CN105193391A - Detecting method for monkey body neurovirulence test - Google Patents
Detecting method for monkey body neurovirulence test Download PDFInfo
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- CN105193391A CN105193391A CN201510703930.8A CN201510703930A CN105193391A CN 105193391 A CN105193391 A CN 105193391A CN 201510703930 A CN201510703930 A CN 201510703930A CN 105193391 A CN105193391 A CN 105193391A
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Abstract
The invention provides a detecting method for a monkey body neurovirulence test. The detecting method comprises the following steps that a monkey body obtained after drug administration is taken, magnetic resonance scanning is conducted on the cross section view, the sagittal view and the coronal view of a monkey brain on the third day, the sixth day and/or the ninth day after drug administration is conducted, and images are read simply. By means of the detecting method, whether drug is accurately injected to a corresponding brain position can be accurately judged in the monkey body neurovirulence test, the accuracy of drug injection can be found in an early stage of the test, the test in a later stage is facilitated, and cost is reduced.
Description
Technical field
The present invention relates to a kind of method detecting monkey neurovirulence Test.
Background technology
Many attenuated live vaccines all derive from the attenuation improvement carried out wild-type strain, and wild-type strain is known has neurotoxicity.In the process of immune human body, do not occur the neurotoxicity remained in order to ensure attenuated live vaccine, the seed culture of viruses (comprising main seed lot or working seed lots) of candidate's attenuated live vaccine needs to carry out clinical front Neurovirulence test.Because attenuated live vaccine is mainly used in the mankind, therefore the clinical front Neurovirulence test of candidate's attenuated live vaccine mainly selects monkey as experimental animal.
Carry out in process in monkey neurovirulence Test, test medicine is expelled to accurately the key point that corresponding cranium brain position is whole test.But, tradition cranium brain medication only relies on brain position finder to locate or relies on clinical experience to carry out, whether accurate injection is to corresponding cranium brain position cannot to determine test medicine, many times only has the phase after experiment, just find after dissection that the injection position of medicine is inaccurate, causing wastes a large amount of manpower and materials.
Summary of the invention
Technical scheme of the present invention there is provided a kind of method detecting monkey neurovirulence Test.
The present invention detects the method for monkey neurovirulence Test, and step is as follows: draw the monkey body after medicine, the 3rd day upon administration, the 6th day and/or the 9th day, carries out magnetic resonance imaging respectively to the cross-section position of monkey brain, sagittal plain and Coronal, reads image.
Preferably, the instrument that described magnetic resonance imaging adopts is the magnetic resonance scanner of BrukerBioSpec7.0TMR.
Preferably, the concrete steps of magnetic resonance imaging are: regioselective scanning sequence, and scanning area position is selected, and obtains Scout scan image; Select T2 weighting sequence according to positioning image again and scan, obtaining scanogram; Finally select T1 sequence and copy T2 weighting sequence position to scan again, obtain scanogram; Finally preserve all above-mentioned scanograms.
Detection method can accurately judge in monkey neurovirulence Test, and whether medicine is expelled to corresponding cranium brain position accurately, at the accuracy of test early discovery drug injection, can be convenient to later stage test, cost-saving.
Below by way of detailed description of the invention, the present invention is described in further detail, but do not limit the present invention, those skilled in the art can make various change and distortion according to the present invention, only otherwise depart from spirit of the present invention, all should belong to the scope of claims of the present invention.
Detailed description of the invention
1, material
1.1 laboratory animal
Kind: Rhesus Macacus;
Grade: regular grade;
Use size of animal and sex: 12, male and female half and half;
Age: 2 ~ 3 years old;
Body weight: 3.10 ~ 4.20kg when buying, 3.45 ~ 4.55kg during grouping;
Source: Green Hao Si bio tech ltd, Chengdu, production licence number: SCXK (river) 2011-013, the Quality of Experimental Animals quality certification is numbered: 0022011;
Quarantine before entering the room: (Detecting Rubella Virus Antibodies In Human Sera is detected voluntarily by consigner to filter out the monkey of Detecting Rubella Virus Antibodies In Human Sera feminine gender, mechanism provides blood serum sample), enter the room in the last week and carry out monkey Mycobacterium tuberculosis, Salmonella, shigella, vermin, toxoplasma examination, qualified rear introducing;
The laundering period main scope of examination: animal general state; Body temperature (anus temperature), food ration, hematology, blood biochemistry, II lead electrocardiogram, urine respectively detect 2 times; Whether the weight of animals reaches the weight range of test requirements document, and 12 monkeys chosen are health (female unpregnancy) monkey and include formal test in.
