CN115044558A - Pig neuron as well as isolated culture method and application thereof - Google Patents

Pig neuron as well as isolated culture method and application thereof Download PDF

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CN115044558A
CN115044558A CN202210698709.8A CN202210698709A CN115044558A CN 115044558 A CN115044558 A CN 115044558A CN 202210698709 A CN202210698709 A CN 202210698709A CN 115044558 A CN115044558 A CN 115044558A
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neurons
tau
porcine
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CN115044558B (en
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闫森
李彩娟
李世华
李晓江
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Jinan University
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Abstract

The invention relates to a porcine neuron and a separation culture method and application thereof. The method for separating and culturing the porcine neurons comprises the following steps: digesting pig brain to prepare dispersed neurons; and inoculating the neurons into an improved culture medium for culturing, wherein the improved culture medium comprises a nerve cell basic culture medium, 1-3% of additive by mass, 1-3 mM of L-glutamine and 15-25 mug/mL of antibiotic, and the additive is B-27 additive or B-27 additive without oxidant. The separation culture method can separate the pig neuron and can see the growth of the neurite in the culture process, and can be used for the research of human neurodegenerative diseases.

Description

Pig neuron and isolated culture method and application thereof
Technical Field
The invention relates to the technical field of cell culture, in particular to a porcine neuron and a separation culture method and application thereof.
Background
Neurodegenerative diseases are a group of diseases that cause dysfunction of the nervous system due to gradual loss or even death of neuronal structure or function, which worsens over time due to loss of neurons or their myelin sheaths, ultimately leading to neurological dysfunction. Common neurodegenerative diseases include alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, and the like. Apart from the few familial neurodegenerative diseases with clear pathogenic genes, the pathogenesis of most neurodegenerative diseases is not clear, and an effective treatment means is also lacking. The common pathological features of neurodegenerative diseases are neuronal damage and loss and intracellular protein aggregation. Therefore, establishing the nerve cell model of the diseases lays a firm foundation for exploring the pathogenic mechanism, the treatment strategy and the like of the diseases.
Microtubule-associated protein tau (tau protein for short), which plays an important role in the development of neurodegenerative diseases, is the major microtubule-associated protein of mature neurons. Tau protein is a protein containing phosphate group, but when the protein is over-phosphorylated, the biological activity of the protein is lost, and various neuropathological phenomena are generated. Neurodegenerative diseases associated with tau protein abnormalities include Alzheimer's disease, frontotemporal dementia, corticobasal syndrome, Parkinson's syndrome, pure dyskinesia with frozen 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, while the striatum of primates and large animals (e.g., pigs) consists of the caudate nucleus and putamen, which are indistinguishable in rodents (e.g., mice); for another example, in brain structure and volume, the mouse brain has no sulcus and is far from the human brain in volume. Moreover, studies have shown that in various neurodegenerative diseases, it is difficult for mouse models to mimic well the phenotypic and pathological characteristics similar to those of the patients with the disease. For example, Huntington's chorea patients have a large number of neurons lost and dead, but no significant neuronal death was observed in the mouse model. In addition, many drugs that are therapeutically effective in mouse studies have been shown to be ineffective in clinical trials in patients, which suggests neuropathological differences between rodents and the human brain and also reflects the need for larger mammals that are more closely related to humans.
In a large animal pig model, the brain of the pig is similar to the structure of the human brain, has a large number of sulci and is closer to the volume of the human brain. Among them, the pig Huntington's disease model not only showed similar chorea-like dyskinesia but also exhibited similar selective neuronal death and striatal atrophy phenomena to the patients. Furthermore, the cardiovascular system, digestive system, skin, nutritional requirements, skeletal development, and mineral metabolism of pigs are very similar to those of humans. In addition, the size and habit of the pig allow for repeated sampling and various surgical procedures. More scientists have begun to look at pigs, especially small pigs.
At present, neurons from rodents such as mice are well-established for isolation and culture, but porcine neurons, although successfully isolated, do not meet the needs of neurodegenerative disease research.
