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

Pig neuron and isolated culture method and application thereof Download PDF

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CN115044558B
CN115044558B CN202210698709.8A CN202210698709A CN115044558B CN 115044558 B CN115044558 B CN 115044558B CN 202210698709 A CN202210698709 A CN 202210698709A CN 115044558 B CN115044558 B CN 115044558B
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neurons
pig
culture
neuron
cell
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CN115044558A (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 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

Pig neuron and isolated culture method and application thereof
Technical Field
The application relates to the technical field of cell culture, in particular to a pig 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 of neuronal structure or function and even death, and worsen over time due to the loss of neurons or their myelin, 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. In addition to the definite causative genes of a few familial neurodegenerative diseases, the pathogenesis of most neurodegenerative diseases is not yet clear, and effective therapeutic means are lacking. A common pathological feature of neurodegenerative diseases is the injury and loss of neurons and intracellular protein aggregation. Therefore, establishing the nerve cell model of the diseases lays a firm foundation for exploring the pathogenesis, 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 main microtubule-associated protein of mature neurons. Tau protein is a protein containing phosphate groups, but when the protein is hyperphosphorylated, it loses its biological activity, and various neuropathological phenomena are generated. Neurodegenerative diseases associated with tau abnormalities include Alzheimer's disease, frontotemporal dementia, corticobasal ganglia 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 is considerable variability between rodents and primates. For example, striatum is the most affected region in huntington's disease, whereas the striatum of primates and large animals (e.g., pigs) consists of caudate and putamen, which are indistinguishable from the striatum of rodents (e.g., mice); as another example, in brain structure and volume, the mouse brain has no sulcus and the volume is far from that of the human brain. Moreover, studies have shown that in various neurodegenerative diseases, mouse models have difficulty in simulating well the phenotypic and pathological characteristics similar to those of the patients with the disease. For example, loss and death of a large number of neurons occurs in the brain of huntington's disease patients, but no significant neuronal death is seen in the mouse model. In addition, many drugs that demonstrated efficacy in mouse experiments, found no efficacy in clinical trials in patients, demonstrated neuropathological differences between rodents and human brains, and also represented the need for research in larger mammals that more closely resemble humans.
In a large animal pig model, the brain is similar to the human brain in structure, has a large number of furrows, and has a volume which is more similar to that of the human brain. Wherein the porcine huntington's disease model may not only exhibit a patient-like chorea movement disorder, but also exhibit a patient-like selective neuronal death and striatal atrophy. Moreover, the cardiovascular system, digestive system, skin, nutritional requirements, skeletal development, mineral metabolism, etc. of pigs are very similar to those of humans. In addition, the size and tame habit of pigs allows for repeated sampling and various surgical procedures to be performed. More scientists have begun to look at pigs, especially small pigs.
At present, neurons of rodents such as mice are very mature, but the neurons of pigs can be successfully isolated, but the requirements of research on neurodegenerative diseases cannot be met.
Disclosure of Invention
Based on this, it is necessary to provide a method for isolated culture of pig neurons to make up for the defect that there is no method for isolated culture of pig neurons that can be used for study of neurodegenerative diseases.
A method for isolated culture of porcine neurons, comprising the steps of:
digesting pig brain to prepare dispersed neurons; a kind of electronic device with high-pressure air-conditioning system
The neurons are inoculated in a modified culture medium for culture, wherein the modified culture medium comprises a nerve cell basal medium, a B-27 additive with the mass percentage of 1% -3%, 1 mM-3 mM L-glutamine and 15 mug/mL-25 mug/mL antibiotics.
The method for separating and culturing the pig neurons comprises the steps of culturing neurons from cerebral cortex by adopting a nerve cell basal medium containing 1% -3% of B-27 additive, 1-3 mM of L-glutamine and 15-25 mug/mL of antibiotics, wherein the culturing time can reach more than 12 days, and the growth of neurites can be seen in the culturing process, so that the method can be used for researching neurodegenerative diseases.
In one embodiment, the improved medium comprises a neural cell basal medium, 5 to 2 mass percent of B-27 additive, 5 to 2.5mM L-glutamine and 15 to 20 mug/mL antibiotic.
In one embodiment, the antibiotic comprises one or more of penicillin, streptomycin, amphotericin B, and gentamicin.
In one embodiment, the step of preparing the dispersed neurons comprises: after digestion of the pig brain, filtering and/or centrifuging the mixture containing cells to remove the incompletely digested tissue mass.