1.2 main agents
1.2.1 test sample
Title or code name: the main seed lot of attenuated rubella live vaccine seed culture of viruses RA27/3 strain, mechanism code name W0037;
Source: Beijing Min Hai Bio-Scientific Inc.;
Character: cheese color loosening body;
Specification: 0.5mL/ bottle after redissolving;
Virus titer: 5.1LgCCID50/mL;
Lot number: M22-201201;
Preservation condition and points for attention :-70 ~-86 DEG C;
Effect duration: permanently effective before redissolving, 1 hour effect duration after redissolving;
Preserve place: mechanism test articles handling portion.
1.2.2 reference substance
Title or code name: the main seed lot reference substance of attenuated rubella live vaccine seed culture of viruses RA27/3 strain, mechanism code name W0037 contrasts;
Source: Beijing Min Hai Bio-Scientific Inc.;
Character: cheese color loosening body;
Specification: 0.5mL/ bottle after redissolving;
Lot number: 201201;
Preservation condition and points for attention :-70 ~-86 DEG C;
Effect duration: permanently effective before redissolving, 1 hour effect duration after redissolving;
Preserve place: mechanism test articles handling portion.
1.2.3 other main agents
Title: pentobarbital sodium;
Source: Sigma company;
Character: white powder;
Specification: 25g/ bottle;
Lot number: 93ET2904K;
Preservation condition: room temperature, airtight preservation;
Effect duration: to 2015 08 month;
Compound method: be mixed with desired concn with 0.9% sodium chloride injection before use.Temporary for subsequent use in 2 ~ 25 DEG C after preparation, 1 month effect duration;
Title or code name: sterilized water for injection;
Source: Kelun Pharm Ind Co., Ltd., Sichuan;
Character: colourless clear liquid;
Specification: 500mL/ bottle;
Lot number: H13040604;
Preservation condition and points for attention: the airtight preservation of room temperature;
Effect duration: 2015 03 month;
Title: lidocaine hydrochloride injection
Source: Zhengzhou Zhuo Feng pharmaceutcal corporation, Ltd
Character: colourless clear liquid
Specification: 2mL/ props up
Content: 0.1g/5mL
Lot number: 12030413
Preservation condition: room temperature, airtight preservation
Effect duration: 2014 02 month
Compound method: directly take, the same day is effective
Title: ketaject injection
Source: animal pharmaceutical factory of Shenyang City
Character: colourless clear liquid
Specification: 2mL:0.3g/ props up
Content: 150mg/mL
Lot number: 20110908;
Preservation condition: room temperature, airtight preservation;
Effect duration: 2013 08 month
Compound method: directly take, the same day is effective.
The preparation of 1.3 main solution
1.3.1 test sample preparation
Compound method: redissolve with sterilized water for injection 0.5mL;
Identification method after preparation: the shipment box of test sample identifies with red-ticket, and indicate test number, substances title, volume, preservation condition, effect duration, person liable and preparation date;
Temporary condition and effect phase after preparation: 2-8 DEG C of preservation after redissolving, directly take injection, use in 1 hour after uncork.
1.3.2 reference substance preparation
Compound method: redissolve with sterilized water for injection 0.5mL;
Identification method after preparation: the shipment box of reference substance identifies with white label, and indicate test number, substances title, volume, preservation condition, effect duration, person liable and preparation date;
Temporary condition and effect phase after preparation: 2-8 DEG C of preservation after redissolving, directly take injection, use in 1 hour after uncork.
1.4 key instrument
Embodiment 1 detection method
(1) respectively at before administration and administration after the 3rd day, the 6th day and/or the 9th day, Rhesus Macacus to be checked is anaesthetized (about 30mg/kg, intravenous injection) first through pentobarbital sodium;
(2) lie low and be fixed on locating slot, and monkey brain is placed in magnetic resonance designated centers position;
(3) run 7T magnetic resonance imaging function software (BrukerBioSpec7.0TMR, manufacturing country: Germany), macaque anesthesia is placed on magnetic resonance animal conveying bed, selects large animal launch and accept coil, prepare scanning;
(4) first carry out monkey brain three-dimensional (cross-section position, sagittal plain, Coronal) location scanning, obtain positioning image; Select the scanning of T2 weighting sequence according to positioning image, obtain scanogram; Finally select T1 sequence and copy the scanning of T2 weighting sequence position, obtaining scanogram; Above-mentioned scanogram is preserved;
(5) the magnetic resonance radiography picture of diagosis more each monkey, judges that whether injection site is accurate.
Embodiment 2 the inventive method detects the neurovirulence of the main seed lot of attenuated rubella live vaccine seed culture of viruses RA27/3 strain
1, administration
The main seed lot of attenuated rubella live vaccine seed culture of viruses RA27/3 strain (mechanism code name W0037) is the Strain of the attenuated rubella live vaccine prepared by Beijing Min Hai Bio-Scientific Inc., for the prevention of rubella, clinical plan intramuscular injection, total virus dosage at least 3.2LgCCID50/ people.
