CN115044558B - Pig neuron and isolated culture method and application thereof - Google Patents

Abstract

The application relates to a pig neuron and a separation culture method and application thereof. The method for separating and culturing the pig neurons comprises the following steps: digesting pig brain to prepare dispersed neurons; and inoculating the neurons into a modified culture medium for culture, wherein the modified culture medium comprises a nerve cell basal medium, an additive with the mass percentage of 1% -3%, 1-3 mM L-glutamine and 15-25 mug/mL antibiotics, and the additive is a B-27 additive or a B-27 additive without an oxidant. The separation culture method can separate pig neurons and can be used for researching human neurodegenerative diseases, and the growth of neurites can be seen in the culture process.

Description

Porcine neuron and its isolation and culture method and application

technical field

The invention relates to the technical field of cell culture, in particular to a pig neuron and its separation and culture method and application.

Background technique

Neurodegenerative diseases are a group of diseases that cause nervous system dysfunction due to the gradual loss of neuronal structure or function or even death. Due to the loss of neurons or their myelin sheath, neurodegenerative diseases worsen over time, eventually leading to Neurological dysfunction. Common neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, etc. Except for a few familial neurodegenerative diseases with clear causative genes, the pathogenesis of most neurodegenerative diseases is still unclear, and effective treatments are lacking. The common pathological features of neurodegenerative diseases are damage and loss of neurons and intracellular protein aggregation. Therefore, the establishment of nerve cell models of such diseases will lay a solid foundation for exploring the pathogenic mechanism and treatment strategies of such diseases.

Microtubule-associated protein tau (microtubule-associated protein tau, referred to as tau protein), which plays an important role in the occurrence and development of neurodegenerative diseases, is the main microtubule-associated protein of mature neurons. Tau protein is a protein containing phosphate groups, but when the protein is hyperphosphorylated, it will lose its biological activity, and then produce various neuropathological phenomena. Neurodegenerative diseases associated with tau abnormalities include Alzheimer's disease, frontotemporal dementia, corticobasal ganglia syndrome, Parkinsonism, pure movement disorders with freezing of gait, and rare motor neuron symptoms or cerebellar ataxia.

The most common animal model of neurodegenerative disease is the mouse model, but there are considerable differences between rodents and primates. For example, the striatum is the most severely affected region in Huntington's disease, whereas the striatum of primates and large animals (such as pigs) consists of the caudate nucleus and putamen, and that of rodents (such as mice) The caudate and putamen of the body are indistinguishable; for another example, in brain structure and volume, the mouse brain has no gyri and its volume is far from that of the human brain. Moreover, studies have shown that in various neurodegenerative diseases, it is difficult for mouse models to simulate the phenotype and pathological characteristics similar to those of patients with the disease. For example, a large number of neurons will be lost and died in the brain of Huntington's disease patients, but no obvious neuron death can be seen in the mouse model. In addition, many drugs that have been shown to be effective in mouse experiments have been found to have no effect on patients in clinical trials, which illustrates the differences in neuropathology between rodent and human brains, and also reflects the effect on large mammals that are closer to humans. research needs.

In the large animal pig model, its brain structure is similar to that of the human brain, with a large number of gyri, and its volume is closer to that of the human brain. Among them, the porcine Huntington's disease model can not only show choreographic movement disorders similar to patients, but also have selective neuron death and striatal atrophy similar to patients. Moreover, the cardiovascular system, digestive system, skin, nutritional requirements, bone development and mineral metabolism of pigs are very similar to those of humans. In addition, the pig's size and docile habits allow for repeated sampling and various surgical procedures. So more scientists began to turn their attention to pigs, especially miniature pigs.

At present, the isolation and cultivation of neurons from rodents such as mice has been very mature, but for pig neurons, although they can be successfully isolated, they still cannot meet the needs of neurodegenerative disease research.