The pig neuron is obtained by culturing the pig neuron by the separation culture method.
A method of preparing a cell for studying a neurodegenerative disease, comprising the steps of:
the pig neuron obtained by the isolated culture method of the pig neuron is infected by the adenovirus containing the tau mutant protein expression element, and the pig neuron infected with the adenovirus is continuously cultured by adopting the improved culture medium to prepare the cell for researching the neurodegenerative disease.
In one embodiment, the tau mutein comprises tau P301L 、tau P301S 、tau V377M 、tau R406W And tau G272V At least one of them.
In one embodiment, the adeno-associated virus is AAV9.
The above-mentioned cell for studying neurodegenerative diseases.
A method for screening or identifying a drug for treating a neurodegenerative disease, the method comprising screening or identifying a drug for treating a neurodegenerative disease using a pig neuron produced by the above-described method for isolated culture of pig neurons or a cell produced by the above-described method for producing cells for studying a neurodegenerative disease.
Drawings
FIG. 1 shows the morphology of neurons isolated from example 1 cultured for 2 days, 5 days, 8 days and 12 days;
FIG. 2 shows the result of immunofluorescence staining of isolated neurons of example 1 by culturing for 8 days;
FIG. 3 shows immunofluorescent staining of neurons after infection with AAV-Tau (P301L) virus in example 1;
FIG. 4 shows the morphology of neurons isolated from example 2 cultured for 2 days, 5 days, 8 days and 12 days;
FIG. 5 shows the morphology of neurons isolated from example 3 cultured for 2 days, 5 days, 8 days and 12 days.
Detailed Description
The present application will be described more fully hereinafter in order to facilitate an understanding of the application, which may be embodied in many different forms and is 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 application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
The term "neuron" is a neuronal cell that is the most fundamental structural and functional unit of the nervous system. Neurons are divided into cell bodies and processes. The cell body consists of cell nucleus, cell membrane and cytoplasm, and has the functions of connecting and integrating input information and transmitting information. The protrusion has two types, namely dendrite and axon. Dendrites are short and branched, and directly expand and protrude from cell bodies to form dendrites, and the dendrites act to receive impulses from other neuron axons and transmit the impulses to the cell bodies. The axon is long and little branched, is a slender protrusion with uniform thickness, and usually plays a role of Yu Zhouqiu, and after receiving external stimulus, the characteristic of transmitting signals is determined by the cell through integrating information, and then the signals are transmitted from the cell body to the axon. The axons, except for the lateral branches, form branch-like nerve endings at their ends. The endings are distributed in certain tissue organs to form various nerve endings. Sensory nerve endings form various receptors; motor nerve endings are distributed in skeletal muscles, forming the final stage of movement.
The term "synapse" refers to a structure of mutual contact in which impulses of one neuron are transferred to another neuron or to another cell. Synapses are functionally related sites between neurons and are also key sites for information transfer. Under the light microscope, it can be seen that the axon terminal of a neuron passes through multiple branches, and finally the end of each branch is enlarged into a cup shape or a sphere shape, called synaptosome. These synaptosomes may contact the cell bodies or dendrites of multiple neurons to form synapses. From electron microscopy, it can be seen that this synapse is made up of three parts, the presynaptic membrane, the synaptic cleft and the postsynaptic membrane.
An embodiment of the present application provides a method for isolated culture of porcine neurons, comprising step S10 and step S20. Specifically:
s10: pig brains were digested to prepare dispersed neurons.
Specifically, digestion is to disperse the pig brain. Alternatively, the pig brain to be digested may be at least one of pig brain cortex tissue, pig striatum, hippocampus and cerebellum. In an alternative specific example, the pig brain that is digested is pig brain cortex tissue. In one embodiment, the digestion uses 0.25% pancreatin as the reagent. It will be appreciated that in other embodiments, the reagents employed for digestion are not limited to 0.25% pancreatin, but may be other reagents. Further, the pig brain was minced to 0.5mm 3 ～1.5mm 3 Digesting the large and small tissue blocks; the digestion time is 12 min-20 min.
Alternatively, the porcine brain is from a porcine embryo. In some embodiments, the above isolated culture method of pig neurons further comprises the step of obtaining pig brain. Optionally, the step of obtaining the pig brain comprises: taking uterus after pregnant pigs are anesthetized about 50 days after pregnant pigs are pregnant, and taking embryos out after splitting the uterus; then cutting a small cortex after dissecting the whole brain of the embryo; after placing the obtained small cortex in a cell culture dish containing cold sterile PBS, removing meninges; the meninged cortex was transferred to a new cell culture dish containing cold PBS and the residual meninges were washed to obtain clean pig brain.