Disclosure of Invention
Therefore, there is a need to provide a method for isolated culture of porcine neurons, so as to overcome the defect that no method for isolated culture of porcine neurons can be used for research of neurodegenerative diseases.
A method for separating and culturing porcine neurons comprises the following steps:
digesting pig brain to prepare dispersed neurons; and
inoculating the neurons into an improved culture medium for culturing, wherein the improved culture medium comprises a nerve cell basic culture medium, 1-3% of B-27 additive by mass, 1-3 mM of L-glutamine and 15-25 mug/mL of antibiotic.
The method for separating and culturing the porcine neurons cultures the neurons from the cerebral cortex by adopting the nerve cell basic culture medium containing 1-3% of B-27 additive, 1-3 mM of L-glutamine and 15-25 mug/mL of antibiotic, the culture time can reach more than 12 days, and the growth of neurites can be seen in the culture process, so that the method can be used for researching neurodegenerative diseases.
In one embodiment, the improved culture medium comprises a nerve cell basic culture medium, 5-2% of B-27 additive by mass, 5-2.5 mM of L-glutamine and 15-20 mu g/mL of antibiotic.
In one embodiment, the antibiotic comprises one or more of penicillin, streptomycin, amphotericin B, and gentamicin.
In one embodiment, the step of preparing discrete neurons comprises: an operation of filtering and/or centrifuging the mixture containing the cells to remove the incompletely digested tissue mass after digestion of the pig brain.
The pig neuron obtained by the separation culture method of the pig neuron is cultured.
A method for preparing a cell for studying a neurodegenerative disease, comprising the steps of:
and infecting the porcine neurons obtained by the isolated culture method of the porcine neurons by adopting the adeno-associated virus containing the tau mutein expression element, continuously culturing the porcine neurons infected with the adeno-associated virus by adopting the improved culture medium, and preparing the cells for researching the neurodegenerative diseases.
In one embodiment, the tau mutein comprises tau P301L 、tau P301S 、tau V377M 、tau R406W And tau G272V At least one of (1).
In one embodiment, the adeno-associated virus is AAV 9.
The cell prepared by the preparation method of the cell for researching neurodegenerative diseases.
A method for screening or identifying a drug for treating neurodegenerative diseases, which comprises using the porcine neurons obtained by the above method for isolated culture of porcine neurons or the cells obtained by the above method for preparation of cells for studying neurodegenerative diseases, and screening or identifying a drug for treating neurodegenerative diseases.
Drawings
FIG. 1 is a graph of the isolated neurons of example 1 cultured for 2 days, 5 days, 8 days, and 12 days;
FIG. 2 shows the immunofluorescence staining of β -tubulin3 with neurons isolated from example 1 cultured for 8 days;
FIG. 3 is the result of immunofluorescence staining of neurons after infection with AAV-Tau (P301L) virus in example 1;
FIG. 4 is a graph of the isolated neurons of example 2 cultured for 2 days, 5 days, 8 days, and 12 days;
FIG. 5 is a graph showing the morphology of the neurons isolated in example 3 cultured for 2 days, 5 days, 8 days, and 12 days.
Detailed Description
The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The terms "first," "second," and the like are used for descriptive purposes only and are not to 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 illustrative. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The term "neuron", i.e., a neuronal cell, is the most fundamental structural and functional unit of the nervous system. Neurons are divided into two parts, the cell body and the processes. The cell body consists of cell nucleus, cell membrane and cytoplasm and has the functions of communicating, integrating input information and transmitting information. There are both dendrites and axons protruding. The dendrites are short and have many branches, and the dendrites directly expand from the cell body to form dendrites, and the dendrites have the function of receiving impulses transmitted by axons of other neurons and transmitting the impulses to the cell body. Axons are long and have few branches, and are thin and uniform projections, often referred to as the axons, which act as information-integrated cellular-specific efferent signals that are transmitted from the cell body to the axons after receiving an external stimulus. Except for lateral branches, the axons have terminal branches that form branch-like nerve endings. The terminals are distributed in certain tissues and organs to form various nerve terminal devices. Sensory nerve endings form various receptors; the motor nerve endings are distributed in skeletal muscles to form the ultimate exercise.
