zebra fish systemic circulating water, the transgenic group adopts transgenic zebra fish juvenile fish to be maintained in zebra fish systemic circulating water, the treatment group with different concentrations of the medicament adopts transgenic zebra fish juvenile fish to be exposed in solutions with different concentrations of the medicament, when the medicament is dissolved by using a solvent other than the zebra fish systemic circulating water, the treatment group is additionally provided with a solvent group, and the solvent group is exposed in a solvent with corresponding concentration;

(4) behavioral trace analysis

After the drug acts, setting experiment condition parameters by using a zebra fish behavior track analysis system, observing and analyzing the behavior tracks of each group of zebra fish, and carrying out quantitative analysis by using speed related variables;

(5) study on brain pathological morphology

After behavior trajectory analysis, selecting a plurality of samples in each group, quickly dissecting on ice after anesthesia, separating fish brains, and performing ultrastructural pathological analysis;

(6) biochemical index detection

Preparing the fish brain obtained by the method in the step (5) into tissue homogenate, and measuring the content change of the Alzheimer disease related index by adopting a molecular biological method.

2. The method for evaluating the efficacy of a drug against Alzheimer's disease using transgenic zebrafish according to claim 1, wherein the Alzheimer's disease-associated genes in step (1) are obtained from GeneCards and have an Alzheimer's disease-associated score of more than 100.

3. The method for evaluating the anti-Alzheimer disease efficacy of a drug by using transgenic zebrafish according to claim 1, wherein the transgenic zebrafish in the step (2) is homozygote, and is prepared by the following steps: the female F obtained in the step (1)₀Mating transgenic zebra fish with wild male zebra fish to obtain F₁Instead, F is₁Selfing the adult zebra fish, sequencing by cutting tail fin, screening heterozygote, and selfing to obtain F₂Generation, selfing to obtain offspring, sequencing by cutting tail fin, and screeningSelecting homozygous transgenic zebrafish.

4. The method for evaluating the efficacy of a drug against Alzheimer's disease using transgenic zebrafish according to claim 1, wherein the diameter of the culture dish in step (2) is 80-90mm, and the density is controlled to 10-50 eggs/dish.

5. The method for evaluating the efficacy of a drug against Alzheimer's disease by using transgenic zebrafish according to claim 1, wherein the embryo culture solution in step (2) is a circulating water system containing 0.5 mg/L methylene blue.

6. The method for evaluating the efficacy of a drug against alzheimer's disease using transgenic zebrafish according to claim 1, wherein in step (2), the conditions of the incubation in the incubator are as follows: culturing in a constant temperature incubator at 28.0-30.0 ℃ to 2-7 dpf.

7. The method for evaluating the efficacy of a drug against alzheimer's disease using transgenic zebrafish according to claim 1, wherein the drug concentration in step (3) is set to 2-3 gradient doses, which is determined by: the transgenic zebra fish juvenile fish is placed in a reaction container and exposed in the medicine with gradient concentration, the juvenile fish shape is observed under a microscope, whether toxic effect occurs or not is judged, the safe concentration range is determined, and 2-3 gradient doses are further determined.

8. The method for evaluating the efficacy of a drug against Alzheimer's disease by using transgenic zebrafish according to claim 1, wherein the experimental condition parameters in the step (4) are as follows: the reaction vessels included 96-well plates, 48-well plates, 24-well plates, 12-well plates, and 6-well plates, the temperature was maintained at 28.0-30.0 ℃, the duration of the behavior trace was 1-24h, the reaction vessels included 3 light/dark cycles per hour, light 10min and dark 10 min.

9. The method for evaluating the efficacy of a drug against alzheimer's disease using transgenic zebrafish of claim 1, wherein the alzheimer's disease-related indicators in step (6) comprise one or more of acetylcholinesterase, malondialdehyde, and superoxide dismutase.

10. The method for evaluating the efficacy of a drug against alzheimer's disease using transgenic zebrafish of claim 1, wherein the molecular biological method in step (6) comprises one or more of immunoblotting, polymerase chain reaction, enzyme-linked immunosorbent assay, immunohistochemistry and immunofluorescence.

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