CN109620971A - A method of screening prevention and treatment anti-parkinson drug - Google Patents
A method of screening prevention and treatment anti-parkinson drug Download PDFInfo
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Abstract
The present invention relates to field of biomedicine technology, and in particular to a method of screening prevention and treatment anti-parkinson drug.It there is now more mature animal model for studying the pathogenesis and therapeutic strategy of Parkinson's disease, but the detection on the relevant molecule basis after the completion of animal model and evaluation method need to be established.The method of screening prevention and treatment anti-parkinson drug provided by the invention includes the foundation of animal model and the foundation of GAP-associated protein GAP detection method, and the animal model is to prepare inclined side PD mouse model using 6-OHDA；The GAP-associated protein GAP detection is the variable quantity with Western blot and qPCR detection Dopamine In Striatum correlation factor；With the variation of TH and Iba1 in immuno-fluorescence assay corpus straitum；The dopamine correlation factor of the detection includes Nurr1, DAT, Pitx3, TH.The method of this screening prevention and treatment anti-parkinson drug provides set of experiment flow method for screening prevention and treatment anti-parkinson drug or operation method, and researcher is facilitated to carry out correlative study.
Description
Technical field
The present invention relates to field of biomedicine technology, and in particular to a method of screening prevention and treatment anti-parkinson drug.
Background technique
Parkinson's disease (Parkinson ' sdisease, PD) is a kind of common nervous system degeneration disease, and the elderly is more
See, average age of onset is 60 years old or so.The illness rate of China over-65s crowd PD is about 1.7%.Most of Parkinson
Patient is Sporadic cases, and only the patient less than 10% has family history.The most important pathological change of Parkinson's disease is that midbrain is black
The denaturation of matter dopamine (dopamine, DA) serotonergic neuron is dead, causes striatum DA level conspicuousness to reduce therefrom final
Lead to disease.Cause the definite cause of disease of this pathological change still unclear at present, inherent cause, environmental factor, age are old
The denaturation that change, oxidative stress etc. can induce PD dopaminergic neuron is dead.
With the increase of Parkinson's disease disease incidence, the drug research for preventing and treating Parkinson's disease becomes the research heat of modern medicine
Point, but relevant medicaments sifting model or method of evaluating drug effect also lack very much, existing Parkinson disease model is common
One is application 6- hydroxyl dopamine (6-OHDA) injection nigrostriatum systems to damage rat substantia nigra-corpus straitum system DOPA
Model made by amine (DA) serotonergic neuron, this model is using relatively broad, but subsequent evaluation process also lacks very much, evaluation side
Method is usually the behavior expression for visually observing experimental animal, and the detection in terms of related biological lacks.
Summary of the invention
In view of the above shortcomings of the prior art, the purpose of the present invention is to provide a kind of screening prevention and treatment anti-parkinson drugs
Method, screening technique of the invention includes building and the experiment detection correlation factor variable quantity of parkinsonian animal models, selected
The PD relevant evaluation factor include Nurr1, DAT, Pitx3, TH, Iba1, pass through Westernblot, PCR and immunofluorescence technique inspection
The content of above-mentioned correlation factor is surveyed, to evaluate drug effectiveness.
In order to achieve the above object, the technical scheme is that
A method of screening prevention and treatment anti-parkinson drug, comprising the following steps:
1. preparing inclined side PD mouse model using 6-OHDA；
2. the variation of Westernblot and RT-PCR detection Dopamine In Striatum correlation factor expression quantity；
3. the variation of TH and Iba1 in Immunofluorescence test corpus straitum.
Further, the step of above-mentioned application 6-OHDA prepares inclined side PD mouse model is as follows:
P of Rats D model is established by nigro-striatal access locating injection 6-OHDA, the injection volume of 6-OHDA is every 4
μl.Postoperative intraperitoneal injection penicillin is anti-infective, and continuous one week.After postoperative 2 weeks, intraperitoneal injection APO induces circling behavior.Injection
After 10min, manual count SD rat rotating cycle records 30min altogether.Mean speed >=7r/min person is defined as the PD positive, even
Continuous 3 weeks positive then to think modeling successfully.
