CN114375902B - Construction method of autoimmune GFAP astrocytopathy rabbit animal model - Google Patents
Construction method of autoimmune GFAP astrocytopathy rabbit animal model Download PDFInfo
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- CN114375902B CN114375902B CN202111496894.4A CN202111496894A CN114375902B CN 114375902 B CN114375902 B CN 114375902B CN 202111496894 A CN202111496894 A CN 202111496894A CN 114375902 B CN114375902 B CN 114375902B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/02—Breeding vertebrates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/05—Detecting, measuring or recording for diagnosis by means of electric currents or magnetic fields; Measuring using microwaves or radio waves
- A61B5/055—Detecting, measuring or recording for diagnosis by means of electric currents or magnetic fields; Measuring using microwaves or radio waves involving electronic [EMR] or nuclear [NMR] magnetic resonance, e.g. magnetic resonance imaging
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/24—Detecting, measuring or recording bioelectric or biomagnetic signals of the body or parts thereof
- A61B5/316—Modalities, i.e. specific diagnostic methods
- A61B5/369—Electroencephalography [EEG]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/40—Detecting, measuring or recording for evaluating the nervous system
- A61B5/4058—Detecting, measuring or recording for evaluating the nervous system for evaluating the central nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B2503/00—Evaluating a particular growth phase or type of persons or animals
- A61B2503/40—Animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B2503/00—Evaluating a particular growth phase or type of persons or animals
- A61B2503/42—Evaluating a particular growth phase or type of persons or animals for laboratory research
Abstract
The invention discloses a construction method of an autoimmune GFAP astrocytopathy rabbit animal model, which comprises the following steps: 1) The preparation method comprises the steps of taking GFAP protein, diluting to 1mg/mL with 2M Urea, PBS and pH=8.5, continuously taking the equal volume of complete Freund's adjuvant, and uniformly mixing on ice by using a refiner until emulsification is complete, so as to prepare a first immune preparation; GFAP protein is taken, 2M Urea, PBS and pH=8.5 are used for diluting to 1mg/mL, an equal volume of incomplete Freund adjuvant is continuously taken, and the complete emulsification is carried out on ice by using a refiner until the complete emulsification is obtained, so that the immunity-enhancing preparation is prepared; 2) The first immunization preparation is subcutaneously injected at 8 points on two sides of the spine of the rabbit in a standard way by paravertebral subcutaneous injection, and 0.1ml is injected at each point to finish the first immunization; 3) The immune boosting preparation is subcutaneously injected into 8 points on two sides of the spine of a rabbit at intervals of two weeks by a paravertebral subcutaneous injection mode, 0.1ml of the immune boosting preparation is injected into each point, three times of immune boosting are needed, and an autoimmune GFAP astrocytopathy rabbit animal model is constructed.
Description
Technical Field
The invention relates to the technical field of animal model construction, in particular to a construction method of an autoimmune GFAP astrocytopathy rabbit animal model.
Background
Autoimmune Glial Fibrillary Acidic Protein (GFAP) astrocytopathy is a rare central nervous system autoimmune disease related to GFAP autoantibodies, but the autoimmune disease is not rare in the neuroimmune disease, and has the characteristics of critical illness state, poor prognosis of partial patients and the like, the disease is quite similar to diseases such as multiple sclerosis, acute disseminated encephalomyelitis, neuromyelitis and the like in clinical manifestation and biochemical detection, the disease is difficult to identify with the autoimmune diseases of the nervous system to cause misdiagnosis, the research on the disease is particularly important, however, in the laboratory research process of the disease, corresponding experimental animal models are lacking at home and abroad at present, the related experiments cannot accurately simulate the morbidity of human related diseases, the pathological process and pathological characteristics of the disease cannot be objectively reflected, and the technical defects such as poor sensitivity, poor disease induction effect, low success rate, complex operation and the like are caused by the existing alternative animal models used in the laboratory, and the waste of experimental animals and the delay of experimental progress are caused.
Disclosure of Invention
The invention aims to overcome the defects of the technology and provide a construction method of an autoimmune GFAP astrocytopathy rabbit animal model.
