CN102551910A - Establishment method of rhesus monkey senile dementia model and drug screening method - Google Patents
Establishment method of rhesus monkey senile dementia model and drug screening method Download PDFInfo
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- CN102551910A CN102551910A CN2010106086987A CN201010608698A CN102551910A CN 102551910 A CN102551910 A CN 102551910A CN 2010106086987 A CN2010106086987 A CN 2010106086987A CN 201010608698 A CN201010608698 A CN 201010608698A CN 102551910 A CN102551910 A CN 102551910A
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- 208000024827 Alzheimer disease Diseases 0.000 title claims abstract description 51
- 206010039966 Senile dementia Diseases 0.000 title claims abstract description 48
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- 238000007877 drug screening Methods 0.000 title description 4
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- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- NNJVILVZKWQKPM-UHFFFAOYSA-N Lidocaine Chemical compound CCN(CC)CC(=O)NC1=C(C)C=CC=C1C NNJVILVZKWQKPM-UHFFFAOYSA-N 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
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Abstract
The invention provides a method for establishing a rhesus monkey senile dementia model. The invention also provides a method for screening the medicine for treating the senile dementia. The method for establishing the animal model adopts the thermosetting probe to destroy the rhesus monkey hippocampi umbrella at fixed points to cause the senile dementia model, has reliable method and good repeatability, can well simulate the symptoms of the senile dementia of human beings to a certain extent, and provides a stable animal model for treating the senile dementia by stem cells.
Description
Technical field
The present invention relates to a kind of method for building up of Rhesus Macacus senile dementia model, belong to drug world.
Background technology
Senile dementia is that to follow aging and cognitive dysfunction occurs be main illness, cognitive function improve sound closely related with the central cholinergic system function; The acupuncture and moxibustion therapy senile dementia has certain curative effect, senile dementia patient's intelligence is improved (improving damage in learning and memory) and clinical symptoms improve tangible effect is all arranged.(Liu Yuxing, etc., acusector experimentation to arched roof-fimbria of hippocampus damage senile dementia learning and memory in rats behavioral implications, " Chinese rehabilitation medicine journal, 2004 19 3 phases of volume)
Summary of the invention
Technical scheme of the present invention has provided a kind of method for building up of Rhesus Macacus senile dementia model.Another technical scheme of the present invention has provided a kind of method of screening treatment alzheimer disease medicine.
The invention provides a kind of method for building up of Rhesus Macacus senile dementia model, it comprises the steps:
A, anaesthesia experiment Rhesus Macacus;
B, postanesthetic experimental monkey groups is fixed on the stereo brain orienting instrument, confirms the position of fimbria of hippocampus;
C, at field of operation subcutaneous injection anesthetis; Before getting coronal suture, the skin of forehead otch of other center line 3cm, boring;
D, the bipolar coagulation pin is inserted Hippocampus, use radiofrequency generator, first electricity irritation is observed and is had or not discomfort, alteration in heart rate, limbs tic; 60 ℃ of damages in advance in 20 seconds of reuse, observation heart rate, blood pressure, limbs muscular strength have or not change, no complication to occur, and then with 75 ℃ of 60 seconds damage target areas, make the damage kitchen range of diameter 4~6mm;
E, extract the radio frequency pin, sew up wound, the wound sterilization gives 800,000 unit benzylpenicillins, can set up Rhesus Macacus senile dementia model.
The invention provides the purposes of animal model in the medicine of screening treatment senile dementia of described method preparation.
The invention provides a kind of method of screening treatment alzheimer disease medicine, it comprises the steps:
A, set up Rhesus Macacus senile dementia animal model;
B, drug candidate is applied to animal model;
C, observe the influence situation of drug candidate, and mark, estimate the medicine of potential treatment senile dementia the quantizating index of senile dementia disease.