Further, above-mentioned dopamine correlation factor includes Nurr1, DAT, Pitx3, TH.
Further, 2. RT-PCR detection Dopamine In Striatum correlation factor the primer sequence is as follows for above-mentioned steps:
The primer sequence of Nurr1 gene:
F:5 '-AAGCCACCTTGCTTGTACCAAA-3 ',
R:5 '-CTTGTAGTAAACCGACCCGCTG-3 '；
The primer sequence of TH gene:
F:5 '-CAGGGCTGCTGTCTTCCTAC-3 ',
R:5 '-GGGCTGTCCAGTACGTCAAT-3 '；
The primer sequence of DAT gene:
F:5 '-TTGCAGCTGGCACATCTATC-3 ',
R:5 '-ATGCTGACCACGACCACATA-3 '；
The primer sequence of Pitx3 gene:
F:5 '-CTTAGTCCCTGCCAGTACGC-3 ',
R:5 '-GTGAGCCAAGGGTGAATTG-3 '.
Further, the method for above-mentioned screening prevention and treatment anti-parkinson drug, rat administration occur 1. to apply 6- in step
OHDA is prepared before or after inclined side PD mouse model.
The utility model has the advantages that
(1) method of screening prevention and treatment anti-parkinson drug provided by the invention is also built while carrying out animal model experiment
The research approach for having found molecular biology mechanism detection GAP-associated protein GAP, supplements the deficiency of existing research means；
(2) GAP-associated protein GAP after detecting animal model in the method for this screening prevention and treatment anti-parkinson drug includes Nurr1,
TH, Pitx3, DAT, Iba1, the occurrence and development that above-mentioned albumen is all found in existing research with Parkinson's disease are related, to it
Expression quantity detection for evaluate effect of drugs more convincing；
(3) present invention utilizes Westernblot, RT-PCR and immunofluorescence techniques as needed to Nurr1, TH,
The expression variable quantity of Pitx3, DAT and Iba1 are detected, by compared with control group, judging drug to be measured or operation to be measured
The effect of method, it is more accurate that multi-method detection judges the result of this method；
(4) method of this screening prevention and treatment anti-parkinson drug can be used for evaluating controlling for oral medicine, injection medicine or treatment operation
Therapeutic effect, use scope are extensive.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with implementation of the invention
Example, technical scheme in the embodiment of the invention is clearly and completely described.Based on the embodiments of the present invention, this field
Those of ordinary skill's every other embodiment obtained without making creative work, belongs to protection of the present invention
Range.
Albumen used in embodiment:
Nurr1: nuclear receptor associated protein 1 is the member of Intracellular transcription factor nuclear receptor family, is maintaining brain DOPA
Key effect is played in amine energy system.The mutation of gene disease relevant to dopaminergic dysfunction is related, including pa gold
Gloomy disease, schizophrenia and manic-depressive psychosis.The protein is considered most important to the development of dopamine phenotype in midbrain.
DAT:dopaminetransport, Dopamine Transporter are located at presynaptic body, reuptake neurotransmitter DOPA
Amine maintains the harmony of synaptic dopamine validity and dopaminergic neuron.
Pitx3: inflammatory factor 3 plays pass in substantia nigra of midbrain dopamine (DA) serotonergic neuron terminal differentiation and existence maintain
Key effect, it is many studies have shown that Pitx3 mononucleotide diversity (SNP) is related with Parkinson's disease (PD).
TH:Tyrosinehydroxylase, tyrosine hydroxylase is the key enzyme of dopamine biosynthetic pathway, and PD is
A kind of neurodegenerative disease as caused by nigrostriatal dopamine wretched insufficiency, the Expression modulation of TH PD development and control
There is important role in treatment.
Iba1: it is specific expressed in macrophage/microglia, it is huge necessary to the film corrugation of M-CSF induction
Phagocyte/microglia specific actin crosslinking protein.