The term "synapse" refers to a structure of mutual contact between an impulse from one neuron to another neuron or between another cell. Synapses are functionally related parts between neurons, and are key parts of information transmission. Under the optical microscope, the axon terminal of a neuron can be seen to pass through multiple branches, and finally, the terminal of each branch is enlarged to form a cup or a sphere, which is called a synaptosome. These synaptic bodies can contact the cell bodies or dendrites of multiple neurons, forming synapses. From the observation under an electron microscope, it can be seen that the synapse is composed of three parts, a presynaptic membrane, a synaptic cleft and a postsynaptic membrane.
An embodiment of the present application provides a method for isolated culture of porcine neurons, which includes steps S10 and S20. Specifically, the method comprises the following steps:
s10: digesting pig brain to prepare dispersed neuron.
In particular, digestion is to disperse the pig brain. Alternatively, the digested porcine brain may be at least one of porcine cerebral cortical tissue, porcine striatum, hippocampus, and cerebellum. In an alternative specific example, the digested porcine brain is porcine cerebral cortical tissue. In one embodiment, the reagent used for digestion is 0.25% pancreatin. It is understood that in other embodiments, the reagent used for digestion is not limited to 0.25% pancreatin, but may be other reagents.Further, the pig brain is cut into 0.5mm 3 ～1.5mm 3 Digesting the tissue blocks with the sizes; the digestion time is 12 min-20 min.
Optionally, the porcine brain is from a porcine embryo. In some embodiments, the above method for culturing porcine neurons further comprises the step of obtaining a porcine brain. Optionally, the step of obtaining a pig brain comprises: anaesthetizing pregnant pigs and taking out the uterus after the pregnant pigs are pregnant for about 50 days, dissecting the uterus and taking out embryos; then cutting out the whole brain of the embryo and cutting out a small cortex; placing the obtained small piece of cortex in a cell culture dish containing cold sterile PBS, and removing meninges; the meningoed cortex was transferred to a new cell culture dish containing cold PBS and the residual meninges were washed to obtain clean porcine brain.
Further, the step of preparing discrete neurons comprises: after digestion of the porcine brain, the cell-containing mixture is filtered and/or centrifuged to remove incompletely digested tissue pieces.
In some embodiments, a tissue cell filter is used to remove incompletely digested tissue pieces from the digested mixture containing cells. Furthermore, the pore size of the histiocyte filter is 40-70 μm. Optionally, after filtering with a 70 μm pore size tissue cell filter, the filtrate is filtered with a 40 μm pore size tissue cell filter. The tissue cell filter with larger pore diameter is adopted to filter larger tissue blocks, and then the tissue cell filter with smaller pore diameter is adopted to further filter, so that the tissue blocks which are not completely digested are further reduced.
In other embodiments, the incompletely digested tissue mass is removed from the digested mixture containing cells by centrifugation. Optionally, the rotation speed of the centrifugation is 1000rpm to 1400rpm, and the centrifugation time is 3min to 5 min.
In other embodiments, the tissue cell filter with a pore size of 70 μm is used for filtration, the filtrate is filtered by the tissue cell filter with a pore size of 40 μm, and finally the filtrate is centrifuged at 1200rpm for 3-5 min.
S20: the neurons are inoculated in the improved culture medium for culture.
Specifically, the improved culture medium comprises a nerve cell basic culture medium, 1-3% of an additive, 1-3 mM of L-glutamine and 15-25 mu g/mL of antibiotic in percentage by mass, wherein the additive is a B-27 additive or an oxidant-free B-27 additive. Although the traditional method for culturing the porcine neurons can separate the porcine neurons, the porcine neurons cannot be cultured for a long time and grow without processes, and the research on neurodegenerative diseases is difficult to satisfy. The research of the inventor of the application finds that the reason why the porcine neurons cannot be cultured for a long time is that the separated porcine neurons are insufficient in energy, and the protrusions of the neurons cannot grow and then die quickly. Therefore, the method for isolated culture of porcine neurons improves the culture medium for culturing the neurons after the separation, introduces additives (B-27 additives or B-27 additives without oxidizing agents) and L-glutamine on the basis of the nerve cell basal medium, and improves the growth of neurites and prolongs the culture time of the neurons by matching the additives and the L-glutamine.
