CN111388684A - Method for evaluating anti-Alzheimer disease efficacy of drug by using transgenic zebra fish - Google Patents
Method for evaluating anti-Alzheimer disease efficacy of drug by using transgenic zebra fish Download PDFInfo
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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
Technical Field
The invention relates to the technical field of drug screening, in particular to a method for evaluating the anti-Alzheimer disease drug effect of a drug by utilizing transgenic zebra fish.
Background
Alzheimer's Disease (AD) is a serious neurodegenerative disease, mainly occurring in the elderly population over 65 years of age, characterized by neurofibrillary tangles and the appearance of senile plaque aggregates, with concomitant neuronal loss and cognitive decline. According to one report, the number of people globally diagnosed with AD will reach 1.3 billion by 2050. Therefore, the development of effective AD drugs is at hand. Despite the extensive research on AD by scientists over the past decades, the complete mechanism of AD remains elusive. In view of the complexity of the etiology of AD, a good animal model is crucial for the study of AD.
The existing AD model mainly comprises a big model and a mouse model, but has the defects of poor pathological simulation, long modeling time, low repeatability, high price and the like, and can not completely meet the requirement of AD research. Studies have found that the neuroanatomy and neurochemical pathways of the brain of the zebra fish (Danio Rerio) show great similarity with the human brain, and the similarity of physiological, emotional and social behavioral patterns among them is well established. Zebrafish are therefore becoming an increasingly successful model for the study of human neurological diseases. Meanwhile, the conventional drug development period is long, the cost is high, and an evaluation method for rapidly and primarily evaluating the anti-AD effect of the drug is lacked, so that the establishment of a simple, efficient and high-repeatability method for evaluating the anti-AD effect of the drug in high flux can greatly reduce the input and consumption of manpower and material resources in the early stage, shorten the development period, and further promote the research and development of the anti-AD drug.
Chinese patent document CN108935231A (application No. CN201810724850.4) discloses a method for establishing a zebra fish senile dementia model and application thereof, the zebra fish is placed in an aqueous culture solution and a glucose culture solution for alternate culture, each culture solution is cultured for 22-26 hours, the culture lasts for 50-60 days, then the obtained zebra fish is cultured in the glucose culture solution and an okadaic acid culture solution for alternate culture, each culture solution is cultured for 22-26 hours, and the culture lasts for 6-10 days. In the invention, the total cultivation molding time is as long as 2 months, and meanwhile, the transfer operation is required every day, which is very inconvenient, and the overlong molding time increases the influence of external factors and increases the consumption of manpower and material resources.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for evaluating the anti-Alzheimer disease effect of a medicament by using transgenic zebra fish, which can quickly and efficiently evaluate whether the medicament has the anti-Alzheimer disease effect.
The technical scheme of the invention is as follows:
a method for evaluating the effect of a drug on Alzheimer's disease by using transgenic zebra fish comprises 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 with the gene fluorescently labeled by injecting zebra fish embryos in a microinjection manner, and culturing positive embryos (showing green fluorescence) to adult 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
Selecting zebra fish juvenile fish, and setting a wild type control group (WT), a transgenic group and a treatment group with different concentrations of medicines, wherein the wild type control group adopts the wild type zebra fish juvenile fish to be maintained in the circulating water of a zebra fish system, the transgenic group adopts the transgenic zebra fish juvenile fish to be maintained in the circulating water of the zebra fish system, the treatment group with different concentrations of medicines adopts the transgenic zebra fish juvenile fish to be exposed in medicine solutions with different concentrations, when a solvent other than the circulating water of the zebra fish system is used for dissolving medicines, a solvent group is additionally arranged, 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 a tissue homogenate from the fish brain obtained by the method in the step (5), and measuring the Alzheimer disease related index change by adopting a molecular biological method;
(7) data statistics and analysis processing
Statistics and data visualization were performed using GraphPad Prism 5 software, comparisons between groups were performed using one-factor analysis of variance, data were expressed as Mean ± standard deviation (Mean ± SD), and P <0.05 was statistically significant.
Preferably, the steps include, but are not limited to, one or more of constructing transgenic zebrafish, culturing transgenic zebrafish embryos, grouping and administering transgenic zebrafish larvae, analyzing behavior tracks, studying brain pathological morphology, detecting biochemical indicators, and performing data statistics and analysis.
According to the 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 all more than 100.