Wherein, the step of the method for building up of described Rhesus Macacus senile dementia animal model is:
A, anaesthesia experiment Rhesus Macacus;
B, postanesthetic experimental monkey groups is fixed on the stereo brain orienting instrument, confirms the position of fimbria of hippocampus;
C, at field of operation subcutaneous injection anesthetis; Before getting coronal suture, the skin of forehead otch of other center line 3cm, boring;
D, the bipolar coagulation pin is inserted Hippocampus, use radiofrequency generator, first electricity irritation is observed and is had or not discomfort, alteration in heart rate, limbs tic; 60 ℃ of damages in advance in 20 seconds of reuse, observation heart rate, blood pressure, limbs muscular strength have or not change, no complication to occur, and then with 75 ℃ of 60 seconds damage target areas, make the damage kitchen range of diameter 4~6mm;
E, extract the radio frequency pin, sew up wound, the wound sterilization gives 800,000 unit benzylpenicillins, can set up Rhesus Macacus senile dementia model.
The method for building up of animal model of the present invention adopts heat to coagulate probe fixed point damage Rhesus Macacus fimbria of hippocampus and causes the senile dementia model; Method is reliable; Good reproducibility, the symptom of good simulating human senile dementia provides stable animal model for the stem-cell therapy senile dementia to a certain extent.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement and the change of other various ways not breaking away under the above-mentioned basic fundamental thought prerequisite of the present invention according to the ordinary skill knowledge and the customary means of this area.
Below, foregoing of the present invention is remake further detailed description through the specific embodiment of embodiment form.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
Fig. 1 perioperatively MRI relatively
Fig. 2 perioperatively normal observation relatively
Fig. 3 perioperatively is got the food success rate relatively
Fig. 4 perioperatively limit time relatively
The specific embodiment
Coagulate probe fixed point damage Rhesus Macacus fimbria of hippocampus through heat, set up Rhesus Macacus senile dementia animal model, for research stem-cell therapy senile dementia provides experiment basis.
1, test material
The 1.5T of Siemens magnetic resonance imager
Shenzhen peace section brain solid positioner (ASA-602S build-in function collection of illustrative plates, monkey adapter)
Shenzhen peace section temperature control heat is coagulated radiofrequency generator (ASA-601T)
2, laboratory animal
2 of regular grade adult healthy Rhesus Macacus, male, age 5-7 one full year of life, weight range is 5-6kg, is introduced by safety animal Study on breeding base, Chengdu, Sichuan (production licence number: SCXK (river) 2004-013).Enter the room and carry out routine blood test, liver function, routine urianlysis the last week.Tuberculin, parasite, Salmonella, the Salmonella of congratulating inspection.Quarantine qualified after, include experiment in.
Rhesus Macacus is raised in conventional animal room, Chinese medicine safety evaluatio center, national Chengdu (experimental animal room occupancy permit number: SYXK (river) 2003-030); Rustless steel monkey cage sub-cage rearing; The raising condition meets GB GB14925-2001,21 ± 5 ℃ of room temperatures (temperature difference per day≤4 ℃), relative humidity 55 ± 15%; Light and shade replaced in 12 hours, rate of ventilation 8~10 times/hour.Monkey mixed feed (Beijing section Australia pull together feed corporation,Ltd production licence SCXK (capital) 2005-0007 is provided) is fed, and reverse osmosis water is freely drunk, and adds fruit every day.
Animal is hung on neck board and the cage and labels as animal identification marking.
3, experimental technique
Dosage with 0.2ml/kg is anaesthetized experimental monkey groups intramuscular injection of ketamine (5%) in advance, and then with the dosage intravenous injection pentobarbital sodium (3%) of 1ml/kg.
Postanesthetic experimental monkey groups is fixed on the stereo brain orienting instrument, on MRI figure, confirms the position of fimbria of hippocampus, confirm D coordinates value with the computer planning system.
(1) at field of operation subcutaneous injection lignocaine.
(2) get before the coronal suture, the skin of forehead otch of other center line 3cm, boring.
(3) the bipolar coagulation pin is inserted Hippocampus, use radiofrequency generator, first electricity irritation is observed and is had or not discomfort, alteration in heart rate, limbs to twitch; 60 ℃ of damages in advance in 20 seconds of reuse, observation heart rate, blood pressure, limbs muscular strength have or not change.No complication occurs, and then with 75 ℃ of 60 seconds damage target areas, approximately makes the damage kitchen range of diameter 4~6mm.
(4) extract the radio frequency pin, sew up wound, the wound sterilization gives 800,000 unit benzylpenicillins, accomplishes operation.
4, O&E
4.1 postoperative observation and detection:
Raise 4.1.1 postoperative is conventional, and monitoring wound healing situation.