The nerve cell basic culture medium is used for providing necessary nutrition for the growth of neurons. Specifically, the neural cell basal medium is a commercially available medium that can be used for culturing neural cells. In one example, the neural cell basal medium is Gibco's Neurolbasel, cat # 21103049.
The additive is used for maintaining the growth and activity of neurons. In some embodiments, the additive is a B-27 additive. The B-27 additive is a serum-free additive. In one embodiment, the B-27 additive is 17504-. In another embodiment, the additive is a B-27 additive free of antioxidants. The B-27 additive without antioxidant is an additive of the B-27 additive after removing five antioxidants (vitamin E, vitamin E acetate, superoxide dismutase, catalase and glutathione), and is designed for researching the effect of free radicals in aging, toxicity, apoptosis and chronic nervous system diseases.
Antibiotics are used to prevent the growth of neurons that are caused by microbial contamination, such as bacteria and/or fungi. Optionally, the antibiotic comprises one or more of penicillin, streptomycin, amphotericin B, and gentamicin. In one embodiment, the antibiotic is penicillin or streptomycin. It is understood that in other embodiments, the antibiotic is not limited to the above.
Further, the improved culture medium comprises a nerve cell basic culture medium, 1.5-2% of B-27 additive by mass percent, 1.5-2.5 mM of L-glutamine and 15-20 mug/mL of antibiotic. In an alternative embodiment, the modified medium comprises a neural cell basal medium, 2% by mass of B-27 supplement, 2mM L-glutamine, and 20 μ g/mL antibiotic. Furthermore, the improved culture medium comprises a nerve cell basic culture medium, 1.8 to 2 mass percent of B-27 additive, 1.5 to 2mM of L-glutamine and 18 to 20 mug/mL of antibiotic.
Specifically, the inoculated neuron cells are cultured in a cell culture box at 37 ℃, half liquid change is carried out every day for the first three days, and then liquid change is carried out every two days. In order to make the cells cleaner, the periphery of the culture dish can be flicked before liquid is changed each time, so that cell impurities, tissue fragments and the like fall off from the wall of the culture dish.
Specifically, the culture dish for culturing neuronal cells is a cell culture dish coated with D-polylysine. In one embodiment, the method for preparing a cell culture dish coated with D-polylysine comprises the following steps: the coating solution is D-polylysine with the final concentration of 100ug/ml, the corresponding volume of the coating solution is added into each culture dish, about 10ml of the coating solution is added into a 10cm culture dish, about 2 ml/hole of the coating solution is added into a six-hole plate, and about 1 ml/hole of the coating solution is added into a twelve-hole plate. Incubate at 37 ℃ overnight. Before using the coated culture dish, the coating solution is recovered, the culture dish is rinsed for three times by PBS without calcium and magnesium ions, and the residual coating solution is washed clean.
It is to be understood that the number of times of filtering in the above step S10 is not limited to one time, and may be multiple times. Multiple filtrations can further reduce incompletely digested tissue mass in the cell suspension. In addition, the filter mesh can be flushed with media during filtration to allow more cells in the filtrate. Of course, the medium may be a modified medium or a neural cell basic medium.
The separation culture method of the pig neurons can successfully separate the pig neurons and carry out primary culture, the culture time can reach more than 12 days, synapses already start to grow well in about 3-4 days, axon growth is very obvious in about 8 days, and the neurons are mutually connected.
In addition, an embodiment of the present application further provides an isolated porcine neuron, wherein the porcine neuron is cultured by the isolated culture method of the porcine neuron according to any one of the above embodiments.