Contents of the invention

Based on this, it is necessary to provide a method for isolating and culturing pig neurons to make up for the defect that there is no method for isolating and culturing pig neurons that can be used for neurodegenerative disease research.

A method for isolating and culturing pig neurons, comprising the following steps:

Dispersed neurons were prepared by digesting porcine brains; and

The neurons are inoculated and cultured in the improved medium, wherein the improved medium includes nerve cell basal medium, B-27 additive with a mass percentage of 1% to 3%, and 1mM to 3mM L-glucose Aminoamide and 15μg/mL～25μg/mL antibiotics.

The method for isolating and culturing the above-mentioned pig neurons is to cultivate neurons from the cerebral cortex by adopting the neuron basal medium containing 1% to 3% of B-27 additive, 1mM to 3mM of L-glutamine and 15 to 25 μg/mL of antibiotics. Neurons can be cultured for more than 12 days, and the growth of neurites can be seen during the culture process, which can be used for the study of neurodegenerative diseases.

In one of the embodiments, the improved medium includes nerve cell basal medium, B-27 additive with a mass percent content of 5%-2%, 5mM-2.5mM L-glutamine and 15-20μg/ mL of antibiotics.

In one embodiment, the antibiotics include one or more of penicillin, streptomycin, amphotericin B and gentamicin.

In one embodiment, the step of preparing dispersed neurons includes: after digesting the pig brain, filtering and/or centrifuging the mixture containing cells to remove incompletely digested tissue pieces.

The pig neurons obtained by the above-mentioned method for separating and culturing pig neurons.

A method for preparing cells for studying neurodegenerative diseases, comprising the following steps:

Using the adeno-associated virus that expresses the tau mutant protein element to infect the porcine neurons obtained by the isolation and culture method of the above-mentioned porcine neurons, and using the improved medium to continue culturing the porcine neurons infected with the adeno-associated virus, to prepare Cells for studying neurodegenerative diseases.

In one of the embodiments, the tau mutein includes at least one of tau ^P301L , tau ^P301S , tau ^V377M , tau ^R406W and tau ^G272V .

In one of the embodiments, the adeno-associated virus is AAV9.

Cells prepared by the above method for preparing cells for studying neurodegenerative diseases.

A method for screening or identifying drugs for the treatment of neurodegenerative diseases, said method using the pig neurons prepared by the above-mentioned method for isolating and culturing pig neurons or the above-mentioned method for preparing cells for studying neurodegenerative diseases The obtained cells are used to screen or identify drugs for the treatment of neurodegenerative diseases.

Description of drawings

Fig. 1 is the morphology of the isolated neurons of Example 1 cultured for 2 days, 5 days, 8 days and 12 days;

Fig. 2 is the β-tubulin3 immunofluorescence staining result of the isolated neurons obtained in Example 1 cultured for 8 days;

Fig. 3 is the immunofluorescence staining result of the neuron after infecting AAV-Tau (P301L) virus in embodiment 1;

Fig. 4 is the morphology of the isolated neurons of Example 2 cultured for 2 days, 5 days, 8 days and 12 days;

Fig. 5 is the morphology of isolated neurons obtained in Example 3 after 2 days, 5 days, 8 days and 12 days of culture.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully below, and the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the present disclosure will be thorough and complete.

The terms "first", "second", etc. are used for descriptive purposes only and should not be construed as indicating or implying relative importance. The term "and/or" includes any and all combinations of one or more of the associated listed items. The term "optionally" means an illustration. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.

The term "neuron" means a neuron cell, which is the most basic structural and functional unit of the nervous system. Neurons are divided into two parts, the cell body and the processes. The cell body is composed of nucleus, cell membrane, and cytoplasm, and has the function of communicating and integrating input information and transmitting information. There are two types of protrusions: dendrites and axons. The dendrites are short and branched, and directly protrude from the cell body to form dendrites. Their function is to receive impulses from the axons of other neurons and transmit them to the cell body. The axon is long and has few branches. It is a slender protrusion with uniform thickness. It often arises from the axon mound. Its function is to receive external stimuli, integrate information and determine the characteristics of the outgoing signal by the cell, and then transmit it from the cell body to the axon. out. In addition to branching out from the axon, its terminal forms a dendrite-like nerve ending. The endings are distributed in certain tissues and organs, forming various nerve ending devices. Sensory nerve endings form various receptors; motor nerve endings are distributed in skeletal muscles to form motor terminals.