Preferably, the alzheimer's disease-related genes in step (1) include, but are not limited to, one or more of APP (relevance score 160.64), PSEN1 (relevance score 146.86), APOE (relevance score 133.71), MAPT (relevance score 110.73) and PSEN2 (relevance score 109.16).
Preferably, the transgenic zebra fish in the step (2) is homozygote, and the preparation method comprises the following steps: the female F obtained in the step (1)0Breeding transgenic zebra fish with wild male zebra fish to obtain F1Selecting heterozygote by sequencing through tailed fin, and selfing to obtainTo F2And (4) carrying out sequencing on progeny obtained by selfing through a tail fin shearing method, and screening the transgenic zebra fish of the homozygote.
Preferably, the zebra fish breeding method in the step (2) comprises the following steps: in the evening, according to the female: placing female parent of zebra fish in a special tank for fish preparation at a ratio of male parent to female parent of zebra fish of 1:1 or 2:1, separating the female parent and the female parent by using a baffle plate, and taking out the baffle plate in the next morning.
According to the invention, the diameter of the culture dish in the step (2) is 80-90mm, and the density is controlled to be 10-50 eggs/dish.
According to the invention, the embryo culture solution in the step (2) is the system circulating water containing 0.5 mg/L methylene blue, and the solution is changed every day to keep the water quality clean.
According to the invention, the conditions of the culture in the incubator are preferably as follows: culturing in a constant-temperature incubator at 28.0-30.0 ℃ until 2-7 dpf (days after fertilization); the constant temperature incubator is not subjected to light period regulation and control.
Preferably, the drug solvent in step (3) includes, but is not limited to, dimethyl sulfoxide (DMSO) and zebra fish systemic circulating water, and is selected according to the physicochemical properties of the drug.
According to the invention, the medicament treatment mode in the step (3) is preferably as follows: precisely weighing quantitative medicine with one ten-thousandth of analytical balance, selecting appropriate solvent according to medicine properties, dissolving, diluting to desired concentration, filtering with 0.22 μm filter head, and storing at-20 deg.C for use.
Preferably, according to the present invention, the drugs in step (3) include, but are not limited to, chemical raw materials, chemical-related preparations, herbal extracts, single herbs, herbal preparations, herbal compounds, and the like.
Preferably according to the invention, the administration period in step (3) is from 3dpf to 7dpf, including but not limited to 3 days, 4 days or 5 days.
According to the invention, preferably, the drug concentration in step (3) is set to 2-3 gradient doses, and the specific determination method is as follows: the transgenic zebra fish juvenile fish is placed in a reaction container, such as a 96-well plate, and is exposed in the drug 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.
According to the invention, the administration in the step (3) is preferably carried out in such a manner that reaction vessels including, but not limited to, 96-well plates, 48-well plates, 24-well plates, 12-well plates and 6-well plates are used, the administration volume is 200 μ L-4 m L, and an equal-concentration equal-volume drug solution is replaced every day to maintain the drug concentration stable.
According to the invention, the experimental condition parameters in the step (4) are: reaction vessels include, but are not limited to, 96-well plates, 48-well plates, 24-well plates, 12-well plates, and 6-well plates, maintained at a temperature of 28.0-30.0 ℃, with a behavior trace time of 1-24h, containing 3 light/dark cycles per hour (10 min light and 10min dark).
The zebra fish behavior trajectory analysis system in the step (4) adopts a Danio Vision zebra fish behavior trajectory analysis system, namely a Danio Vision zebra fish behavior trajectory tracking system.
According to the present invention, the motion related variables in step (4) include, but are not limited to, the movement distance within 1h and the average movement speed within 1 h.
According to the present invention, the microstructure analysis in step (5) includes, but is not limited to, various methods such as transmission lens observation.
In the step (5), the plurality of samples is 3 to 10 samples.
Preferably, according to the present invention, the alzheimer's disease related index in the step (6) includes, but is not limited to, one or more of acetylcholinesterase (ChE), acetylcholinesterase (ChAT), Malondialdehyde (MDA) and superoxide dismutase (SOD).
According to the present invention, the molecular biological method in step (6) includes, but is not limited to, one or more of immunoblotting (Western Blot), Polymerase Chain Reaction (PCR), enzyme-linked immunosorbent assay (E L ISA), Immunohistochemistry (IHC) and Immunofluorescence (IF).