Embodiment one: a method of screening prevention and treatment anti-parkinson drug
The foundation of 1.PD rat model
Male SD rat is selected to carry out the foundation of model, referring to " the rat brain stereotactic figure of professor Paxinos chief editor
Spectrum " determine injection position: first injection position: 0.7mm before bregma, middle line right 3.0mm, Subdural space 4.5mm；Second point injection
Position: 0.2mm after bregma, middle line right 2.6mm, Subdural space 6.0mm.Tip position is adjusted, carries out sphenotresia using dental burr,
With 10 μ l micro syringes, two predetermined target spots of intracerebral are injected separately into 6-OHDA, every 4 μ l to the right.Postoperative intraperitoneal injection mould
Element is anti-infective, and continuous one week.After postoperative 2 weeks, intraperitoneal injection APO (apomorphine) induces circling behavior.After injecting 5-10min,
Manual count SD rat rotating cycle, records 30min altogether.Mean speed >=7 turn/min person is defined as the PD positive, selects PD positive
Rat continues drug administration or operative treatment.Every group 6, randomly select 6 only injecting normal saline as negative control.
2 drug administrations to be measured carry out treatment operation
Drug to be measured is administered according to its property, and curative drug drug treatment, preventive medicine after modeling success are being made
Drug treatment before mould.Treatment operation such as cell transplantation can carry out after modeling success.
Observation after 3 treatments
3w, 6w, 9w and 12w APO are injected intraperitoneally the rat after modeling and induce rotation, record rotation time after transplanting
Number.
The variation of DA correlation factor in 4 detection corpus straitums
After terminating experiment as needed, respectively with Nurr1, DAT, Pitx3 between Westernblot and RT-PCR detection each group
And the variation of TH expression, above-mentioned factor expression up-regulation then show that drug to be measured plays the role of alleviating PD.
Total serum IgE (every group of n=is extracted from tissue using TRIzol common reagent (TiangenBiotech, BeiJing, China)
4), and complementary DNA (cDNA) is synthesized.Nurr1, TH, Pitx3, DAT use following primer amplification cDNA segment:
The primer sequence of Nurr1 gene:
F:5 '-AAGCCACCTTGCTTGTACCAAA-3 ',
R:5 '-CTTGTAGTAAACCGACCCGCTG-3 '；
The primer sequence of TH gene:
F:5 '-CAGGGCTGCTGTCTTCCTAC-3 ',
R:5 '-GGGCTGTCCAGTACGTCAAT-3 '；
The primer sequence of DAT gene:
F:5 '-TTGCAGCTGGCACATCTATC-3 ',
R:5 '-ATGCTGACCACGACCACATA-3 '；
The primer sequence of Pitx3 gene:
F:5 '-CTTAGTCCCTGCCAGTACGC-3 ',
R:5 '-GTGAGCCAAGGGTGAATTG-3 '.
RT-PCR reaction condition are as follows: 94 DEG C, 3 minutes, 35 circulations；94 DEG C, 45 seconds；58 DEG C, 45 seconds；72 DEG C, 1 minute；
Finally extend 72 DEG C, 10 minutes.After reaction, PCR product is taken to analyze through 1% agarose gel electrophoresis.To target gene with
The mRNA of reference gene GAPDH carries out real-time PCR analysis.
The total protein of the striatum for the treatment of group and control rats is extracted with RIPA lysis buffer,
Westernblot method detects Nurr1, TH, Pitx3, the albumen variable quantity of DAT, using β-actin as internal reference reference protein, to egg
White testing result is analyzed.
The variation of 5 Immunofluorescence test corpus straitum TH and Iba1
After terminating experiment as needed, 10% chloraldurate intraperitoneal injection of anesthesia SD rat, through physiological saline and 4% poly
Brain is taken after formaldehyde perfusion, carries out frozen section after saccharose gradient dehydration.The brain section made passes through fixation, permeable membrane, closed
Journey, is added rabbit-anti TH (1:200, Abcam) and 4 DEG C of the anti-Iba1 of mouse (1:200, Abcam) is incubated overnight.Next day sucks respectively
State primary antibody, after PBS develops a film 3 times, corresponding fluorescence secondary antibody be added and is incubated for 2h, after developed a film 3 times using PBS, be added dropwise and contain DAPI
Mountant after covered mounting, just setting fluorescence microscopy under the microscope and take pictures.