The term "synapse" refers to the structure by which impulses from one neuron travel to another neuron or to another cell in contact with each other. Synapse is the functional link between neurons and the key part of information transmission. Under an optical microscope, it can be seen that the axon terminal of a neuron has gone through multiple branches, and finally the end of each small branch expands into a cup or ball shape, called a synaptosome. These synaptosomes can make contact with the cell bodies or dendrites of multiple neurons, forming synapses. Observed under an electron microscope, it can be seen that this synapse is composed of three parts: the presynaptic membrane, the synaptic cleft, and the postsynaptic membrane.

One embodiment of the present application provides a method for isolating and culturing porcine neurons, the method for isolating and culturing comprises step S10 and step S20. specifically:

S10: Digest the pig brain to prepare dispersed neurons.

Specifically, digestion is to disperse the pig brains. Optionally, the digested pig brain can be at least one of pig cerebral cortex tissue, pig striatum, hippocampus and cerebellum. In an alternative specific example, the digested porcine brain is porcine cerebral cortex tissue. In one embodiment, the reagent used for digestion is 0.25% trypsin. It can be understood that, in other embodiments, the reagent used for digestion is not limited to 0.25% trypsin, and can also be other reagents. Further, the porcine brain is cut into pieces with a size of 0.5 mm ³ to 1.5 mm ³ for digestion; the digestion time is 12 min to 20 min.

Optionally, the pig brain is from a pig embryo. In some embodiments, the above method for isolating and culturing pig neurons further includes the step of obtaining pig brains. Optionally, the step of obtaining the pig brain includes: taking the uterus after anesthetizing the pregnant pig at about 50 days of pregnancy, cutting the uterus to take out the embryo; then cutting out the whole brain of the embryo and cutting a small piece of cortex; After placing a small piece of cortex in a cell culture dish containing cold sterile PBS, remove the meninges; transfer the removed meninges to a new cell culture dish containing cold PBS, and wash the remaining meninges to obtain a clean pig brain.

Further, the step of preparing dispersed neurons includes: after digesting the pig brain, filtering and/or centrifuging the mixture containing cells to remove incompletely digested tissue pieces.

In some embodiments, a tissue cell strainer is used to remove incompletely digested tissue pieces in the digested cell-containing mixture. Furthermore, the tissue cell filter has a pore size of 40 μm to 70 μm. Optionally, after filtering with a tissue cell filter with a pore size of 70 μm, the filtrate is filtered with a tissue cell filter with a pore size of 40 μm. First use a tissue cell filter with a larger pore size to filter larger tissue pieces, and then use a tissue cell filter with a smaller pore size for further filtration to further reduce incompletely digested tissue pieces.

In other embodiments, centrifugation is used to remove incompletely digested tissue pieces in the digested mixture containing cells. Optionally, the rotational speed of the centrifugation is 1000 rpm to 1400 rpm, and the centrifugation time is 3 min to 5 min.

In other embodiments, the filtrate is filtered through a tissue cell filter with a pore size of 40 μm, and finally the filtrate is centrifuged at 1200 rpm for 3 minutes to 5 minutes.

S20: Inoculate neurons in the improved medium and culture them.