Chengdu Huaxi Haiqi Medical Technology Co., Ltd. (National Chengdu Center For Safety Evaluation of Drugs) produces the related request of calibrating by " drug registration management method " and biological product, carry out Rhesus Macacus mound intracerebral injection attenuated rubella live vaccine seed culture of viruses RA27/3 strain main seed lot monkey neurovirulence Test, observe it and whether monkey central nervous system is hidden have neurovirulence.
This test establishes 2 groups altogether, is respectively matched group and administration group, matched group 2 monkeys, administration group 10 monkeys, equal male and female half and half.All monkeys are Detecting Rubella Virus Antibodies In Human Sera feminine gender through examination.Each group of monkey all carries out mound intracerebral injection by aseptic operation method, administration group monkey each single injection in left and right side cerebral hemisphere thalamus gives the main seed lot of attenuated rubella live vaccine seed culture of viruses RA27/3 strain and is about 0.4mL (virus titer is about 5.1LgCCID50/mL), and namely every monkey dosage is about 5.0LgCCID50; Matched group monkey each single injection in left and right side cerebral hemisphere thalamus gives 0.4mL attenuated rubella live vaccine seed culture of viruses RA27/3 strain main seed lot reference substance.
2, detect
Administration was defined as test the 1st day the same day.Test the accuracy adopting magnetic resonance imaging to determine mound intracerebral injection on the 3rd day.
After administration, every day at the upper and lower noon respectively observes a monkey general status and whether limb paralysis or other nervous symptoms occurs, and administration group observes 21 days, and matched group observes 31 days; Test the 1st (before administration), within 7 and 21 days, respectively measure 1 respectively group monkey body weight; Test and respectively measure a monkey food ration on the 7th, 20 day; Test the 1st (after administration 1 hour), within 2,3,6,9,12,15,18,21 days, respectively measure 1 anus temperature; Observe and terminate to dissect monkey, carry out histopathological examination to brain and spinal cord, check point comprises frontal lobe, temporal lobe, top, occipital lobe, precentral gyrus, postcentral gyrus, island leaf, parahippocampal gyrus, Hippocampus, diencephalon (thalamus), caudatum, lentiform nucleus, claustrum, amygdaloid body, cerebellum, midbrain, pons, oblongata and neck, breast and waist marrow.Wherein, neck myeloid tissue gets 5 positions with interval, cervical enlargement place, and 3 positions are got at breast myeloid tissue interval; Myeloid tissue gets 2 positions between the first spinal cord, the second spinal cord and waist and expands 4 positions at place.Above-mentioned tissue of drawing materials adopts HE dyeing row Histopathology assessment.Select one-sided (left side) each position cerebral tissue, bilateral thalamus tissue (injection site) and the first myeloid tissue else and carry out Weil iron alum haematoxylin and Masson dyeing, and carry out microexamination; The tissue that microscopy notes abnormalities for doing HE dyeing, adds and does above-mentioned two kinds of specific stains and carry out pathological evaluation.
3, result
Magnetic resonance imaging checks
After adopting the inventive method to check, can confirm through magnetic resonance imaging inspection, administration group and all monkeys of matched group confirm that injection site is all within the scope of thalamus after thalamus injection.
General status
To observing end after administration group and the injection of matched group monkey thalamus, spiritual expression is normal, and upper and lower extremities motion freely, normally can jump, run in cage, appetite, ingest normal; Irritant reaction is sensitive to external world, there are no drowsiness, stupor, convulsions, epilepsy, ataxia, upper and lower extremities trembles and nystagmus, singly quiver, the neurotoxic symptoms such as hemiplegia.
Body weight, food ration and anus temperature
Administration group and matched group monkey thalamus were injected in the rear observation period, and its body weight, food ration and body temperature are showed no abnormal change.
Rubella antibody testing result
Before entering the room, all monkey Detecting Rubella Virus Antibodies In Human Seras are detected as feminine gender.
Test the 21st day, matched group monkey Detecting Rubella Virus Antibodies In Human Sera is detected as feminine gender; The immuno positive rate that administration group monkey Detecting Rubella Virus Antibodies In Human Sera detects is 100%.
Test the 31st day, matched group monkey Detecting Rubella Virus Antibodies In Human Sera is detected as feminine gender.
