CN115089613A - Application of neural stem cell transplantation in neurodegenerative diseases - Google Patents
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Abstract
The invention discloses an application of neural stem cell transplantation in neurodegenerative diseases, relates to the field of neural stem cell transplantation, and aims at the problem that the existing treatment mode has poor treatment effect on the neurodegenerative diseases when the existing neurodegenerative diseases are treated. The application of the neural stem cell transplantation in the neurodegenerative diseases is characterized in that the neural stem cells are extracted and cultured, and meanwhile, a treatment experiment is carried out by adopting a rat relative to the neural stem cells, so that the effect of the neural stem cells on the neurodegenerative diseases is judged, and the optimal implantation time is judged.
Description
Technical Field
The invention relates to the field of neural stem cell transplantation, in particular to application of neural stem cell transplantation in neurodegenerative diseases.
Background
Neurodegenerative diseases are caused by loss of neurons or myelin sheaths thereof, and deteriorate with time, so that dysfunction occurs, the neurodegenerative diseases comprise cerebral ischemic stroke, cerebral hemorrhage, Alzheimer disease, Parkinson disease and the like, and the treatment effect is poor when the neurodegenerative diseases are treated at present, and certain disadvantages exist.
Aiming at the problem that the existing treatment mode has poor treatment effect on the neurodegenerative diseases when the neurodegenerative diseases are treated at present, the application of neural stem cell transplantation in the neurodegenerative diseases is provided.
Disclosure of Invention
The application of neural stem cell transplantation in neurodegenerative diseases provided by the invention solves the problem that the existing treatment mode has poor treatment effect on the neurodegenerative diseases when the neurodegenerative diseases are treated at present.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of neural stem cell transplantation in neurodegenerative diseases comprises the culture of neural stem cells and the experiment of the neural stem cell transplantation in the neurodegenerative diseases, wherein the culture of the neural stem cells is used for extracting brain tissues in the brain of an SD rat, and the experiment of the neural stem cell transplantation in the neurodegenerative diseases is used for observing the effect of the neural stem cells in the neurodegenerative diseases;
the culture method of the neural stem cells comprises the following steps:
s1: taking brain tissue of a newborn SD rat, and fully rinsing the brain tissue by D-Hanks liquid, wherein the brain tissue needs to be extracted in a sterile environment;
s2: stripping off the meninges under a dissecting microscope, and separating the hippocampus;
s3: cutting the hippocampus by using an ophthalmic scissors, and transferring the hippocampus to the inside of a serum-free culture medium, wherein the serum-free culture medium is a mixed solution of DMEM/F12 culture medium, leukocyte antigen and basic fibroblast growth factor;
s4: preparing single cell suspension through mechanical separation, dyeing and counting single cells, meanwhile, placing the single cells in a culture plate for culture, and meanwhile, adding 500 mu L of cell suspension into the culture plate;
s5: until neurospheres formed in the plates, they were again mechanically isolated, clonally passaged, and so on, repeating the above steps continuously, and keeping the repetition interval for one week while collecting stem cell balls.
In a further preferred embodiment of the present invention, the number of times of rinsing in step S1 is three, the rinsing environment is a sterile environment, and the rinsing time is 10 min.
In a more preferred embodiment of the present invention, the mixed solution in the step S3 is prepared by mixing: DMEM/F12 medium: leukocyte antigen: basic fibroblast growth factor-2: 1: 1.
As a further preferable embodiment of the present invention, the cell suspension described in the above step S4 is DMEM/F12 medium containing 10% fetal bovine serum.
In a more preferred embodiment of the present invention, the density of the cell pellet in the step S5 is 5 × 104 cells/ml.
The application of the neural stem cell transplantation in the neurodegenerative diseases including cerebral ischemic stroke, cerebral hemorrhage, Alzheimer disease and Parkinson disease comprises the following steps that the experimental object is a rat which is adult and clean and has the body weight of 180-220 g.
As a further preferable scheme of the invention, the application in treating cerebral ischemic stroke comprises the following steps:
s1: taking two groups of rats, wherein the two groups of rats are in cerebral ischemia two-week state;
s2: implanting a group of rats with 24 μ L of neural stem cells at a concentration of 25000/μ L into ischemic lateral striatal region and cortex, and observing for 18 weeks;
s3: after 18 weeks the rats had returned to virtually pre-ischemic levels of sensory and motor disturbances;
s4: repeating the above steps to implant 24 μ L of neural stem cells with a concentration of 25000/. mu.L into the CA1 area of hippocampal striatum of another group of rats, and observing for 18 weeks;
s5: eighteen weeks later, 1% -3% of transplanted cells were found to survive for a long period in the rat hippocampal striatal CA1 region, with 3% -9% of the cells differentiating into neurons and the spatial cognitive function of the rat improved.