Specifically, the improved medium includes nerve cell basal medium, additives with a mass percentage of 1%-3%, 1mM-3mM L-glutamine and 15-25μg/mL antibiotics, and the additives are B-27 additives Or B-27 additive without oxidizer. Although the traditional method of culturing porcine neurons can isolate porcine neurons, it cannot be cultured for a long time without neurite growth, which is difficult to meet the needs of neurodegenerative disease research. However, the inventors of the present application have found that the reason why pig neurons cannot be cultured for a long time may be that isolated pig neurons have insufficient energy and the processes of neurons cannot grow, and then die soon. Therefore, the above-mentioned isolation and culture method of pig neurons improves the culture medium of isolated neurons, and introduces additives (B-27 additives or B-27 additives without oxidant) on the basis of nerve cell basal medium. And L-glutamine, through the combination of additives and L-glutamine, it can improve neurite growth and prolong the culture time of neurons.

Neuronal Basal Medium is used to provide the necessary nutrients for the growth of neurons. Specifically, the nerve cell basal medium is a commercially available medium for culturing nerve cells. In one embodiment, the neurol base medium is Gibco's Neurolbasel, the product number of which is 21103049.

Supplements are used to maintain neuronal growth and activity. In some embodiments, the additive is B-27 additive. B-27 Supplement is a serum-free supplement. In one example, the B-27 additive is Gibco's 17504-044. In another embodiment, the additive is B-27 additive without antioxidant. Antioxidant-free B-27 Supplement is B-27 Supplement after removal of five antioxidants (vitamin E, vitamin E acetate, superoxide dismutase, catalase, and glutathione), designed for research Designed for the role of free radicals in aging, toxicity, apoptosis and chronic neurological diseases.

Antibiotics are used to prevent microbial contamination, such as bacteria and/or fungi, from preventing neurons from growing. Optionally, the antibiotics include one or more of penicillin, streptomycin, amphotericin B and gentamicin. In one embodiment, the antibiotics are penicillin and streptomycin. It is understood that in other embodiments, the antibiotics are not limited to the above.

Further, the improved medium includes nerve cell basal medium, B-27 additive with a mass percentage of 1.5% to 2%, 1.5mM to 2.5mM L-glutamine and 15μg/mL to 20μg/mL of antibiotics . In an optional specific example, the improved medium includes nerve cell basal medium, 2% by mass B-27 supplement, 2mM L-glutamine and 20 μg/mL of antibiotics. Further, the improved medium includes nerve cell basal medium, B-27 additive with a mass percentage of 1.8% to 2%, 1.5mM to 2mM L-glutamine and 18μg/mL to 20μg/mL of antibiotics .

Specifically, the inoculated neuronal cells were cultured in a 37° C. cell culture incubator, half of the medium was changed every day for the first three days, and the medium was changed every two days thereafter. In order to make the cells cleaner, lightly tap the surroundings of the culture dish before each liquid change, so that cell impurities and tissue fragments fall off the walls of the culture dish.

Specifically, the culture dish used for culturing neuronal cells is a cell culture dish coated with D-polylysine. In one of the embodiments, the preparation method of the D-polylysine-coated cell culture dish comprises the following steps: the coating solution contains D-polylysine at a final concentration of 100ug/ml, and each culture Add the corresponding volume of coating solution to the dish, add about 10ml of coating solution to a 10cm petri dish, add about 2ml/well of coating solution to a six-well plate, and add about 1ml/well of coating solution to a twelve-well plate. 37°C incubator overnight. Before using the coated culture dish, recover the coating solution, and rinse the culture dish three times with PBS without calcium and magnesium ions to wash away the residual coating solution.

It can be understood that the number of times of filtering in the above step S10 is not limited to one time, but may also be multiple times. Multiple filtrations can further reduce incompletely digested tissue clumps in the cell suspension. In addition, when filtering, the filter screen can also be washed with medium to make more cells in the filtrate. Of course, the medium here can be an improved medium or a basic medium for nerve cells.

The above method of isolation and culture of pig neurons can successfully isolate pig neurons and carry out primary culture. The culture time can reach more than 12 days, and the synapses have begun to grow well in about 3 to 4 days, and the axons have begun to grow well in about 8 days. The growth is extremely pronounced and the neurons are interconnected.