Further, the step of preparing the dispersed neurons comprises: after digestion of the pig brain, the cell-containing mixture is filtered and/or centrifuged to remove pieces of tissue that are not completely digested.
In some embodiments, a tissue cell filter is used to remove pieces of tissue that are not completely digested from the digested mixture containing cells. Further, the pore size of the histiocyte filter is 40 μm to 70. Mu.m. Optionally, the filtrate is filtered by a tissue cell filter with a pore size of 70 μm after the filtration is performed by a tissue cell filter with a pore size of 40 μm. The tissue block with larger pore size is filtered by the tissue cell filter with smaller pore size, and then the tissue block is further filtered by the tissue cell filter with smaller pore size, so that the incompletely digested tissue block is further reduced.
In other embodiments, the non-digested tissue mass is removed from the digested cell-containing mixture by centrifugation. Optionally, the rotational speed of the centrifugation is 1000 rpm-1400 rpm, and the centrifugation time is 3 min-5 min.
In other embodiments, the filtrate is filtered using a tissue cell filter having a pore size of 70 μm, and the filtrate is filtered using a tissue cell filter having a pore size of 40 μm, and finally centrifuged at 1200rpm for 3min to 5min.
S20: neurons were inoculated in modified medium for culture.
Specifically, the improved culture medium comprises a nerve cell basal culture medium, an additive with the mass percent of 1% -3%, 1 mM-3 mM L-glutamine and 15-25 mug/mL antibiotics, wherein the additive is a B-27 additive or a B-27 additive without an oxidant. Although the traditional method for culturing the pig neurons can separate the pig neurons, the pig neurons cannot be cultured for a long time without protruding growth, and the research on neurodegenerative diseases is difficult to satisfy. The research of the inventor of the application shows that the reason why the pig neurons cannot be cultured for a long time is probably that the separated pig neurons have insufficient energy and the protrusions of the neurons cannot grow, so that the pig neurons die quickly. Therefore, the isolated culture method of the pig neurons improves the culture medium of the neurons after the isolated culture, introduces additives (B-27 additive or B-27 additive without oxidant) and L-glutamine on the basis of the basic culture medium of the neurons, and improves the neurite outgrowth and prolongs the culture time of the neurons through the cooperation of the additives and the L-glutamine.
The neural cell basal medium is used to provide necessary nutrients 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 embodiment, the neural cell basal medium is Gibco's Neurolbasel under the accession number 21103049.
The additive is used for maintaining neuron growth and activity. 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 17504-044 of Gibco. In another embodiment, the additive is an antioxidant-free B-27 additive. 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 specially designed for researching the effect of free radicals in aging, toxicity, apoptosis and chronic nervous system diseases.
Antibiotics are used to prevent the inability of neurons to grow due to microbial contamination by bacteria and/or fungi. Optionally, the antibiotic comprises one or more of penicillin, streptomycin, amphotericin B and gentamicin. In one embodiment, the antibiotics are penicillin and streptomycin. It is to be understood that in other embodiments, the antibiotics are not limited to the foregoing.
Further, the improved culture medium comprises a nerve cell basal culture medium, a B-27 additive with the mass percentage of 1.5% -2%, 1.5 mM-2.5 mM L-glutamine and 15 mug/mL-20 mug/mL antibiotics. In an alternative specific example, the modified medium comprises a neural cell basal medium, 2% by mass of B-27 supplement, 2mM L-glutamine, and 20 μg/mL antibiotic. Further, the improved culture medium comprises a nerve cell basal culture medium, a B-27 additive with the mass percent of 1.8% -2%, 1.5-2 mM L-glutamine and 18-20 mug/mL antibiotics.
Specifically, the inoculated neuron cells are cultured in a cell culture box at 37 ℃, and half liquid exchange is carried out every day for the first three days, and then liquid exchange is carried out every two days. In order to make the cells cleaner, the periphery of the culture dish can be gently tapped before each liquid change, so that cell impurities, tissue fragments and the like fall off from the wall of the culture dish.