Compared with the prior art, the invention has the following advantages:
the invention constructs a drug screening model of transgenic zebra fish with Alzheimer's disease, uses juvenile fish as a research object, cultures the juvenile fish in a solution with a specific drug concentration, detects relevant biochemical indexes of Alzheimer's disease and researches brain pathological forms by analyzing behavior tracks of the juvenile fish, and preliminarily judges 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.
Drawings
FIG. 1 is a flow diagram of a screening technique;
wherein, 1, constructing transgenic zebra fish; 2. culturing a transgenic zebra fish embryo; 3. grouping and administering; 4. analyzing a behavior track; 5. researching brain pathological morphology; 6. detecting biochemical indexes; 7. data statistics and analysis processing;
FIG. 2 is a graph of the wild type control group (WT) and the 30s internal moving trajectory (A) and 30s internal moving hotspot (B) of the APPsw transgenic genome;
FIG. 3 is an electron microscopy micrograph of the wild type control (WT) (A, C) and APPsw transgenic (B, D) brain structures;
FIG. 4 is a graph showing the distance traveled in 1h for examples 1, 2, 3 and 4;
FIG. 5 is a graph comparing the average moving speed of examples 1, 2, 3 and 4 over 1 h;
FIG. 6 is a graph comparing the activity of acetylcholinesterase (AChE) for examples 1, 2, 3 and 4;
FIG. 7 is a graph comparing the activity of acetylcholine transesterification enzyme (ChAT) of examples 1, 2, 3 and 4;
FIG. 8 is a graph comparing the Malondialdehyde (MDA) content of examples 1, 2, 3 and 4;
FIG. 9 is a graph comparing the activity of superoxide dismutase (SOD) in examples 1, 2, 3 and 4.
Detailed Description
In order to clarify the technical solution and the outstanding advantages of the present invention, the following examples are given for further illustration. It should be understood that the specific examples are set forth herein for purposes of illustration only and are not intended to be limiting.
Example 1:
1.1 construction of transgenic Zebra fish
Constructing a target vector containing an APP gene rapture mutant (APPsw) sequence, and obtaining a transgenic zebra fish model with the gene fluorescently labeled through zebra fish embryo microinjection. Culturing the positive embryo (showing green fluorescence) to adult fish to obtain F0And (4) generation.
1.2 culture of transgenic Zebra fish embryos
F is to be0Mating female transgenic zebra fish with wild male zebra fish to obtain F1Selecting heterozygote by sequencing through a tail fin shearing machine, and selfing to obtain F2And (3) cutting tail fins, sequencing the progeny obtained by selfing, screening homozygotes, placing the homozygote zebra fish in a special fish preparation tank according to a ratio of male to female of 1:1 in the evening, separating the zebra fish by using a baffle plate, removing the baffle plate in the morning of the next day, collecting fertilized fish eggs, removing dead eggs and impurities, placing the fertilized fish eggs and impurities in a culture dish with the diameter of 80mm, controlling the density to be 25 eggs/dish, changing the solution every day by using system circulating water containing 0.5 mg/L methylene blue, keeping the water quality clean, and culturing the fertilized fish eggs in a constant-temperature culture box at 28.5 ℃ in a dark period to 6dpf (days after fertilization).
1.3 behavioral Trace analysis
At 7dpf, 12 zebra fish were randomly selected from wild type control group (WT) and APPsw transgenic genomes and placed in a 96-well plate, a Danio Vision zebra fish behavior trajectory analysis system was used, experimental condition parameters were set, the behavior trajectories of the zebra fish in each group were observed and analyzed, and quantitative analysis was performed using two parameters of movement distance within 1h and average movement speed within 1 h.
1.4 study of the pathological morphology of the brain
After behavior trajectory analysis, 3 samples are selected for each group, after anesthesia, the samples are quickly dissected on ice, fish brains are separated, and the fish brains are photographed under an electron microscope for ultrastructural pathological analysis.
1.5 Biochemical index detection
The WT group and the APPsw genome-transformed zebra fish brains are prepared into tissue homogenate, and the content or activity of acetylcholinesterase (AChE), acetylcholinesterase (ChAT), Malondialdehyde (MDA) and superoxide dismutase (SOD) is determined by adopting an E L ISA method.
1.6 data statistics and analysis processing
Statistics and data visualization were performed using GraphPad Prism 5 software, comparisons between groups were performed using one-factor analysis of variance, data were expressed as Mean ± standard deviation (Mean ± SD), and P <0.05 was statistically significant.