As a further preferred embodiment of the present invention, the application in treating cerebral hemorrhage comprises the following steps:
s1: taking out four groups of rats, wherein the first group is one week after cerebral hemorrhage, the second group is three weeks after cerebral hemorrhage, the third group is seven weeks after cerebral hemorrhage, and the fourth group is nine weeks after cerebral hemorrhage;
s2: implanting neural stem cells into bleeding areas of four groups of rats respectively, wherein the content of the implanted neural stem cells is 23000/mu L, the implantation amount is 20 mu L, and observing for two weeks;
s3: the survival rate of the implanted neural stem cells of the first group is 28%, the motor function of the rat is improved, the survival rate of the implanted neural stem cells of the second group is 67%, the motor function of the rat is obviously improved and effectively migrates to the lesion site, the survival rate of the implanted neural stem cells of the third group is 83%, the motor function of the rat is obviously improved and migrates to the lesion site in a large amount.
As a further preferred embodiment of the present invention, the application in treating Alzheimer disease comprises the following steps:
s1: taking two groups of rats, wherein the two groups of rats are rats with Alzheimer disease, implanting neural stem cells into brains of the rats, the implanted neural stem cells are 21000/mu L, the implantation amount is 25 mu L, and observing for one month;
s2: the implanted neural stem cells survive in frontal lobe and hippocampus in brain of rat, differentiate into neurons, and establish connection with host, and simultaneously cause increase of acetylcholinesterase fiber density, improve learning and memory ability of rat, and reduce deposition of senile plaque in hippocampus and formation of neuron fiber tangle.
As a further preferred embodiment of the invention, the application in treating Parkinson's disease comprises the following steps:
s1: taking two groups of rats, wherein the two groups of rats have Parkinson's disease, implanting 500 neural stem cells into the brains of the rats, implanting the neural stem cells once a week for two weeks, and observing for one month;
s2: the implanted rat has normal daily activities, improved motor function, improved myotonia and tremor, reduced complications and continuous degeneration of dopamine system.
The invention has the beneficial effects that:
the device judges the effect of the neural stem cells on neurodegenerative diseases and judges the optimal implantation time by extracting and culturing the neural stem cells and simultaneously adopting a relative rat to perform a treatment experiment.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
The invention provides the following technical scheme: the application of neural stem cell transplantation in neurodegenerative diseases comprises the culture of neural stem cells and the experiment of the neural stem cell transplantation in the neurodegenerative diseases, wherein the culture of the neural stem cells is used for extracting brain tissues in the brain of an SD rat, and the experiment of the neural stem cell transplantation in the neurodegenerative diseases is used for observing the effect of the neural stem cells in the neurodegenerative diseases;
the culture of the neural stem cells comprises the following steps:
s1: taking brain tissue of a newborn SD rat, and fully rinsing the brain tissue by D-Hanks liquid, wherein the brain tissue needs to be extracted in a sterile environment; rinsing for three times, wherein the rinsing environment is a sterile environment, and the rinsing time is 10 min;
s2: stripping off the meninges under a dissecting microscope, and separating the hippocampus;
s3: cutting the hippocampus by using an ophthalmic scissors, and transferring the hippocampus to the inside of a serum-free culture medium, wherein the serum-free culture medium is a mixed solution of DMEM/F12 culture medium, leukocyte antigen and basic fibroblast growth factor; the mixed solution comprises the following mixing ratio: DMEM/F12 medium: leukocyte antigen: basic fibroblast growth factor 2:1: 1;
s4: preparing single cell suspension through mechanical separation, dyeing and counting single cells, meanwhile, placing the single cells in a culture plate for culture, and meanwhile, adding 500 mu L of cell suspension into the culture plate; the cell suspension is DMEM/F12 culture medium containing 10% fetal bovine serum;
s5: until forming neurospheres in the culture plate, mechanically separating the neurospheres again, carrying out cloning passage, repeating the steps repeatedly and keeping the repetition interval for one week, and collecting stem cell spheres; the density of the cell balls was 5 x 104 cells/ml.
An application of neural stem cell transplantation in neurodegenerative diseases, wherein the neurodegenerative diseases comprise cerebral ischemic stroke, cerebral hemorrhage, Alzheimer disease and Parkinson disease, the experimental object is a rat, the rat is an adult female, a clean grade and the weight of the rat is 180-fold-220 g.
