CN105878251B - The preparation method and its usage of animal model for parkinsonism - Google Patents
The preparation method and its usage of animal model for parkinsonism Download PDFInfo
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- CN105878251B CN105878251B CN201610257341.6A CN201610257341A CN105878251B CN 105878251 B CN105878251 B CN 105878251B CN 201610257341 A CN201610257341 A CN 201610257341A CN 105878251 B CN105878251 B CN 105878251B
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Abstract
The present invention relates to field of biomedicine technology, and in particular to a kind of preparation method and its usage of animal model for parkinsonism.The preparation method of animal model for parkinsonism in the present invention includes: (1) in every animal subcutaneous injection cannabinoid receptor antagonists, once a day, continuous 5~15 days；(2) animal specimen obtained in step (1) is carried out to the experiment of detection sports coordination；(3) brain tissue of animal is detected to get animal model for parkinsonism after taking step (2) to assess.The animal model of preparation method preparation in through the invention has modeling success rate high, the advantages of without other toxic side effects, and the animal model for parkinsonism obtained can be used for studying the mechanism of the pathogenesis of cannabinoid system and Parkinson's disease, the drug for delaying, treating Parkinson's disease is screened in turn, is had a good application prospect.
Description
Technical field
The present invention relates to field of biomedicine technology, specifically, the present invention relates to a kind of systems of animal model for parkinsonism
Preparation Method and application thereof.
Background technique
Parkinson's disease (Parkinson's disease, PD) is most common after being ill with black substance densification after Alzheimer
The denaturation of portion's dopamine (DA) serotonergic neuron, the neurodegenerative disease that missing is main pathological change, it is old at 65-69 years old
Disease incidence accounts for about 0.5%-1% in year crowd, and disease incidence can reach 1%-3% in 80 years old or more the elderly.Although close
Nian Lai, as that studies Parkinson's disease gos deep into, having some treatment methods can reduce certain symptoms of disease, but all reach
Less than the purpose thoroughly cured, therefore, deeper into research PD pathogenesis and treatment method it is necessary, and stability and high efficiency
Animal model be further investigate the disease premise.
At present there are mainly two types of the more generally acknowledged methods for establishing PD model: one is apply 6- hydroxyl dopamine (6-
OHDA nigrostriatum system is injected) to damage mould made by rat substantia nigra-corpus straitum system dopamine (DA) serotonergic neuron
Type, the modeling need 3 D stereo orientation injection, and technical operation is more demanding, exists compared with high failure rate；Another kind is with 1-
Methyl 4-phenyl -1,2,3,6- tetrahydropyridines (MPTP) prepare primate models and the C57B mouse PD models such as monkey, still
MPTP equally has the neurotoxicity of unrepairable to the mankind, and operation needs stringent security control.And both of which cannot
Lewy body (Lewy body) does not occur in the pathologic process of accurate simulation Parkinson's disease, such as MPTP model.Above-mentioned model
Existing shortcoming significantly limits the application of these models.It is dynamic there is an urgent need in the art to develop more reliable Parkinson's disease
Object model.
Summary of the invention
The object of the present invention is to provide a kind of preparation method and its usages of animal model for parkinsonism, solve existing skill
Modeling success rate is low in art, the insecure problem of model.
In order to solve the above technical problems, embodiments of the present invention provide a kind of preparation side of animal model for parkinsonism
Method, comprising the following steps: (1) in every animal it is subcutaneously injected cannabinoid receptor antagonists, once a day, continuous 5~15 days, i.e.,
Obtain animal specimen 1；(2) animal specimen 1 is subjected to the experiment of detection sports coordination to get animal specimen 2；(3) institute is taken
The brain tissue for stating animal specimen 2 is detected to get animal model for parkinsonism.
Another object of the present invention is to be to provide the purposes of the Parkinson disease model of above method preparation, wherein should
Parkinson disease model is used for research and the drug development of the pathologic process mechanism of cannabinoid system participation Parkinson's disease.
The present invention in terms of existing technologies, injects cannabinoid receptor antagonists in animal body, and animal is made pa occur
The gloomy sick sample symptom of gold, by detecting to obtain reliable and effective pa to Parkinson's disease sample animal progress Behavior evaluation and brain tissue
The gloomy sick animal model of gold, and improve the success rate of modeling.