In addition, an embodiment of the present application also provides an isolated porcine neuron, which is cultured by the method for isolating and culturing porcine neurons in any of the above embodiments.

In addition, an embodiment of the present application also provides a method for preparing cells for studying neurodegenerative diseases, the preparation method comprising:

S100: using an adeno-associated virus containing an element expressing a tau mutant protein to infect the porcine neurons obtained by the isolation and culture method of the porcine neurons in any one of the above embodiments.

Tau protein is a microtubule-associated protein. The cellular function of tau protein in the normal brain is to bind to tubulin and promote its polymerization to form microtubules. The tau protein is a phosphate-containing protein. The tau protein in the normal human brain contains 2-3 phosphate groups per molecule, while the tau protein in the brain of Alzheimer's disease patients is abnormally hyperphosphorylated, and each molecule of the tau protein can contain 5-9 phosphate groups. a phosphate group and lose its normal biological function. Optionally, the tau mutein includes at least one of tau ^P301L , tau ^P301S , tau ^V377M , tau ^R406W and tau ^G272V . It should be noted that tau ^P301L refers to the mutation of P (proline) at the 301st position from the N-terminus of the human tau protein to L (leucine); tau ^P301S refers to the 301st position from the N-terminus of the human tau protein The P (proline) of the tau mutant protein was mutated to S (serine); the rest of the tau mutant proteins were deduced by analogy.

Adreno-Associated Virus (AAV) is a common human parvovirus with natural defects, no envelope and no pathogenicity. When AAV infects mouse neurons, the infection efficiency is low, and a large number of neurons die after infection. However, the porcine neurons obtained by the method for isolating and culturing pig neurons in any of the above embodiments are in good cell state and can resist the toxic effect of the virus itself. Further, the adeno-associated virus is AAV9. AAV9 has a specific infection efficiency for neurons, and the efficiency is high. The empty vector capable of expressing tau mutant protein is not limited to AAV, and may also be adenovirus or other viral vectors. Other viral vectors include, but are not limited to, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, herpetic stomatitis virus vectors, retrovirus vectors, and lentivirus vectors.

More specifically, the preparation method of the adeno-associated virus containing the tau mutein element is a routine method in the art. For example, it can be obtained by incubating a vector loaded with an element expressing tau mutant protein and related to packaging of adeno-associated virus.

In some embodiments, porcine neurons are infected with an adeno-associated virus containing an element expressing a tau mutein when there is significant synapse outgrowth. When there is obvious synapse growth, the growth of neurons tends to be stable, has a certain ability to withstand viruses, and will not die due to virus infection. In addition, when neurons are cultured until the axons are stable, the metabolism of the cells will slow down at this time, which will easily lead to low infection efficiency.

S200: Culture porcine neurons infected with adeno-associated virus, and prepare cells for studying neurodegenerative diseases.

Specifically, continue to use the above-mentioned modified medium to culture infected porcine neurons, and observe the changes in cell state and cell morphology.

It has been verified that the above-mentioned method for preparing cells for studying neurodegenerative diseases produces cells with obvious axonal rupture, which well simulates the pathological death characteristics of human neurons.

In addition, an embodiment of the present application also provides a cell for studying neurodegenerative diseases, the cell has obvious axonal rupture, and can well simulate the pathological death characteristics of human neurons. Specifically, the cells are prepared by the method for preparing cells for studying neurodegenerative diseases in any of the above embodiments.

In addition, an embodiment of the present application also provides a use of the above-mentioned cells for the study of neurodegenerative diseases or pig neurons cultured by the method of isolating and culturing pig neurons in any of the above-mentioned embodiments, and the use is for screening or identifying Drugs used to treat neurodegenerative diseases. Further, an embodiment of the present application also provides a method for screening or identifying drugs for the treatment of neurodegenerative diseases, the method uses the pig neurons cultured by the above-mentioned isolation and culture method of pig neurons or the above-mentioned pig neurons for research Cells prepared by the cell preparation method for neurodegenerative diseases are used to screen or identify drugs for treating neurodegenerative diseases.

specific embodiment

The following will be described in detail in conjunction with specific embodiments. The following examples do not include other components except unavoidable impurities unless otherwise specified. The reagents and instruments used in the examples are all conventional choices in the art unless otherwise specified. The experimental methods for which specific conditions are not indicated in the examples are implemented according to conventional conditions, such as the conditions described in literature, books or the method recommended by the manufacturer.