6 interpretations of result
Above-mentioned experimental result is for statistical analysis, and the APO intraperitoneal injection for being respectively compared treatment group and control rats is made
The change of Nurr1, DAT, Pitx3 and TH expression of the number of revolutions after rat, Westernblot and RT-PCR detection after mould
Change and Immunofluorescence test corpus straitum TH and Iba1 variation, after treatment rat occur number of revolutions reduction, Nurr1, DAT,
Pitx3 and TH expression quantity increase and TH positive cell increased significantly, and Iba1 positive cell reduce phenomena such as, then show this to
It surveys drug or treatment means is effectively, to can be developed further into the drug for the treatment of PD.
Claims (5)
1. a kind of method of screening prevention and treatment anti-parkinson drug, it is characterised in that: the following steps are included:
1. preparing inclined side PD mouse model using 6-OHDA；
2. the expression quantity of Western blot and RT-PCR detection Dopamine In Striatum correlation factor；
3. the variation of TH and Iba1 in Immunofluorescence test corpus straitum.
2. it is according to claim 1 screening prevention and treatment anti-parkinson drug method, it is characterised in that: the step 1. in answer
The step of preparing inclined side PD mouse model with 6-OHDA is as follows:
P of Rats D model is established by nigro-striatal access locating injection 6-OHDA, the injection volume of 6-OHDA is every 4 μ L；
Postoperative intraperitoneal injection penicillin is anti-infective；Postoperative 2 weeks, intraperitoneal injection APO induced circling behavior, after injecting 10min, recorded rat
Rotating cycle records 30min altogether；Mean speed >=7 turn/min person be defined as PD the positive, continuous 3 weeks the positive then think modeling
Success.
3. the method for screening prevention and treatment anti-parkinson drug according to claim 1, it is characterised in that: the dopamine is related
The factor includes Nurr1, DAT, Pitx3, TH.
4. it is according to claim 3 screening prevention and treatment anti-parkinson drug method, it is characterised in that: described bar of amine correlation because
Sub- the primer sequence is as follows:
The primer sequence of Nurr1 gene:
F:5 '-AAGCCACCTTGCTTGTACCAAA-3 ',
R:5 '-CTTGTAGTAAACCGACCCGCTG-3 '；
The primer sequence of TH gene:
F:5 '-CAGGGCTGCTGTCTTCCTAC-3 ',
R:5 '-GGGCTGTCCAGTACGTCAAT-3 '；
The primer sequence of DAT gene:
F:5 '-TTGCAGCTGGCACATCTATC-3 ',
R:5 '-ATGCTGACCACGACCACATA-3 '；
The primer sequence of Pitx3 gene:
F:5 '-CTTAGTCCCTGCCAGTACGC-3 ',
R:5 '-GTGAGCCAAGGGTGAATTG-3 '.
5. the method for screening prevention and treatment anti-parkinson drug according to claim 1, it is characterised in that: rat administration occurs
1. step applies 6-OHDA to prepare inclined side PD mouse model before or after.
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| CN110331192A (en) * | 2019-06-17 | 2019-10-15 | 西安交通大学 | The detection method and its application of TH gene SNP site relevant to Opioid dependence |
| CN110663646A (en) * | 2019-10-21 | 2020-01-10 | 四川农业大学 | Chronic food-borne Parkinson disease mouse model and establishment method and application thereof |
| CN112430667A (en) * | 2020-11-18 | 2021-03-02 | 天津市康婷生物工程集团有限公司 | Method for evaluating treatment effect of mesenchymal stem cells in Parkinson's disease |
| CN115569202A (en) * | 2022-10-19 | 2023-01-06 | 苏州捷乐思生物科技有限公司 | Animal model of parkinsonism, drug screening method and therapeutic drug |
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| CN112430667A (en) * | 2020-11-18 | 2021-03-02 | 天津市康婷生物工程集团有限公司 | Method for evaluating treatment effect of mesenchymal stem cells in Parkinson's disease |
| CN115569202A (en) * | 2022-10-19 | 2023-01-06 | 苏州捷乐思生物科技有限公司 | Animal model of parkinsonism, drug screening method and therapeutic drug |
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