In addition, an embodiment of the present application also provides a method for preparing a cell for studying a neurodegenerative disease, the method comprising:
s100: the porcine neurons obtained by the isolated culture method of infecting the porcine neurons of any of the above embodiments with adeno-associated virus containing elements expressing tau muteins are cultured.
tau protein is a microtubule-associated protein. The cellular function of tau in the normal brain is to bind tubulin and promote its polymerization to form microtubules. tau protein is phospho-group-containing protein, tau protein in normal human brain contains 2-3 phosphate groups per molecule, while tau protein in Alzheimer's disease patient brain is abnormally over-phosphorylated, and tau protein can contain 5-9 phosphate groups per molecule and lose normal biological function. Optionally, the tau mutein comprises tau P301L 、tau P301S 、tau V377M 、tau R406W And tau G272V At least one of (1). Note that tau is P301L Refers to the mutation of P (proline) at the 301 st position from the N end of the human tau protein into L (leucine); tau (tau) P301S P (proline) at position 301 from the N-terminus of human tau protein is mutated to S (serine); the rest of the tau muteins were analogized in turn.
adeno-Associated Virus (AAV) is a common human parvovirus, naturally defective, non-enveloped and non-pathogenic. AAV infects mouse neurons, but the infection is inefficient and there is a massive death of neurons after infection. The porcine neurons prepared by the isolated culture method of the porcine neurons of any embodiment have good cell states and can resist the toxic effects of the viruses. Further, the adeno-associated virus is AAV 9. AAV9 has specific infection efficiency on neuron, and high efficiency. The empty vector capable of expressing the tau mutein is not limited to AAV, but may be an adenovirus, or other viral vectors. Other viral vectors include, but are not limited to: an alphavirus vector, a herpesvirus vector, a measles virus vector, a poxvirus vector, an herpetic stomatitis virus vector, a retrovirus vector, and a lentivirus vector.
More specifically, the preparation of adeno-associated virus containing elements expressing tau muteins is a routine procedure in the art. For example, the tau mutein expression vector can be obtained by incubating the vector with the packaging related adenovirus.
In some embodiments, in the presence of significant synaptic growth, porcine neurons are infected with an adeno-associated virus containing an element that expresses a tau mutein. When obvious synapse growth exists, the neuron growth tends to be stable, the neuron has certain capacity of bearing viruses, and the neuron can not die due to the infection of the viruses. In addition, when neurons are cultured until axons are stable, the metabolism of cells is slowed, which tends to result in inefficient infection.
S200: culturing pig neuron infected with adeno-associated virus, and preparing cell for researching neurodegenerative disease.
Specifically, the infected porcine neurons were cultured with the improved medium described above, and the cell state and cell morphology changes were observed.
Proved by verification, the cells prepared by the preparation method for researching the neurodegenerative diseases have obvious axon breakage and well simulate the pathological death characteristics of human neurons.
In addition, the embodiment of the application also provides a cell for researching the neurodegenerative disease, the cell has obvious axon breakage phenomenon, and the pathological death characteristics of human neurons can be well simulated. Specifically, the cell is prepared by the preparation method of the cell for researching neurodegenerative diseases in any embodiment.
In addition, an embodiment of the present application further provides a use of the above cell for studying neurodegenerative diseases or the above isolated culture method of porcine neurons of any of the above embodiments for culturing porcine neurons, for screening or identifying a drug for treating neurodegenerative diseases. Further, an embodiment of the present application further provides a method for screening or identifying a drug for treating neurodegenerative diseases, the method using the porcine neurons cultured by the above method for isolated culture of porcine neurons or the cells prepared by the above method for preparing cells for studying neurodegenerative diseases, and screening or identifying a drug for treating neurodegenerative diseases.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except for inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.
Example 1
1. The day before the experiment the following materials were prepared:
(1) d-polylysine coated cell culture dishes: the coating solution is sterile distilled water containing D-polylysine with the final concentration of 100 mug/mL; the corresponding volume of coating solution was added to each dish, approximately 10mL of coating solution was added to a 10cm dish, 2 mL/well of coating solution was added to a six-well plate, 1 mL/well of coating solution was added to a twelve-well plate, and the incubator was incubated overnight at 37 ℃.