Example 1

1. Prepare the following materials the day before the experiment:

(1) D-polylysine-coated cell culture dishes: the coating solution is sterilized distilled water containing D-polylysine at a final concentration of 100 μg/mL; add a corresponding volume of coating to each culture dish Add about 10mL of coating solution to a 10cm petri dish, add 2mL/well of coating solution to a six-well plate, add 1mL/well of coating solution to a twelve-well plate, and incubate overnight at 37°C.

(2) Improved medium: Neurolbasel (Gibco, 15140-122) containing 2% B-27 (Gibco, 17504044), 2mM L-Glutamine (Gibco, 25030-081) and 20 μg/mL penicillin-streptomycin (Gibco, 15140-122) , 21103049) medium.

(3) Calcium and magnesium ion-free PBS containing 20 μg/mL penicillin-streptomycin double antibody;

(4) Two pointed tweezers, two straight tweezers, two curved tweezers and two scissors after autoclaving.

2. On the day of surgery, prepare as follows:

(1) Soak the scalpel, surgical scissors and tweezers in 75% alcohol;

(2) prepare anesthetic;

(3) Recover the coating solution, rinse the coated cell culture dish three times with PBS without calcium and magnesium ions, and wash away the residual coating solution;

(4) Prepare a 10cm cell culture dish, add 30mL PBS without calcium and magnesium ions, then prepare a 6cm cell culture dish, add 10mL PBS without calcium and magnesium ions, and place the above cell culture dish on ice.

After the above preparatory work is completed, surgical materials and cell separation and culture are carried out. The specific steps are as follows:

S1: After the pregnant pig is pregnant for 50 days, the pregnant pig is anesthetized and the uterus is taken out, and the uterus is opened to take out the embryo. Then cut out the whole brain of the embryo and cut a small piece of cerebral cortex, and place it in a 10cm cell culture dish containing cold sterile PBS, remove the meninges; then transfer the removed cortex to a new cold PBS containing In a cell culture dish, the residual meninges were cleaned and transferred to a new PBS-free cell culture dish, and then the clean cortex was cut into pieces of 1 mm ³ with scissors.

S2: Add 0.25% trypsin to the cerebral cortex fragments obtained in step S1, pipette evenly, and digest in a 37°C cell culture incubator for 15 minutes.

S3: Add 1 times the prepared modified medium to stop the digestion.

S4: Put a 70μm tissue cell filter into a 50mL centrifuge tube, wet it with the modified medium first, then place the digested tissue on the filter for filtration, and finally wash the filter once with the modified medium to obtain more Cell.

S5: Put a 40 μm tissue cell filter in a 50 mL centrifuge tube, wet it with the modified medium first, and then put the filtrate obtained in step S4 into the filter for filtration to further reduce incompletely digested tissue pieces.

S6: Centrifuge the filtrate in the 50 mL centrifuge tube at 1200 rpm for 5 min.

S7: Discard the supernatant and collect the cell pellet. Then, the modified medium was added to the pellet to resuspend the cells, counted under a microscope, and the cells were inoculated at an appropriate concentration of 1×10 ⁴ /cm ² in a cell culture dish coated with D-polylysine.

S8: The seeded cells were cultured in a 37°C cell incubator, and the medium was half changed every day for the first three days. Then change the fluid every two days. In order to make the cells more clean, lightly pat the surroundings of the culture dish before changing the medium each time to make the cell impurities and tissue fragments fall off the wall of the culture dish. The morphology of some cells during the culture process is shown in Figure 1. It can be seen from Figure 1 that there are clearly visible neurons on the 8th day of cell culture, and the cell bodies and axons are obvious.