An application of neural stem cell transplantation in treating cerebral ischemic stroke comprises the following steps:
s1: taking two groups of rats, wherein the two groups of rats are in cerebral ischemia two-week state;
s2: implanting a group of rats with 24 μ L of neural stem cells at a concentration of 25000/μ L into ischemic lateral striatal region and cortex, and observing for 18 weeks;
s3: after 18 weeks the rats had returned to virtually pre-ischemic levels of sensory and motor disturbances;
s4: repeating the above steps to implant 24 μ L of neural stem cells with a concentration of 25000/. mu.L into the CA1 area of hippocampal striatum of another group of rats, and observing for 18 weeks;
s5: eighteen weeks later, 1% -3% of transplanted cells were found to survive for a long time in the hippocampal striatum CA1 region of the rat, wherein 3% -9% of the cells differentiated into neurons and the spatial cognitive function of the rat was improved;
by observing the behavioral expression of the rat, it can be seen that the implantation of corresponding neural stem cells into the hippocampal striatum CA1 region, the ischemic side striatum region and the cortex of the rat is beneficial to the treatment of cerebral ischemic stroke.
The application of neural stem cell transplantation in treating cerebral hemorrhage comprises the following steps:
s1: taking out four groups of rats, wherein the first group is one week after cerebral hemorrhage, the second group is three weeks after cerebral hemorrhage, the third group is seven weeks after cerebral hemorrhage, and the fourth group is nine weeks after cerebral hemorrhage;
s2: implanting neural stem cells into bleeding areas of four groups of rats respectively, wherein the content of the implanted neural stem cells is 23000/mu L, the implantation amount is 20 mu L, and observing for two weeks;
s3: the survival rate of the implanted neural stem cells of the first group is 28%, the motor function of the rat is improved, the survival rate of the implanted neural stem cells of the second group is 67%, the motor function of the rat is obviously improved and effectively migrates to the lesion site, the survival rate of the implanted neural stem cells of the third group is 83%, the motor function of the rat is obviously improved and migrates to the lesion site in a large amount;
as can be seen from the cell survival rate in the rat brain, the effect of implanting the neural stem cells after seven weeks of cerebral hemorrhage is the best.
The application of neural stem cell transplantation in treating Alzheimer disease comprises the following steps:
s1: taking two groups of rats, wherein the two groups of rats are rats with Alzheimer disease, implanting neural stem cells into brains of the rats of one group, the content of the implanted neural stem cells is 21000/mu L, the implantation amount is 25 mu L, and observing for one month;
s2: the implanted neural stem cells survive in frontal lobe and hippocampus in brain of rat, differentiate into neurons, establish connection with host, cause acetylcholinesterase fiber density to increase, improve learning and memory ability of rat, reduce deposition of senile plaque in hippocampus and formation of neuron fiber tangle;
as can be seen from the behavior of the rat implanted with the neural stem cells, the neural stem cells have certain effect on treating the Alzheimer disease.
The application of neural stem cell transplantation in treating Parkinson's disease comprises the following steps:
s1: taking two groups of rats, wherein both the two groups of rats have Parkinson's disease, implanting 400-500 neural stem cells into the brains of one group of rats, implanting once a week, continuously implanting for two weeks, and observing for one month;
s2: the implanted rat has normal daily activities, improved motor function, improved myotonia and tremor, reduced complications and continuous degeneration of dopamine system;
one month after the implantation of the neural stem cells in the rat, it can be seen that the neural stem cells have a certain effect on the treatment of the Parkinson's disease.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent alternatives or modifications according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.
Claims (10)
1. The application of neural stem cell transplantation in neurodegenerative diseases comprises the culture of neural stem cells and the experiment of the neural stem cell transplantation in neurodegenerative diseases, wherein the culture of the neural stem cells is used for extracting brain tissues in the brain of SD rats, and the experiment of the neural stem cell transplantation in neurodegenerative diseases is used for observing the effect of the neural stem cells in the neurodegenerative diseases;
the culture method of the neural stem cells comprises the following steps:
s1: taking brain tissue of a newborn SD rat, and fully rinsing the brain tissue by D-Hanks liquid, wherein the brain tissue needs to be extracted in a sterile environment;
s2: stripping off the meninges under a dissecting microscope, and separating the hippocampus;
s3: cutting the hippocampus by using an ophthalmic scissors, and transferring the hippocampus to the inside of a serum-free culture medium, wherein the serum-free culture medium is a mixed solution of DMEM/F12 culture medium, leukocyte antigen and basic fibroblast growth factor;
s4: preparing single cell suspension through mechanical separation, dyeing and counting single cells, meanwhile, placing the single cells in a culture plate for culture, and meanwhile, adding 500 mu L of cell suspension into the culture plate;
s5: until neurospheres formed in the plates, they were again mechanically isolated, clonally passaged, and so on, repeating the above steps continuously, and keeping the repetition interval for one week while collecting stem cell balls.