(2) Improving a culture medium: neurolbasesel (Gibco, 21103049) medium containing 2% B-27(Gibco, 17504044), 2mM L-Glutamine (Gibco, 25030-.
(3) Calcium-magnesium ion-free PBS containing 20 μ g/mL penicillin-streptomycin double antibody;
(4) the high-pressure sterilized sharp forceps, the straight forceps, the bent forceps and the scissors are respectively two.
2. The day of surgery was prepared preoperatively as follows:
(1) soaking a scalpel, surgical scissors and tweezers in 75% alcohol;
(2) preparing an anesthetic;
(3) recovering the coating solution, rinsing the coated cell culture dish for three times by using PBS (phosphate buffer solution) without calcium and magnesium ions, and washing out the residual coating solution;
(4) a10 cm cell culture dish was prepared, 30mL of PBS free from calcium and magnesium ions was added, a 6cm cell culture dish was prepared, 10mL of PBS free from calcium and magnesium ions was added, and the cell culture dish was placed on ice.
After the preparation work is finished, surgical material taking and cell separation and culture are carried out, and the method specifically comprises the following steps:
s1: after the pregnant pig is pregnant for 50 days, the pregnant pig is anesthetized, the uterus is taken out, and the uterus is dissected to take out the embryo. Cutting out whole embryo brain, cutting a small cerebral cortex, placing in a 10cm cell culture dish containing cold sterile PBS, and removing meninges; then the meningeal cortex was transferred to a new cell culture dish containing cold PBS, the residual meninges were washed clean and transferred to a new PBS-free cell culture dish, and the clean cortex was cut to 1mm with scissors 3 Fragments of (a).
S2: 0.25% pancreatin was added to the cortical fragment obtained in step S1, and the mixture was whipped to uniformity and digested in a cell culture chamber at 37 ℃ for 15 minutes.
S3: digestion was stopped by adding 1-fold prepared modified medium.
S4: a70 μm tissue cell filter was placed in a 50mL centrifuge tube and first wetted with modified media, then the digested tissue was placed in the filter for filtration and finally the filter was washed once with modified media to obtain more cells.
S5: a40 μm tissue cell filter was placed in a 50mL centrifuge tube, first wetted with modified media, and then the filtrate from step S4 was placed in the filter for filtration to further reduce incompletely digested tissue pieces.
S6: the filtrate in a 50mL centrifuge tube was centrifuged at 1200rpm for 5 min.
S7: abandonAnd (4) supernatant and collecting cell precipitates. The cells were then resuspended by adding modified media to the pellet, counted under a microscope, and the cells were aligned at the appropriate concentration of 1X 10 4 /cm 2 And inoculating the cells in a cell culture dish coated by D-polylysine.
S8: the cultured cells were cultured in a cell culture chamber at 37 ℃ and half-fluid changes were made every day for the first three days. The solution was changed every two days. In order to make the cells cleaner, the periphery of the culture dish is lightly tapped before liquid is changed each time, so that cell impurities, tissue fragments and the like fall off from the wall of the culture dish, and the shape of part of cells in the culture process is shown in figure 1. As can be seen from FIG. 1, neurons, cell bodies and axons were clearly visible at day 8 of cell culture.
To verify whether the cells we isolated were truly neuronal, we stained the neuronal marker β -tubulin3, the results of which are shown in fig. 2.
S9: after 4 days of cell culture and when there was significant synaptic growth, AAV-Tau (P301L) virus was infected, and cell status and cell morphology changes were observed daily after virus infection. Wherein the AAV-Tau (P301L) virus contains Tau mutant protein Tau P301L AAV for expression elements was purchased from Peizhou Pachy Biotechnology, Inc.