1.7 results of the experiment
By using a zebra fish behavior track tracking system, the swimming track of the zebra fish within 30s is counted, and the swimming track of the zebra fish in the APPsw transgenic group is obviously smaller than that of the WT group (figure 2A). Counting the hot zone patterns of zebra fish in 30s, wherein the more red indicates the longer the zebra fish stay in the area, it can be seen that the zebra fish in the APPsw group stay in the same area for a significantly longer time than the WT group (fig. 2B). Meanwhile, the moving distance (P <0.001) within 1h and the average moving speed (P <0.001) within 1h (FIG. 5) of the APPsw genome-transformed zebrafish were both significantly reduced.
The ultrastructure of the brain of the zebra fish is observed by using a transmission electron microscope, the nucleus of the neuron cell of the WT group of zebra fish is circular, the nuclear membrane is clear, and organelles such as mitochondria, endoplasmic reticulum and the like in cytoplasm are abundant (fig. 3A, C). The nucleus of the APPsw transgenic zebra fish neuron is irregular, and some neurons have cell contents which are disintegrated and damaged, endoplasmic reticulum is swollen and deformed, and more vacuoles.
Biochemical index detection shows that compared with the WT group, the activity of the APPsw genome-transformed zebrafish AchE is remarkably increased (P <0.001) (figure 6), the activity of ChAT is remarkably reduced (P <0.001) (figure 7), the MDA content is remarkably increased (P <0.05) (figure 8), and the SOD activity is remarkably reduced (P <0.001) (figure 9).
In conclusion, the APPsw transgenic zebra fish model is successfully constructed.
Example 2:
2.1 construction of transgenic Zebra fish
Constructing a target vector containing an APP gene rapture mutant (APPsw) sequence, and obtaining a transgenic zebra fish model with the gene fluorescently labeled through zebra fish embryo microinjection. The positive embryoCulturing embryo (showing green fluorescence) to adult fish, namely F0And (4) generation.
2.2 culture of transgenic Zebra fish embryos
F is to be0Mating female transgenic zebra fish with wild male zebra fish to obtain F1Selecting heterozygote by cutting tail fin sequencing, and selfing to obtain F2And 3, sequencing the offspring obtained by selfing through a tail fin shearing machine, screening homozygotes, placing the homozygote zebra fish into a special fish preparation tank according to the ratio of male to female being 1:1 in the evening, separating the zebra fish by using a baffle plate, taking out the baffle plate in the morning of the next day, collecting fertilized fish eggs, removing dead eggs and impurities, placing the fertilized fish eggs and the impurities in a culture dish with the diameter of 80mm, controlling the density to be 25 eggs/dish, changing the solution every day by using system circulating water containing 0.5 mg/L methylene blue, keeping the water quality clean, and culturing the fertilized fish eggs in a constant-temperature culture box at the temperature of 28.0 ℃ in a dark period regulation and control mode to reach 2dpf (days after fertilization).
2.3 grouping and administration
2dpf of zebrafish larvae were selected, and a wild type control group (WT), an APPsw transgenic genome and a huperzine A (HUPA) group were set, and 30 fish were placed in each group in a 6-well plate. Precisely weighing quantitative huperzine A with ten-thousandth analytical balance, preparing into 200nM and 800nM solution with zebra fish system circulating water, and filtering with 0.22 μm filter head. Wild type control (WT) and APPsw transgenic genomes were maintained in circulating water of zebrafish system and huperzine A (HUPA) groups were exposed to 200nM and 800nM drug solutions.
2.4 behavioral Trace analysis
The action time of the medicine is 3dpf-6dpf, half of the medicine liquid is changed every day, 12 zebra fishes are randomly selected from 200nM huperzine and 800nM concentration at 7dpf and placed in a 96-well plate, a Danio Vision zebra fish behavior trajectory analysis system is used, experiment condition parameters are set, behavior trajectories of all groups of zebra fishes are observed and analyzed, and quantitative analysis is carried out by using two parameters of moving distance within 1h and average moving speed within 1 h.
2.5 data statistics and analysis processing
Statistics and data visualization were performed using GraphPad Prism 5 software, comparisons between groups were performed using one-factor analysis of variance, data were expressed as Mean ± standard deviation (Mean ± SD), and P <0.05 was statistically significant.