4.1.2 the record operation record is included the experimental record archives in.
4.2 model success judgment criteria
4.2.1 postpone to get the food training
All laboratory animals are carried out behavioristics's training in advance before operation.
(1) every day 1 time was carried out in experiment weekly 5 days.
(2) outside the monkey cage, put the training devices, four cribs are arranged on the plate, can be in interior placement food particle, a removable plank lid is put in the crib top, and removable lid covers or exposes crib.The training monkey is placed with the crib of food through touch, pushes lid aside to obtain food.
(3) in the experimental monkey groups visible range, put food into arbitrary crib at random.Cover all cribs, place experimental monkey groups can reach scope the training devices then, make it freely to select, the record once success rate has only and selects correct side to give the food award.This postpones for 0s, trains 10 times at every turn, after success rate reaches 100%, carries out next time period training.
(4) still in the experimental monkey groups visible range, place food to arbitrary crib at random, cover all cribs and device is placed the experimental monkey groups invisible area, go above-mentioned training behind the 5s again, a week, average success rate need reach 90%-100%.This postpones for 10s, trains 10 times at every turn.
(5) step that postpones according to 5s is proceeded the training that be 20s, 30s, 60s, 90s, 120s time delay, and success rate need reach 85%-95% respectively, 75%-85%, and 65%-75%, 55%-65%, one week of 40%-55% is average.
(6) after the above-mentioned training of completion, carry out the training of limit time, the maximum duration that can reach 20% success rate with experimental monkey groups is a limit time, and training is full after 3 months, the limit time and the calculating mean value in one week of continuous record experimental monkey groups.
(7) after the operation, train once more, record success rate and the limit time of monkey in whole experiment and compares before the art.
(8) after the operation, to the 1st the end of month, carry out once above-mentioned experiment every day from postoperative the 1st day, the record time, and with art before compare.After 1 month, weekly above-mentioned experiment is carried out once in the experimental monkey groups activity, be recorded to experiment always and stop (experiment stops comprising: animal dead, the complete obiteration of laboratory animal symptom or performed the operation back 6 months).
(9) the successful judge of model: with the postoperative maximum delay difference of blanking time, available own control analysis if statistical significance is arranged, is explained the model success before every animal comparison art.
4.2.2 conventional behavioristics is observed
(1) the experimental monkey groups active situation was observed in the 7th day, the 14th day in the back of raising in cages.Art was observed the experimental monkey groups active situation in preceding 2 days once more, confirmed no abnormally, can carry out modeling.
(2) the 1st day after surgery to the 1st the end of month, and the experimental monkey groups active situation is observed every day.After 1 month, weekly the experimental monkey groups active situation is observed, stops (experiment stops comprising: animal dead, the complete obiteration of laboratory animal symptom or performed the operation back 6 months) until experiment, and with art before relatively.Unusual performance in the observation period, occurs like experimental monkey groups, should carry out video record.
5, experimental result is seen Fig. 1.
Tangible cicatrization appears in the visible psalterium umbrella place of postoperative, and prompting heat is damaged successfully with fixed attention, sees Fig. 2.After operation wound recovers; It is thus clear that the experimental monkey groups attention concentrates ability obviously to descend; Personality generation significant change shows as lowering one's guard property, no matter is that raising and trainer or strange personnel all can initiatively get close to, get food speed and obviously slow down and often ignore and disturb down still independently feed.Often there is idle exercise to occur, holds foot and twitch, frequently limbs stretched out cage and do not know to regain so that occur scratch etc. outward like backhand.See Fig. 3, visible from figure, postoperative, two monkeys are under the situation of no time delay, and success rate does not have big decline, but after postponing, all occurs very significantly reducing, and difference has statistical significance (P<0.01).Delay after the 30s, two monkeys all can't be accomplished.Visible from Fig. 4, the limit time that postoperative two monkeys can be accomplished training obviously reduces, and difference has statistical significance (P<0.01), and this result proves the modeling success.
Experimental result can be known; Animal model of the present invention is to adopt heat to coagulate probe fixed point damage Rhesus Macacus fimbria of hippocampus to cause the senile dementia model; Can be to a certain extent the good symptom of simulating human senile dementia; For the stem-cell therapy senile dementia stable animal model is provided, this modeling method is reliable, good reproducibility.