In particular, the culture dish for culturing neuronal cells is a cell culture dish coated with D-polylysine. In one embodiment, the method of preparing a cell culture dish coated with D-polylysine comprises the steps of: the coating liquid is D-polylysine with the final concentration of 100ug/ml, the coating liquid with the corresponding volume is added into each culture dish, about 10ml of the coating liquid is added into the culture dish with 10cm, about 2 ml/hole of the coating liquid is added into the six-hole plate, and about 1 ml/hole of the coating liquid is added into the twelve-hole plate. Incubator at 37 ℃ overnight. Before using the coated dishes, the coating solution was recovered and the dishes were rinsed three times with calcium-magnesium ion free PBS, and the residual coating solution was washed clean.
It is to be understood that the number of times of filtering in the above step S10 is not limited to one but may be a plurality of times. Multiple filtration may further reduce the amount of incompletely digested tissue pieces in the cell suspension. In addition, during filtration, the filter screen may also be rinsed with media to allow for more cells in the filtrate. Of course, the medium may be modified medium or neural cell basal medium.
The method for separating and culturing the pig neurons can successfully separate the pig neurons and perform primary culture, the culture time can reach more than 12 days, synapses start to grow well about 3-4 days, axon growth is obvious about 8 days, and the neurons are connected with each other.
In addition, an embodiment of the application also provides an isolated pig neuron, which is obtained by culturing the pig neuron by the isolated culture method of any example.
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 culturing the porcine neurons of any of the above examples were infected with adeno-associated virus containing elements expressing tau mutant.
tau protein is a microtubule-associated protein. The cellular function of tau protein in normal brain is to bind to tubulin and promote its polymerization to form microtubules. tau protein is phospho-containing protein, and in normal human brain tau protein contains 2-3 phosphate groups per molecule, and in brain of Alzheimer disease patient tau protein is abnormally hyperphosphorylated, and per molecule tau protein can contain 5-9 phosphate groups, and its normal biological function is lost. Alternatively, the tau mutein comprises tau P301L 、tau P301S 、tau V377M 、tau R406W And tau G272V At least one of them. It should be noted that tau P301L Refers to the mutation of P (proline) at position 301 from the N-terminus of human tau protein to L (leucine); tau P301S Refers to the mutation of P (proline) at position 301 from the N-terminus of human tau protein to S (serine); the remaining tau muteins are analogized sequentially.
adeno-Associated Virus (AAV) is a common human parvovirus, naturally defective, non-enveloped and non-pathogenic. AAV infects mouse neurons, but the efficiency of infection is low and neurons die extensively after infection. The pig neuron prepared by the method for separating and culturing pig neurons of any embodiment has good cell state and can resist the toxic effect of viruses. Further, the adeno-associated virus is AAV9.AAV9 has specific infection efficiency to neurons and high efficiency. The empty vector that expresses tau muteins is not limited to AAV, but may be an adenovirus, or other viral vector. Other viral vectors include, but are not limited to: alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retrovirus vectors, and lentivirus vectors.
More specifically, methods for preparing adeno-associated viruses containing elements expressing tau muteins are routine in the art. For example, by incubating the vector loaded with the tau-expressing mutant protein element in association with the packaging of the adeno-associated virus.
In some embodiments, porcine neurons are infected with adeno-associated virus containing elements expressing tau muteins in the presence of significant synaptic growth. When there is obvious synaptic growth, the neuron growth tends to be stable, has a certain capacity of bearing virus, and does not die due to infection of the virus. In addition, when neurons are cultured until axons stabilize, the metabolism of cells is slowed down at this time, which tends to result in low infection efficiency.
S200: pig neurons infected with adeno-associated virus were cultured and cells for studying neurodegenerative diseases were prepared.
Specifically, the above-described modified medium was continuously used to culture the swine neurons after infection, and the cell state and cell morphology change were observed.
Proved by verification, the cell prepared by the preparation method for researching the cell of the neurodegenerative disease has obvious axon fracture, and well simulates the pathological death characteristics of human neurons.
In addition, an embodiment of the application also provides a cell for researching neurodegenerative diseases, which has obvious axon rupture phenomenon and can well simulate the pathological death characteristics of human neurons. Specifically, the cell is prepared by the preparation method of the cell for researching the neurodegenerative disease of any one of the above examples.
In addition, an embodiment of the present application also provides a use of the above-described cells for studying neurodegenerative diseases or the pig neurons cultured by the isolated culture method of pig neurons of any of the above examples for screening or identifying a drug for treating neurodegenerative diseases. Further, an embodiment of the present application provides a method for screening or identifying a drug for treating a neurodegenerative disease, which comprises culturing the obtained pig neuron or the cell prepared by the above method for preparing a cell for studying a neurodegenerative disease using the above isolated culture method of pig neuron, and screening or identifying a drug for treating a neurodegenerative disease.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following is a detailed description of specific embodiments. The following examples are not specifically described but do not include other components than the unavoidable impurities. Reagents and apparatus used in the examples, unless otherwise specified, are all routine choices in the art. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer.