2.6 results of the experiment
Compared with the APPsw transgenic group, the huperzine A at the concentration of 200nM and the huperzine A at the concentration of 800nM can obviously improve the moving distance (P <0.05) in 1h (figure 4) and the average movement speed (P <0.001) in 1h (figure 5) of the zebra fish, and the effect of improving the Alzheimer disease of the huperzine A at the concentration is proved.
Example 3:
3.1 construction of transgenic Zebra fish
Constructing a target vector containing an APP gene rapture mutant (APPsw) sequence, and obtaining a transgenic zebra fish model with the gene fluorescently labeled through zebra fish embryo microinjection. Culturing the positive embryo (showing green fluorescence) to adult fish to obtain F0And (4) generation.
3.2 culture of transgenic Zebra fish embryos
F is to be0Mating female transgenic zebra fish with wild male zebra fish to obtain F1Selecting heterozygote by cutting tail fin sequencing, and selfing to obtain F2And (3) performing sequencing on progeny obtained by selfing through a tail fin shearing device, screening homozygotes, placing the homozygote zebra fish into a special fish preparation tank according to a ratio of male to female being 1:1 in the evening, separating the zebra fish with a baffle plate, taking out the baffle plate in the morning of the next day, collecting fertilized fish eggs, removing dead eggs and impurities, placing the fertilized fish eggs and the impurities in a culture dish with the diameter of 80mm, controlling the density at 40 eggs/dish, changing the solution every day by using system circulating water containing 0.5 mg/L methylene blue, keeping the water quality clean, and performing dark period regulation culture in a constant-temperature culture box at the temperature of 28.0 ℃ until 2dpf (days after fertilization).
3.3 grouping and administration
2dpf of zebrafish larvae were selected, and a wild type control group (WT), an APPsw transgenic group and donepezil hydrochloride (DPZ) group were set, 30 per group, in a 6-well plate. Precisely weighing quantitative donepezil hydrochloride by a ten-thousandth analytical balance, preparing 2 mu M, 4 mu M and 8 mu M solutions by circulating water of a zebra fish system, and filtering by a 0.22 mu M filter head. Wild type control (WT) and APPsw transgenic genomes were maintained in systemic circulating water of zebrafish and donepezil hydrochloride groups were exposed to 2, 4 and 8 μ M drug solutions, respectively.
3.4 behavioral Trace analysis
The action time of the medicine is 3dpf-6dpf, half of the medicine liquid is changed every day, 12 zebra fishes are randomly selected from donepezil hydrochloride under the concentration of 2 μ M and 4 μ M and placed into a 96-well plate at 7dpf, a Danio Vision zebra fish behavior locus analysis system is used, experiment condition parameters are set, behavior loci of all groups of zebra fishes are observed and analyzed, and quantitative analysis is carried out by using two parameters of the moving distance within 1h and the average moving speed within 1 h.
3.5 Biochemical index detection
The method comprises preparing tissue homogenate of fish brain of zebra fish with donepezil hydrochloride concentration of 8 μ M, and determining the content or activity of acetylcholinesterase (AChE), acetylcholinesterase (ChAT), Malondialdehyde (MDA) and superoxide dismutase (SOD) by E L ISA method.
3.6 data statistics and analysis processing
Statistics and data visualization were performed using GraphPad Prism 5 software, comparisons between groups were performed using one-factor analysis of variance, data were expressed as Mean ± standard deviation (Mean ± SD), and P <0.05 was statistically significant.
3.7 results of the experiment
Compared with the APPsw transgenic genome, both the donepezil hydrochloride under the concentration of 2 mu M and the donepezil hydrochloride under the concentration of 4 mu M can obviously improve the moving distance (P <0.05) in 1h (figure 4) and the mean movement speed (P <0.001) in 1h (figure 5) of the zebrafish, and the donepezil hydrochloride under the concentration of 8 mu M can obviously reduce the activity of the AchE (P <0.001) (figure 6) and increase the activity of the ChAT (P <0.01) (figure 7), and meanwhile, the donepezil hydrochloride under the concentration has the action trends of reducing the MDA content and increasing the SOD activity (figures 8 and 9), thereby proving that the donepezil hydrochloride under the concentration has the effect of improving the Alzheimer disease.
Example 4:
4.1 construction of transgenic Zebra fish
Constructing a target vector containing an APP gene rare type mutant (APPsw) sequence, and performing microinjection on zebra fish embryos to obtain transgenic zebra fish with the gene fluorescently labeledA fish model. Culturing the positive embryo (showing green fluorescence) to adult fish to obtain F0And (4) generation.