Further, cannabinoid receptor antagonists are preferably BML-190, and BML-190 may pass through blood-brain barrier, pass through inhibition
CB2 receptor inhibits the activity of Parkinson's disease GAP-associated protein GAP Parkin/PINK1, inhibits PI3K/AKT signal transduction pathway, in turn
The death for aggravating dopaminergic neuron, makes animal the symptom of Parkinson's disease occur.
Further, in order to make the animal for being injected BML-190 the symptom of Parkinson's disease, the quality of BML-190 occur
Concentration is set as 20~40mg/kg.
Further, detect sports coordination experiment be turn-club test, revolving speed be 4~40rpm, the time be 200~
400s, record mouse fall the time, are repeated 3 times.
Further, animal brain tissue includes Substantia Nigra tissue, hippocampus tissue, brain stem district's groups are knitted, cortical area is organized,
The main brain area that Substantia Nigra tissue, hippocampus tissue, brain stem district's groups are knitted, cortical area tissue is Parkinson's disease change, takes above-mentioned tissue
It can preferably detect the lesion of Parkinson's lesion tissues following MCAO in rats.
Further, animal is non-human mammal.
Further, non-human mammal is selected from mouse, rat.
Further, the mouse is C57B mouse, and C57B mouse is often established with Parkinson disease model small for laboratory
Mouse.
Further, cannabinoid receptor antagonists are injected in the abdominal cavity position of C57B mouse, the drug that the present invention chooses
Injection site is abdominal cavity, mouse peritoneal portion have the advantages that inject range it is big, it is easy to operate, be easily achieved.
Detailed description of the invention
Fig. 1 is the reality that the turn-club test analysis BML-190 according to the present invention in embodiment one influences mice behavior
Test result figure；
Fig. 2 be according to the present invention the western blot analysis BML-190 in embodiment one to mouse effect back brain CB2 by
Body protein and the variation of Pakin protein expression.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing to each reality of the invention
The mode of applying is explained in detail.However, it will be understood by those skilled in the art that in each embodiment of the present invention,
In order to make the reader understand this application better, many technical details are proposed.But even if without these technical details and base
In the various changes and modifications of following embodiment, each claim of the application technical side claimed also may be implemented
Case.
The first embodiment of the present invention is related to a kind of preparation methods of animal model for parkinsonism, comprising the following steps:
(1) cannabinoid receptor antagonists are subcutaneously injected in every animal, once a day, continuous 5~15 days to get animal specimen 1；(2)
The animal specimen 1 is subjected to the experiment of detection sports coordination to get animal specimen 2；(3) brain of the animal specimen 2 is taken
Portion's tissue is detected to get animal model for parkinsonism.
As a kind of non-limiting embodiment, animal can be non-human mammal, be preferably selected from mouse, rat.
As a kind of non-limiting embodiment, cannabinoid receptor antagonists can be BML-190.
For example: a kind of preparation method of parkinsonian mouse model.
The C57B mouse of weight (0.02g) equalization is randomly divided into 4 groups, it may be assumed that A group, B group, C group, D group, every group is 12
The phosphate buffered saline solution (PBS) of 100 μ l is injected intraperitoneally in mouse, respectively A group mouse daily, is Normal group；B group mouse
The MPTP of 100 μ l is injected intraperitoneally in BML-190, C group mouse of 100 μ l of intraperitoneal injection daily daily；D group mouse is injected intraperitoneally daily
The MPTP of the BML-190 of 100 μ l and 100 μ l；Wherein, the time of BML-190 and MPTP injection is separated by 6h.After continuous injection 12 days
(note: daily regular time administration, wherein 9 points of injection MPTP of the morning, afternoon 3 points of injections BML-190, PBS).It is small by 4 groups
Mouse carries out turn-club test analysis, and the mouse that sports coordination ability in turn-club test is decreased obviously is considered as parkinsonian mouse mould
Type, wherein the modeling success rate of B group mouse is up to 92%.
In addition, claiming the weight of a mouse in experiment every four days, wherein the mouse of Parkinson's disease modeling group is more right than health
According to group weight low 8.9%, and observe mouse hair color.When experimental endpoints, mouse turn-club test carries out sports coordination ability analysis, will
The speed of transfer rod is trained from 4 modulations to 40 turns, by all mouse in advance in the rotating device to reach a stable behavior, record
Mouse can rest on the time on swingle, be repeated 3 times.(Parkinson's disease degree, pa are judged with the shortening that mouse falls the time
The gloomy disease of gold is more serious, and the locomitivity of mouse is poorer, and it is slower to react, i.e., residence time is shorter on the spinning device).