Embodiment 2 utilizes the classical medicine checking senile dementia model of the present invention of treatment senile dementia and tentatively sets up related drugs screening system
A, set up Rhesus Macacus senile dementia animal model by embodiment 1 described method;
B, medicine, the drug candidate of classic treatment senile dementia is applied to animal model of the present invention respectively;
C, compare, observe the influence situation of drug candidate, and mark, estimate the medicine of potential treatment senile dementia the quantizating index of senile dementia disease with the classical medicine of treatment senile dementia; Simultaneously verified that also senile dementia animal model of the present invention is used for the feasibility of drug screening.
Through the evaluation of step c, classical medicine can obviously improve the symptom of model monkeys, proves monkey senile dementia model modeling success of the present invention, has proved that also this drug screening system is effective simultaneously.
Through the evaluation of step c, if drug candidate has improvement to the symptom of above-mentioned monkey senile dementia, and reach the standards of grading of similar classic treatment medicine, prove that promptly this drug candidate has the effect of treatment senile dementia.
Claims (4)
1. the method for building up of a Rhesus Macacus senile dementia model, it comprises the steps:
A, anaesthesia experiment Rhesus Macacus;
B, postanesthetic experimental monkey groups is fixed on the stereo brain orienting instrument, confirms the position of fimbria of hippocampus;
C, at field of operation subcutaneous injection anesthetis; Before getting coronal suture, the skin of forehead otch of other center line 3cm, boring;
D, the bipolar coagulation pin is inserted Hippocampus, use radiofrequency generator, first electricity irritation; 60 ℃ of damages in advance in 20 seconds of reuse then with 75 ℃ of 60 seconds damage target areas, are made the damage kitchen range of diameter 4~6mm;
E, extract the radio frequency pin, sew up wound, the wound sterilization gives 800,000 unit benzylpenicillins, can set up Rhesus Macacus senile dementia model.
2. the purposes of the animal model of the described method of claim 1 preparation in the medicine of screening treatment senile dementia.
3. one kind is screened the method for treating the alzheimer disease medicine, and it comprises the steps:
A, set up Rhesus Macacus senile dementia animal model;
B, drug candidate is applied to animal model;
C, observe the influence situation of drug candidate, and mark, estimate the medicine of potential treatment senile dementia the quantizating index of senile dementia disease.
4. the method for screening of medicaments according to claim 3, it is characterized in that: the step of the method for building up of described Rhesus Macacus senile dementia animal model is:
1., anaesthesia experiment Rhesus Macacus;
2., postanesthetic experimental monkey groups is fixed on the stereo brain orienting instrument, confirm the position of fimbria of hippocampus;
3., at field of operation subcutaneous injection anesthetis; Before getting coronal suture, the skin of forehead otch of other center line 3cm, boring;
4., the bipolar coagulation pin is inserted Hippocampus, use radiofrequency generator, first electricity irritation; 60 ℃ of damages in advance in 20 seconds of reuse then with 75 ℃ of 60 seconds damage target areas, are made the damage kitchen range of diameter 4~6mm;
5., extract the radio frequency pin, sew up wound, the wound sterilization gives 800,000 unit benzylpenicillins, can set up Rhesus Macacus senile dementia model.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN105663109A (en) * | 2015-12-29 | 2016-06-15 | 浙江旭晨医药科技有限公司 | Application of triglyceride compound in preparation of medicine for treating neurodegenerative diseases |
CN110827950A (en) * | 2019-11-25 | 2020-02-21 | 四川大学华西医院 | Method for determining dosage of anesthetic, method for training model, and model |
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2010
- 2010-12-28 CN CN2010106086987A patent/CN102551910A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN105663109A (en) * | 2015-12-29 | 2016-06-15 | 浙江旭晨医药科技有限公司 | Application of triglyceride compound in preparation of medicine for treating neurodegenerative diseases |
CN110827950A (en) * | 2019-11-25 | 2020-02-21 | 四川大学华西医院 | Method for determining dosage of anesthetic, method for training model, and model |
CN110827950B (en) * | 2019-11-25 | 2022-10-28 | 四川大学华西医院 | Method for determining dosage of anesthetic, method for training model, and model |
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Application publication date: 20120711 |