4.2 culture of transgenic Zebra fish embryos
F is to be0Mating female transgenic zebra fish with wild male zebra fish to obtain F1Selecting heterozygote by sequencing through a tail fin shearing machine, and selfing to obtain F2And 3, performing sequencing on offspring obtained by selfing through tail fin shearing, screening homozygotes, placing the homozygote zebra fish in a special fish preparation tank according to a ratio of male to female of 2:1 in the evening, separating the zebra fish with a baffle plate, taking out the baffle plate in the morning of the next day, collecting fertilized fish eggs, removing dead eggs and impurities, placing the fertilized fish eggs and the impurities in a culture dish with the diameter of 90mm, controlling the density at 30 eggs/dish, changing the liquid with system circulating water containing 0.5 mg/L methylene blue every day, keeping the water quality clean, and performing non-light period regulation and control culture in a constant-temperature culture box at 28.5 ℃ until 2dpf (days after fertilization).
4.3 grouping and administration
2dpf of zebrafish larvae were selected, and wild type control (WT), APPsw subgenomic and memantine ammonioate (Mem) groups were set up at 30 in 6-well plates. A ten-thousandth analysis Tianping is adopted to accurately weigh quantitative memantine ammonium hydrochloride, zebra fish system circulating water is used for preparing 50 mu M and 100 mu M solutions, and a 0.22 mu M filter head is used for filtering. Wild type control (WT) and APPsw transgenic genomes were maintained in systemic circulating water of zebrafish and memantine hydrochloride groups were exposed to 50 μ M and 100 μ M drug solutions.
4.4 behavioral trajectory analysis
The action time of the medicine is 3dpf-6dpf, half of the medicine liquid is changed every day, 12 zebra fishes are randomly selected from memantine ammonium hydrochloride with the concentration of 50 μ M and 100 μ M at 7dpf and placed in a 96-well plate, a Danio Vision zebra fish behavior locus analysis system is used, experiment condition parameters are set, behavior loci of all groups of zebra fishes are observed and analyzed, and quantitative analysis is carried out by using two parameters of the moving distance within 1h and the average moving speed within 1 h.
4.5 Biochemical index detection
The method comprises preparing tissue homogenate from fish brain of zebra fish with memantine ammonium hydrochloride concentration of 50 μ M, and determining the content or activity of acetylcholinesterase (AChE), acetylcholinesterase (ChAT), Malondialdehyde (MDA) and superoxide dismutase (SOD) by E L ISA method.
4.6 data statistics and analysis processing
Statistics and data visualization were performed using GraphPad Prism 5 software, comparisons between groups were performed using one-factor analysis of variance, data were expressed as Mean ± standard deviation (Mean ± SD), and P <0.05 was statistically significant.
4.7 results of the experiment
Compared with the APPsw transgenic group, both the memantine hydrochloride at the concentration of 50 mu M and the memantine hydrochloride at the concentration of 100 mu M can obviously improve the moving distance (P <0.05) in 1h (figure 4) and the average movement speed (P <0.01) in 1h (figure 5) of the zebrafish, and the memantine hydrochloride at the concentration of 50 mu M can obviously reduce the activity of AchE (P <0.001) (figure 6) and increase the activity of ChAT (P <0.01) (figure 7) and has the action trends of reducing the MDA content and increasing the SOD activity (figures 8 and 9), thereby proving that the memantine hydrochloride at the concentration has the effect of improving the Alzheimer 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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| CN112870384A (en) * | 2021-02-04 | 2021-06-01 | 山东省科学院生物研究所 | Method for screening anti-Alzheimer's disease drugs based on behavior and biochemical indexes and drugs screened by method |
| CN114487315A (en) * | 2021-12-21 | 2022-05-13 | 苏州大学 | Method for screening drugs influencing biorhythm behaviors by using juvenile zebra fish |
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| CN108938667A (en) * | 2018-09-28 | 2018-12-07 | 山东省科学院生物研究所 | A kind of construction method of the alzheimer's disease model for drug screening |
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| CN112870384A (en) * | 2021-02-04 | 2021-06-01 | 山东省科学院生物研究所 | Method for screening anti-Alzheimer's disease drugs based on behavior and biochemical indexes and drugs screened by method |
| CN114487315A (en) * | 2021-12-21 | 2022-05-13 | 苏州大学 | Method for screening drugs influencing biorhythm behaviors by using juvenile zebra fish |
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