Example 1
1. The following materials were prepared the day before the experiment:
(1) D-polylysine coated cell culture dishes: the coating liquid is sterilized distilled water containing D-polylysine with the final concentration of 100 mug/mL; coating liquid with corresponding volume is added into each culture dish, about 10mL of coating liquid is added into a 10cm culture dish, 2 mL/hole of coating liquid is added into a six-hole plate, 1 mL/hole of coating liquid is added into a twelve-hole plate, and the culture is carried out overnight at 37 ℃.
(2) Improving a culture medium: neurolbasel (Gibco, 21103049) medium containing 2% B-27 (Gibco, 17504044), 2mM L-Glutamine (Gibco, 25030-081) and 20. Mu.g/mL penicillin-streptomycin (Gibco, 15140-122).
(3) PBS containing 20 μg/mL penicillin-streptomycin diabody without calcium magnesium ions;
(4) The two forceps are respectively a sharp forceps, a straight forceps, a curved forceps and a scissors after high-pressure sterilization.
2. The day of surgery was prepared as follows:
(1) Immersing the scalpel, the surgical scissors and the forceps in 75% alcohol;
(2) Preparing a gunny;
(3) Recovering the coating liquid, and rinsing the coated cell culture dish for three times by using PBS without calcium and magnesium ions, and cleaning the residual coating liquid;
(4) A10 cm cell culture dish was prepared, 30mL of PBS free of calcium and magnesium ions was added, a 6cm cell culture dish was further prepared, 10mL of PBS free of calcium and magnesium ions was added, and the cell culture dish was placed on ice.
After the preparation is completed, the surgical material taking and the separation and the culture of cells are carried out, and the specific steps are as follows:
s1: after pregnant pigs are pregnant for 50 days, the pregnant pigs are anesthetized, the uterus is taken out, and the uterus is dissected to take out embryos. Then dissecting the whole brain from the embryo, cutting a small brain cortex, placing in a 10cm cell culture dish containing cold sterile PBS, and removing brainA membrane; the meninged cortex was then transferred to a new cell culture dish containing cold PBS, the residual meninges were washed clean and transferred to a new cell culture dish without PBS, and the clean cortex was then sheared with scissors to 1mm 3 Is a fragment of (c).
S2: adding 0.25% pancreatin into the cortex fragment obtained in step S1, blowing uniformly, and digesting for 15 min at 37deg.C in cell incubator.
S3: digestion was terminated by adding 1-fold of the prepared modified medium.
S4: the 70 μm tissue cell filter was placed in a 50mL centrifuge tube, wetted with modified medium, the digested tissue was placed in the filter for filtration, and the filter was rinsed once with modified medium to obtain more cells.
S5: the 40 μm tissue cell filter was placed in a 50mL centrifuge tube, wetted with the modified medium, and the filtrate obtained in step S4 was placed in the filter for filtration, further reducing incompletely digested tissue pieces.
S6: the filtrate in the 50mL centrifuge tube was centrifuged at 1200rpm for 5min.
S7: the supernatant was discarded and the cell pellet was collected. Then adding modified culture medium to the pellet to re-suspend the cells, counting under a microscope, and then adding the cells to a proper concentration of 1×10 4 /cm 2 Inoculated into a cell culture dish coated with D-polylysine.
S8: the seeded cells were cultured in a 37℃cell incubator, and half-changed every day for the first three days. After which the fluid was changed every two days. In order to make the cells cleaner, the periphery of the culture dish is gently tapped before each liquid change, so that cell impurities, tissue fragments and the like fall off from the wall of the culture dish, and part of the cell morphology in the culture process is shown in figure 1. As can be seen from FIG. 1, neurons, cell bodies and axons were clearly visible on day 8 of cell culture.
To verify whether our isolated cells were truly neurons, we stained the marker protein β -tubulin3 of neurons, the staining results are shown in fig. 2.
S9: after the cell culture was carried out for 4 days,when there was significant synaptic growth, AAV-Tau (P301L) virus was infected, and the cell status and cell morphology were observed daily after virus infection. Wherein the AAV-Tau (P301L) virus is Tau comprising Tau muteins P301L AAV for expression elements was purchased from guangzhou pie biotechnology limited.