In order to solve the technical problems, the technical scheme provided by the invention is a construction method of an autoimmune GFAP astrocytopathy rabbit animal model, which comprises the following steps:
1) The preparation method comprises the steps of taking GFAP protein, diluting the GFAP protein to 1mg/mL by using 2MUrea, PBS and pH=8.5, continuously taking the complete Freund adjuvant with equal volume, and uniformly mixing the GFAP protein with ice by using a refiner until emulsification is complete, so as to prepare a first immune preparation;
the GFAP protein is taken, diluted to 1mg/mL by 2MUrea, PBS and pH=8.5, the incomplete Freund's adjuvant with equal volume is continuously taken, and the mixture is uniformly mixed on ice by a refiner until the emulsification is complete, so as to prepare the immunity enhancing preparation;
2) The first immunization preparation is subcutaneously injected at 8 points on two sides of the spine of the rabbit in a standard way by paravertebral subcutaneous injection, and 0.1ml is injected at each point to finish the first immunization;
3) The immune boosting preparation is subcutaneously injected into 8 points on two sides of the spine of a rabbit at intervals of two weeks by a paravertebral subcutaneous injection mode, 0.1ml of the immune boosting preparation is injected into each point, three times of immune boosting are needed, and an autoimmune GFAP astrocytopathy rabbit animal model is constructed.
Further, the injection amount of the first immunization preparation is 800 mug; the injection amount of the booster preparation is 400 mug.
Further, the concentration of the first immune preparation and the booster immune preparation is 0.5mg/mL.
Further, the rabbit is New Zealand rabbit.
Compared with the prior art, the invention has the advantages that: the invention can provide an ideal experimental animal platform for diagnosing autoimmune GFAP astrocytopathy, dividing subtypes, optimizing related intervention means, improving prognosis, treatment level and other experimental designs of related patients, effectively solves the scientific research problem that the current research process of GFAP astrocytopathy lacks corresponding experimental animal models, and simultaneously adopts rabbits as platform carriers, so that various problems of sensitivity of experimental animals to GFAP antibodies, poor disease induction effect and the like in the existing experiments can be effectively improved, related experimental cost can be reduced, time of experimental operators is saved, and an experimental foundation is provided for carrying out deep research on the occurrence mechanism of diseases.
Drawings
FIG. 1 is an electroencephalogram detection chart of a method for constructing an autoimmune GFAP astrocytopathy rabbit animal model according to the present invention;
wherein A is the electroencephalogram of a rabbit in the control group, and B is the electroencephalogram of a rabbit with a GFAP model;
FIG. 2 is a diagram of MR detection of a GFAP model rabbit of a method for constructing an autoimmune GFAP astrocytopathy rabbit animal model of the present invention;
wherein C is MR and shows brain enhancement focus, D is MR spectrum and suggests NAA to decline;
FIG. 3 is a flow chart of the construction of a GFAP model rabbit of the method for constructing an autoimmune GFAP astrocytopathy rabbit animal model of the present invention;
FIG. 4 is a GFAP model rabbit cerebrospinal fluid GFAP antibody map of a method for constructing an autoimmune GFAP astrocytopathy rabbit animal model of the present invention;
FIG. 5 is a diagram of inflammatory cells in the brain of a GFAP model rabbit, which is a method for constructing an autoimmune GFAP astrocytopathy rabbit animal model according to the present invention.
FIG. 6 is a graph showing the results of mental evaluation of a GFAP model rabbit according to the method for constructing an autoimmune GFAP astrocytopathy rabbit animal model of the present invention.
Detailed Description
The method for constructing an autoimmune GFAP astrocytosis rabbit animal model according to the present invention is described in further detail below with reference to examples.
Example 1
1. The construction method of the autoimmune GFAP astrocytopathy rabbit animal model comprises the following steps:
1) The preparation method comprises the steps of taking GFAP protein, diluting to 1mg/mL with 2M Urea, PBS and pH=8.5, continuously taking the equal volume of complete Freund's adjuvant, and uniformly mixing on ice by using a refiner until emulsification is complete, so as to prepare a first immune preparation;
GFAP protein is taken, 2M Urea, PBS and pH=8.5 are used for diluting to 1mg/mL, an equal volume of incomplete Freund adjuvant is continuously taken, and the complete emulsification is carried out on ice by using a refiner until the complete emulsification is obtained, so that the immunity-enhancing preparation is prepared;
2) The first immunization preparation is subcutaneously injected at 8 points on two sides of the spine of the rabbit in a standard way by paravertebral subcutaneous injection, and 0.1ml is injected at each point to finish the first immunization;
3) The immune boosting preparation is subcutaneously injected into 8 points on two sides of the spine of a rabbit at intervals of two weeks by a paravertebral subcutaneous injection mode, 0.1ml of the immune boosting preparation is injected into each point, three times of immune boosting are needed, and an autoimmune GFAP astrocytopathy rabbit animal model is constructed.