In order to verify whether the cells we isolated are really neurons, we stained the neuron marker protein β-tubulin3, and the staining results are shown in Figure 2.

S9: After the cells were cultured for 4 days, when there was obvious synapse growth, they were infected with AAV-Tau (P301L) virus, and the cell state and cell shape changes were observed every day after virus infection. Wherein, the AAV-Tau (P301L) virus is an AAV containing tau mutant protein tau ^P301L expression element, purchased from Guangzhou Paizhen Biotechnology Co., Ltd.

S10: On the 12th day of cell culture, the virus-infected cells were taken for immunofluorescence staining identification, the infected neurons were labeled with P-tau, and all neurons were labeled with β-tubulin3. The results are shown in Figure 3. In Figure 3, "WT" is the neuron control group not infected with the virus, and "AAV-Tau" is the neuron group after the virus infection (the bottom two rows are parallel samples, both of which are the neuron group after the virus infection) .

As can be seen from Figure 3, the tau mutant protein is expressed, and the AAV-Tau (P301L) virus has a high infection efficiency, and it is found that the neurons transfected with the AAV-Tau (P301L) virus have obvious axonal rupture (Figure 3). Arrow), well simulated the pathological death characteristics of human neurons, indicating that the cells used to study neurodegenerative diseases were successfully constructed. This also shows that the cell model of tau disease can be successfully established in only 12 days by using the above method, which greatly shortens the pathological onset time of neurodegenerative disease cells, and thus greatly shortens the experimental period.

Example 2

The isolation and culture method of neurons in this example is roughly the same as that in Example 1, the difference is that the improved medium in this example does not contain B-27 additive, and the other components are the same as the improved medium in Example 1.

Partial cell morphology of neurons cultured in this example is shown in FIG. 4 . It can be seen from Figure 4 that the neurites cannot grow well, and at 12 days, the cells have aged and the neurites have shrunk.

Example 3

The method for isolating and culturing neurons in this example is roughly the same as that in Example 1, the difference being that the improved medium in this example does not contain L-glutamine, and the other components are the same as the improved medium in Example 1.

Partial cell morphology of neurons cultured in this example is shown in FIG. 5 . It can be seen from Fig. 5 that the growth of neuronal processes is slow, and the cells show the aging phenomenon of malnutrition.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, which are convenient for a specific and detailed understanding of the technical solution of the present invention, but should not be construed as limiting the protection scope of the invention patent. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. It should be understood that technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the patent for the present invention shall be determined by the content of the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims (3)

1. a method for preparing pig neurons for studying neurodegenerative diseases, characterized in that, comprising the following steps: Digest pig brains to prepare dispersed neurons; The step of preparing dispersed porcine neurons includes: after digesting the porcine brain, filtering and/or centrifuging the mixed solution containing porcine neurons to remove incompletely digested tissue pieces; and After the dispersed pig neurons were inoculated in the improved medium and cultured for 4 days, when there was obvious synapse growth, the pig neurons were infected with the adeno-associated virus AAV-Tau ^P301L containing the expression tau mutant protein element Tau ^P301L , and using the improved medium to continue culturing the pig neurons infected with the adeno-associated virus until the 12th day to prepare pig neurons for studying neurodegenerative diseases; The modified medium includes nerve cell basal medium, B-27 supplement with a mass percent content of 2%, 2mM L-glutamine and 20 μg/mL penicillin-streptomycin. 2. The porcine neurons obtained by the method for preparing porcine neurons for studying neurodegenerative diseases according to claim 1. 3. A method for screening or identifying drugs for the treatment of neurodegenerative diseases, characterized in that the method uses the porcine neuron according to claim 2 to screen or identify drugs for treating neurodegenerative diseases.