S10: on day 12 of cell culture, virus-infected cells were identified by immunofluorescent staining, P-tau-labeled infected neurons, and beta-tubulin 3-labeled all neurons, and the results are shown in FIG. 3. In FIG. 3, "WT" is a control group of neurons that are not infected with virus, and "AAV-Tau" is a group of neurons after infection with virus (the two lower panels are parallel, both are groups of neurons after infection with virus).
As can be seen from FIG. 3, tau mutant protein is expressed, AAV-Tau (P301L) virus has high infection efficiency, and obvious axon rupture of neurons transfected with AAV-Tau (P301L) virus (arrow in FIG. 3) is found, which well simulates the pathological death characteristics of human neurons, and shows that the cell construction for researching neurodegenerative diseases is successful. This also demonstrates that the cell model of tau disease can be successfully established by using the method for only 12 days, which greatly shortens the pathological onset time of cells of neurodegenerative diseases, and further greatly shortens the experimental period.
Example 2
The method for isolating and culturing neurons of this example was substantially the same as in example 1, except that the modified medium of this example did not contain B-27 additive, and the other composition was the same as that of example 1.
A partial cell morphology of the neurons cultured in this example is shown in FIG. 4. As can be seen from fig. 4, the neuronal processes do not grow well and at day 12 the cells have aged and the processes atrophy.
Example 3
The method for isolating and culturing neurons of this example was substantially the same as in example 1, except that the modified medium of this example did not contain L-glutamine, and the other composition was the same as in example 1.
A partial cell morphology of the neurons cultured in this example is shown in FIG. 5. As can be seen from fig. 5, the neuronal processes grow slowly and the cells exhibit a dystrophic aging phenomenon.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. It should be understood that, based on the technical solutions provided by the present application, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims (3)

1. A method of preparing a porcine neuron for use in the study of neurodegenerative disease comprising the steps of:
digesting pig brain to prepare dispersed neurons;
the step of preparing the dispersed porcine neurons comprises: an operation of filtering and/or centrifuging the mixed liquor containing pig neurons to remove incompletely digested tissue pieces after digestion of the pig brain; a kind of electronic device with high-pressure air-conditioning system
When the dispersed pig neurons are inoculated in a modified culture medium for 4 days to culture, and obvious synapse growth exists, tau containing elements expressing Tau mutant protein are adopted P301L Adeno-associated virus AAV-Tau of (A) P301L Infecting the pig neurons and continuing the culture with a modified mediumRaising porcine neurons infected with the adeno-associated virus to day 12, preparing porcine neurons for studying neurodegenerative diseases;
the improved culture medium comprises a nerve cell basal medium, 2% of B-27 additive, 2mM of L-glutamine and 20 mug/mL of penicillin-streptomycin.
2. The porcine neuron of claim 1 prepared by the method for preparing porcine neuron for studying neurodegenerative disease.
3. A method of screening or identifying a drug for treating a neurodegenerative disease, wherein the method uses the porcine neuron of claim 2 to screen or identify a drug for treating a neurodegenerative disease.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6140116A (en) * 1994-11-08 2000-10-31 Diacrin, Inc. Isolated and modified porcine cerebral cortical cells
CN102978162A (en) * 2012-12-24 2013-03-20 黄柏胜 Neuron separation and culture method and reagent
CN102994452A (en) * 2012-12-24 2013-03-27 黄柏胜 Method for efficiently separating and culturing neurons
US9481864B1 (en) * 2015-06-30 2016-11-01 University Of South Florida Conversion of non-neuronal cells into neurons

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1795895A1 (en) * 2005-12-08 2007-06-13 KeyNeurotek AG A tissue-based assay system for Alzheimer-specific degeneration and pathology
JP7429404B2 (en) * 2019-08-06 2024-02-08 慶應義塾 Method for producing tau-related disease models

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6140116A (en) * 1994-11-08 2000-10-31 Diacrin, Inc. Isolated and modified porcine cerebral cortical cells
CN102978162A (en) * 2012-12-24 2013-03-20 黄柏胜 Neuron separation and culture method and reagent
CN102994452A (en) * 2012-12-24 2013-03-27 黄柏胜 Method for efficiently separating and culturing neurons
US9481864B1 (en) * 2015-06-30 2016-11-01 University Of South Florida Conversion of non-neuronal cells into neurons

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