2. Clinical scoring
1) Scoring criteria (reference Benson scoring criteria)
0 point: no symptoms of disease;
1, the method comprises the following steps: weight loss or light toddler gait < ataxia);
2, the method comprises the following steps: the occurrence of a toddler gait (ataxia) with mild hind-hand or weakness of the forelimb (recovery after passive eversion);
3, the method comprises the following steps: the patient can not recover after the patient turns over passively when the patient suffers from serious hind limb or front limb weakness (limb dragging), but can move after the patient is stimulated by the patient;
4, the following steps: complete paralysis of the two hind limbs, paralysis disease of the forelimbs or weakness of muscle strength and incontinence of urine;
5, the method comprises the following steps: dying state or death.
Note that: symptoms were calculated as 0.5 between the two criteria.
2) Scoring requirements and results
From the first immunization, control and control rabbits were scored daily, while the control and control rabbits were evaluated for mental performance, and the average weekly score of each group of animals was tabulated as shown in the following table:
| Time | control group score (weekly average within the group) | Test group score (weekly average within the group) |
| First week of | 0±0.5 | 0 |
| Second week | 0±0.5 | 0 |
| Third week | 0±0.5 | 0 |
| Fourth periphery | 0±0.5 | 0 |
| Fifth week | 0±0.5 | 0 |
| Sixth week | 0±0.5 | 0 |
| Seventh week | 0±0.5 | 0 |
As can be seen from the above table, the clinical score of the test group was 0, but the rabbits of the test group showed mood changes and were easy to irritate from 1 week after the first booster immunization; compared with the control group, the obvious emotion after the second boosting is violent, and the hypnotic test is not easy to pacify; 3-4 days after the third boost, the rabbits in the test group showed active aggression.
3. Magnetic resonance examination
Carrying out three-plane positioning map scanning on test group rabbits after modeling is completed by using a Philips 3.0T MRI scanner (a special coil for rabbits), then carrying out T1WI, T2WI and T2WI-FLAIR sequence scanning, carrying out magnetic resonance spectroscopy analysis and examination after confirming that focuses appear on brains of the rabbits, adopting a two-dimensional multi-voxel 2D-PRESS sequence, TR 2000ms,TE 144ms,FOV 9mm X9mm, and having a layer thickness of 5mm, wherein the rest parameters are completed by using automatic presetting; and a metabolic profile was reconstructed using the MR spectra View software package in the philips post-processing ISP workstation, comparing each metabolite (Cho, cr, cr, lip, NAA, etc.) profile with each metabolite ratio (Cho/Cr, cho/NAA, NAA/Cr, etc.) profile, wherein brain enhancement lesions of the test group rabbits were shown (fig. 2-C), and MR spectra suggested a decrease in NAA of the test group rabbits (fig. 2-D).
4. Electroencephalogram examination
Meanwhile, the control group rabbits and the test group rabbits after modeling are subjected to electroencephalogram examination by using an EEG-1200c type electroencephalogram instrument produced by Japanese photoelectric company, wherein the electroencephalogram instrument adopts silver chloride columnar electrodes, the sampling frequency is 250Hz, an average electrode is adopted as a reference lead, the sensitivity is 10mV/mm, the paper feeding speed is 30mm/s, filtering is carried out for 0.3-30 Hz, and electroencephalograms are respectively drawn, wherein the electroencephalogram (figure 1-B) of the test group rabbits is locally different from the electroencephalogram (figure 1-A) of the control group rabbits.
5. Other item inspection
Meanwhile, rabbits in the test group are respectively checked by adopting a conventional checking scheme and adopting a ELISA, TBA, CBA and other checking methods, and GFAP antibody diagrams (figure 4) and brain inflammatory cell diagrams (figure 5) of the GFAP model rabbits are respectively provided.