Histopathological examination
HE chromoscopy result shows, and matched group has caudatum, the parahippocampal gyrus of 2 (2/2 ratio) monkey brain; Administration group has the diencephalon of 4 (4/10 ratio) monkey brain, precentral gyrus, postcentral gyrus, temporal lobe, caudatum, the visible place of parahippocampal gyrus or many places stove cerebral tissue degeneration necrosis/glial cells hyperplasia and gitter cell to infiltrate.Confirm through Weil dyeing and Masson dyeing, locally there is the damage of spared nerve myelin and the prosthetic reaction of gtelatinous fibre hypertrophy in above-mentioned site morbidity; under diseased region all dyes with HE, stove cerebral tissue degeneration necrosis accompanies glial cells hyperplasia positions are conformed to each other, and prompting finding pathological changes should be the local response caused by mechanical stimulus of injection.
Slight-slight xanthein deposition is there is in the visible diencephalon of another visible administration group 2 monkeys (2/10 ratio) or the administration such as precentral gyrus, parahippocampal gyrus needle tracking reactive site, color after preparing with test sample is close, should be the pigmentation of test sample self.
In addition, administration group monkey brain, spinal cord are respectively organized and are had no other pathological change relevant to test sample.
Under this experimental condition, Rhesus Macacus gives the main seed lot of attenuated rubella live vaccine seed culture of viruses RA27/3 strain by 0.4mL/ side single injection in bilateral thalamus, and (dosage is only about 5.0LgCCID50/, lot number is M22-201201), monkey has no dead, neurobehavioral, the motor function of all monkeys, ingest, the ordinary circumstance such as body temperature is good; After immunity, administration group monkey Detecting Rubella Virus Antibodies In Human Sera positive rate is 100%; Magnetic resonance imaging confirms all visible needle tracking reaction of every monkey in conjunction with pathologyofbraintissue inspection; Cerebral tissue and myeloid tissue have no the abnormal structure pathological change relevant to the main seed lot of attenuated rubella live vaccine seed culture of viruses RA27/3 strain.Illustrate that the main seed lot of attenuated rubella live vaccine seed culture of viruses RA27/3 strain does not have neurovirulence to monkey body.
Binding antibody testing result and histopathological examination, can also illustrate that medicine is accurately injected in thalamus really.Experimental result illustrates, whether the medicine that detection method can accurately detect really is accurately injected into corresponding cranium brain position.
To sum up, detection method can accurately judge in monkey neurovirulence Test, and whether medicine is expelled to corresponding cranium brain position accurately, at the accuracy of test early discovery drug injection, can be convenient to later stage test, cost-saving.
Claims (3)
1. one kind is detected the method for monkey neurovirulence Test, it is characterized in that: step is as follows: draw the monkey body after medicine, the 3rd day upon administration, the 6th day and/or the 9th day, respectively magnetic resonance imaging is carried out to the cross-section position of monkey brain, sagittal plain and Coronal, read image.
2. method according to claim 1, is characterized in that: the instrument that described magnetic resonance imaging adopts is the magnetic resonance scanner of BrukerBioSpec7.0TMR.
3. method according to claim 1, is characterized in that: the concrete steps of magnetic resonance imaging are: regioselective scanning sequence scans, and obtains Scout scan image; T2 weighting sequence is selected again to scan according to Scout scan image; Finally select T1 sequence and copy T2 weighting sequence position to scan; Finally preserve above-mentioned all scanograms.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101361659A (en) * | 2008-09-19 | 2009-02-11 | 王任直 | Cynomolgus monkey magnetic resonance scanning method |
| CN103933550A (en) * | 2014-03-13 | 2014-07-23 | 上海浦灵生物科技有限公司 | Method for establishing Macaca fascicularis experimental autoimmune encephalomyelitis model and application thereof |
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- 2015-10-26 CN CN201510703930.8A patent/CN105193391A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101361659A (en) * | 2008-09-19 | 2009-02-11 | 王任直 | Cynomolgus monkey magnetic resonance scanning method |
| CN103933550A (en) * | 2014-03-13 | 2014-07-23 | 上海浦灵生物科技有限公司 | Method for establishing Macaca fascicularis experimental autoimmune encephalomyelitis model and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| 尹雅姝等: "猴体神经毒力试验在减毒活疫苗安全性评价中的应用", 《中国生物制品学杂志》 * |
| 屈哲等: "猴体神经毒力实验组织病理学评价方法的建立", 《中国新药杂志》 * |
| 李尤等: "猴体神经毒理试验方法", 《中国药理学与毒理学杂志》 * |
| 李萍萍等: "麻疹-流行性腮腺炎-流行性乙型脑炎联合减毒活疫苗猴体神经毒力研究", 《中国疫苗和免疫》 * |
| 李龙等: "EV71灭活疫苗（人二倍体细胞）在恒河猴婴猴模型中的免疫保护性分析", 《中国科学：生命科学》 * |
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