S10: on day 12 of cell culture, virus-infected cells were identified by immunofluorescence staining, with P-tau labeling of infected neurons and β -tubulin3 labeling of all neurons, as shown in figure 3. In FIG. 3, "WT" is a neuron control group not infected with a virus, and "AAV-Tau" is a neuron group after infection with a virus (the two lowermost rows are parallel samples, and both are neuron groups after infection with a virus).
As can be seen from FIG. 3, Tau mutein is expressed, AAV-Tau (P301L) virus has high infection efficiency, and significant axonal breakage (arrow in FIG. 3) of neurons transfected with AAV-Tau (P301L) virus is found, which well simulates pathological death characteristics of human neurons, indicating successful cell construction for studying neurodegenerative diseases. This also shows that the cell model of tau disease can be successfully established only in 12 days by adopting the method, thus greatly shortening the pathological attack time of cells of neurodegenerative diseases and further greatly shortening the experimental period.
Example 2
The neuron isolation culture method of this example is substantially the same as that of example 1, except that the modified medium of this example does not contain the B-27 additive, and the composition is otherwise the same as that of the modified medium of example 1.
The cell morphology of a part of the neurons cultured in this example is shown in FIG. 4. As can be seen from fig. 4, the neuronal processes did not grow well and at 12 days, the cells had aged and the processes shriveled.
Example 3
The method for isolated culture of neurons in this example is substantially the same as in example 1, except that the modified medium of this example does not contain L-glutamine, and the composition is otherwise the same as that of the modified medium of example 1.
The cell morphology of a part of the neurons cultured in this example is shown in FIG. 5. As can be seen from fig. 5, the neurite outgrowth was slow and the cells showed the phenomenon of undernourished aging.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, which is convenient for specific and detailed understanding of the technical solutions of the present invention, but the present invention should not be construed as being limited to the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.

Claims (10)

1. A pig neuron separation culture method is characterized by comprising the following steps:
digesting pig brain to prepare dispersed neurons; and
inoculating the neurons into an improved culture medium for culturing, wherein the improved culture medium comprises a nerve cell basic culture medium, an additive with the mass percentage of 1-3%, 1-3 mM of L-glutamine and 15-25 mug/mL of antibiotic, and the additive is a B-27 additive or a B-27 additive without an oxidant.
2. The isolated culture method according to claim 1, wherein the modified culture medium comprises a nerve cell basal medium, 1.5-2% by mass of B-27 additive, 1.5-2.5 mM of L-glutamine, and 15-20 μ g/mL of antibiotic.
3. The isolated culture method of claim 1, wherein the antibiotic comprises one or more of penicillin, streptomycin, amphotericin B, and gentamicin.
4. The isolated culture method according to any one of claims 1 to 3, wherein the step of preparing the scattered neurons comprises: an operation of filtering and/or centrifuging the mixture containing the cells to remove the incompletely digested tissue mass after digestion of the pig brain.
5. The porcine neuron cultured by the isolated culture method of the porcine neuron according to any one of claims 1 to 4.
6. A method for preparing a cell for studying a neurodegenerative disease, comprising the steps of:
infecting the porcine neurons obtained by the isolated culture method of the porcine neurons according to any one of claims 1 to 4 with an adeno-associated virus containing element for expressing tau mutein, and continuously culturing the porcine neurons infected with the adeno-associated virus with the improved culture medium to prepare cells for studying neurodegenerative diseases.
7. The method of claim 6, wherein the tau mutein comprises tau P301L 、tau P301S 、tau V377M 、tau R406W And tau G272V At least one of (1).
8. The method of claim 7, wherein the adeno-associated virus is AAV 9.
9. The cell according to any one of claims 6 to 8, which is obtained by the method for producing a cell for studying a neurodegenerative disease.
10. A method for screening or identifying a drug for treating neurodegenerative disease, which comprises screening or identifying a drug for treating neurodegenerative disease using the porcine neurons obtained by the isolation culture method for porcine neurons according to any one of claims 1 to 4 or the cells obtained by the preparation method for cells for studying neurodegenerative disease according to any one of claims 6 to 8.
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