The invention can provide an ideal experimental animal platform for diagnosing autoimmune GFAP astrocytopathy, dividing subtypes, optimizing related intervention means, improving prognosis, treatment level and other experimental designs of related patients, effectively solves the scientific research problem that the current research process of GFAP astrocytopathy lacks corresponding experimental animal models, and simultaneously adopts rabbits as platform carriers, so that various problems of sensitivity of experimental animals to GFAP antibodies, poor disease induction effect and the like in the existing experiments can be effectively improved, related experimental cost can be reduced, time of experimental operators is saved, and an experimental foundation is provided for carrying out deep research on the occurrence mechanism of diseases.
The invention and its embodiments have been described above without limitation. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present invention.
Claims (2)
1. The method for constructing the autoimmune GFAP astrocytopathy rabbit animal model is characterized by comprising the following steps of:
1) The preparation method comprises the steps of taking GFAP protein, diluting to 1mg/mL with 2M Urea, PBS and pH=8.5, continuously taking the equal volume of complete Freund's adjuvant, and uniformly mixing on ice by using a refiner until emulsification is complete, so as to prepare a first immune preparation;
the GFAP protein is taken, diluted to 1mg/mL by 2M Urea, PBS and pH=8.5, the incomplete Freund's adjuvant with equal volume is continuously taken, and the mixture is uniformly mixed on ice by a homogenizer until the emulsification is complete, so as to prepare the immunity enhancing preparation;
2) The first immunization preparation is subcutaneously injected at 8 points on two sides of the spine of a rabbit in a standard way by paravertebral subcutaneous injection, wherein each point is 0.1mL, the first immunization is completed, the injection quantity of the first immunization preparation is 800 mug, and the concentration of the first immunization preparation is 0.5mg/mL;
3) The method comprises the steps of injecting the boosting preparation into 8 points on two sides of the spine of a rabbit in a standard subcutaneous injection mode at intervals of two weeks, performing boosting by 0.1mL at each point, and constructing an autoimmune GFAP astrocytopathy rabbit animal model, wherein the injection amount of the boosting preparation is 400 mug, and the concentration of the boosting preparation is 0.5mg/mL;
after the animal model of the sick rabbit is constructed, carrying out clinical grading, magnetic resonance examination, electroencephalogram examination and other examinations, wherein the magnetic resonance examination is to firstly carry out three-plane positioning map scanning on rabbits in a test group by using a scanner, then carry out sequence scanning, and carry out magnetic resonance spectrum analysis examination after confirming that the brain of the rabbit has focus; the electroencephalogram examination is to use an electroencephalogram instrument to carry out electroencephalogram examination on rabbits in a test group; the other tests are that rabbits in the test group are tested by adopting a conventional test scheme and adopting a ELISA, TBA, CBA test method respectively, and GFAP antibody maps and brain inflammatory cell maps of the GFAP model rabbits are provided respectively.
2. The method for constructing an autoimmune GFAP astrocytopathy rabbit animal model as claimed in claim 1, wherein the method comprises the following steps: the rabbit is New Zealand rabbit.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108997496A (en) * | 2018-10-16 | 2018-12-14 | 无锡傲锐东源生物科技有限公司 | Anti- GFAP protein monoclonal antibody and application thereof |
| CN110604098A (en) * | 2019-09-23 | 2019-12-24 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Method for constructing animal model of rheumatoid arthritis combined with interstitial lung disease |
| CN110903389A (en) * | 2019-12-13 | 2020-03-24 | 福州迈新生物技术开发有限公司 | Monoclonal antibody and cell line for resisting GFAP protein, and preparation method and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108997496A (en) * | 2018-10-16 | 2018-12-14 | 无锡傲锐东源生物科技有限公司 | Anti- GFAP protein monoclonal antibody and application thereof |
| CN110604098A (en) * | 2019-09-23 | 2019-12-24 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Method for constructing animal model of rheumatoid arthritis combined with interstitial lung disease |
| CN110903389A (en) * | 2019-12-13 | 2020-03-24 | 福州迈新生物技术开发有限公司 | Monoclonal antibody and cell line for resisting GFAP protein, and preparation method and application thereof |
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