It is worth noting that, mouse residence time on transfer rod is to judge the goldstandard experiment of Parkinsonian symptoms,
As shown in Figure 1, BML-190 administration B group parkinsonian mouse transfer rod fall the time be substantially less than (P < 0.001) A group normally it is right
According to the level of group mouse, and to fall the time then lower for the D group mouse transfer rod of BML-190 joint MPTP administration, this illustrate CB2 by
Body antagonist can induce exacerbation parkinson symptom.
Influence for analysis BML-190 to mouse brain Neuron Apoptosis, in the present embodiment, by above-mentioned four groups of mouse
It puts to death, and the tissue in each area of the brains such as black substance, hippocampus, brain stem, cortex for taking four groups of mouse, extracts the total protein in each tissue
By Western blotting analysis detection dopamine correlative protein expression situation, as shown in Fig. 2, the mouse after BML-190 administration, suppression
The expression of Substantia Nigra and hippocampus CB2 has been made, the activity of Substantia Nigra and hippocampus Parkin/PINK1 is inhibited, has inhibited PI3K/AKT
Access illustrates that CB2 small molecular antagonists can induce Parkinson's disease in Pathological levels, it should be noted that the Control in Fig. 2
As PBS Normal group.
It will be understood by those skilled in the art that the respective embodiments described above are to realize specific embodiments of the present invention,
And in practical applications, can to it, various changes can be made in the form and details, without departing from the spirit and scope of the present invention.
Claims (6)
1. a kind of preparation method of animal model for parkinsonism, which comprises the following steps:
(1) cannabinoid receptor antagonists are subcutaneously injected in every animal, once a day, continuous 5~15 days to get animal specimen 1；
(2) animal specimen 1 is subjected to the experiment of detection sports coordination to get animal specimen 2；
(3) brain tissue of the animal specimen 2 is taken to be detected to get animal model for parkinsonism；
Wherein, the cannabinoid receptor antagonists are BML-190, and the mass concentration of the BML-190 is 20~40mg/kg；
The experiment of the detection sports coordination is turn-club test, and revolving speed is 4~40rpm, and the time is 200~400s, is recorded small
Mouse falls the time, is repeated 3 times.
2. the preparation method of animal model for parkinsonism according to claim 1, which is characterized in that the animal brain group
It is woven to Substantia Nigra tissue, hippocampus tissue, brain stem district's groups are knitted, cortical area tissue.
3. the preparation method of animal model for parkinsonism according to claim 1, which is characterized in that the animal is inhuman
Mammal.
4. the preparation method of animal model for parkinsonism according to claim 3, which is characterized in that the inhuman lactation is dynamic
Object is selected from mouse, rat.
5. the preparation method of animal model for parkinsonism according to claim 4, which is characterized in that the mouse is C57B
Mouse.
6. the preparation method of animal model for parkinsonism according to claim 5, which is characterized in that the Cannabined receptor
Antagonist is injected in the abdominal cavity position of the C57B mouse.
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| CN112931397B (en) * | 2021-01-25 | 2024-02-27 | 江南大学 | Construction method of parkinsonism animal model |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1809566A (en) * | 2003-04-23 | 2006-07-26 | 辉瑞产品公司 | Cannabinoid receptor ligands and uses thereof |
| CN1816536A (en) * | 2003-06-26 | 2006-08-09 | 武田药品工业株式会社 | Regulator of cannabinoid receptor |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1809566A (en) * | 2003-04-23 | 2006-07-26 | 辉瑞产品公司 | Cannabinoid receptor ligands and uses thereof |
| CN1816536A (en) * | 2003-06-26 | 2006-08-09 | 武田药品工业株式会社 | Regulator of cannabinoid receptor |
Non-Patent Citations (3)
| Title |
|---|
| Neuroprotective Potential of Adenosine A2A and Cannabinoid CB1 Receptor Antagonists in an Animal Model of Parkinson Disease;Silvia Cerri 等;《J Neuropathol Exp Neurol》;20140531;第73卷(第5期);第414-424页 |
| 内源性大麻素系统与帕金森病关系的研究进展;邹梓良 等;《广东医学》;20150531;第36卷(第9期);第1449-1451页 |
| 大麻素系统与帕金森病及其运动并发症关系的研究进展;马雅萍 等;《中国临床神经科学》;20081231;第16卷(第1期);第99-102页 |
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