2. The use of claim 1, wherein the number of rinsing steps in step S1 is three, the rinsing environment is sterile, and the rinsing time is 10 min.
3. The use of claim 1, wherein the mixed solution of step S3 is prepared by mixing the following components in the following ratio: DMEM/F12 medium: leukocyte antigen: basic fibroblast growth factor 2:1: 1.
4. The use of claim 1, wherein the cell suspension of step S4 is DMEM/F12 medium containing 10% fetal bovine serum.
5. The use of the neural stem cell transplant of claim 1, wherein said density of said cytospheres in step S5 is 5 x 104 cells/ml.
6. The use of the neural stem cell transplantation of claim 1, in neurodegenerative diseases, wherein the neurodegenerative diseases include cerebral ischemic stroke, cerebral hemorrhage, Alzheimer disease and Parkinson disease, the experimental subjects are rats which are adult females and are clean and the body weight of the rats is 180-220 g.
7. The application of the neural stem cell transplantation in neurodegenerative diseases according to claim 6, is characterized in that the application in treating cerebral ischemic stroke comprises the following steps:
s1: taking two groups of rats, wherein the two groups of rats are in cerebral ischemia two-week state;
s2: implanting 24 μ L of neural stem cells at a concentration of 25000/. mu.L into an ischemic lateral striatum region and a cortex of a group of rats, and observing for 18 weeks;
s3: after 18 weeks the rats had returned to virtually pre-ischemic levels of sensory and motor disturbances;
s4: repeating the above steps to implant 24 μ L of neural stem cells with a concentration of 25000/. mu.L into the CA1 area of hippocampal striatum of another group of rats, and observing for 18 weeks;
s5: eighteen weeks later, 1% -3% of transplanted cells were found to survive for a long period in the rat hippocampal striatal CA1 region, with 3% -9% of the cells differentiating into neurons and the spatial cognitive function of the rat improved.
8. The use of the neural stem cell transplant in neurodegenerative disease as claimed in claim 6, wherein the use in treating cerebral hemorrhage comprises the following steps:
s1: taking out four groups of rats, wherein the first group is one week after cerebral hemorrhage, the second group is three weeks after cerebral hemorrhage, the third group is seven weeks after cerebral hemorrhage, and the fourth group is nine weeks after cerebral hemorrhage;
s2: implanting neural stem cells into bleeding areas of four groups of rats respectively, wherein the content of the implanted neural stem cells is 23000/mu L, the implantation amount is 20 mu L, and observing for two weeks;
s3: the survival rate of the implanted neural stem cells of the first group is 28%, the motor function of the rat is improved, the survival rate of the implanted neural stem cells of the second group is 67%, the motor function of the rat is obviously improved and effectively migrates to the lesion site, the survival rate of the implanted neural stem cells of the third group is 83%, the motor function of the rat is obviously improved and migrates to the lesion site in a large amount.
9. The use of the neural stem cell transplant in neurodegenerative disease according to claim 6, wherein the use in treating Alzheimer's disease comprises the following steps:
s1: taking two groups of rats, wherein the two groups of rats are rats with Alzheimer disease, implanting neural stem cells into brains of the rats, the implanted neural stem cells are 21000/mu L, the implantation amount is 25 mu L, and observing for one month;
s2: the implanted neural stem cells survive in frontal lobe and hippocampus in brain of rat, differentiate into neurons, and establish connection with host, and simultaneously cause increase of acetylcholinesterase fiber density, improve learning and memory ability of rat, and reduce deposition of senile plaque in hippocampus and formation of neuron fiber tangle.
10. The use of the neural stem cell transplantation of claim 6, in the treatment of Parkinson's disease, comprising the following steps:
s1: taking two groups of rats, wherein the two groups of rats have Parkinson's disease, implanting 500 neural stem cells into the brains of the rats, implanting the neural stem cells once a week for two weeks, and observing for one month;
s2: the implanted rat has normal daily activities, improved motor function, improved myotonia and tremor, reduced complications and continuous degeneration of dopamine system.
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