CN110367188A - Myocardium transfects the construction method of the mouse model of slow virus - Google Patents
Myocardium transfects the construction method of the mouse model of slow virus Download PDFInfo
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- CN110367188A CN110367188A CN201910268287.9A CN201910268287A CN110367188A CN 110367188 A CN110367188 A CN 110367188A CN 201910268287 A CN201910268287 A CN 201910268287A CN 110367188 A CN110367188 A CN 110367188A
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- 241000700605 Viruses Species 0.000 title claims abstract description 24
- 210000004165 myocardium Anatomy 0.000 title claims abstract description 19
- 238000010276 construction Methods 0.000 title claims abstract description 8
- 238000010172 mouse model Methods 0.000 title claims abstract description 8
- 238000002347 injection Methods 0.000 claims abstract description 43
- 239000007924 injection Substances 0.000 claims abstract description 43
- 210000003205 muscle Anatomy 0.000 claims abstract description 14
- 238000001890 transfection Methods 0.000 claims abstract description 11
- 210000005240 left ventricle Anatomy 0.000 claims abstract description 7
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 56
- 210000000038 chest Anatomy 0.000 claims description 10
- 230000029058 respiratory gaseous exchange Effects 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 230000002107 myocardial effect Effects 0.000 claims description 8
- 241000255925 Diptera Species 0.000 claims description 7
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- 238000005303 weighing Methods 0.000 claims description 3
- 239000003814 drug Substances 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 8
- 238000002474 experimental method Methods 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 3
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- 230000002526 effect on cardiovascular system Effects 0.000 description 3
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- 238000010255 intramuscular injection Methods 0.000 description 1
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- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000002406 microsurgery Methods 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/30—Animals modified by surgical methods
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0337—Animal models for infectious diseases
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
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- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicinal Preparation (AREA)
Abstract
The present invention relates to a kind of construction methods of myocardium transfection slow virus mouse model, and the homogeneity and stability of cardiac muscle administration can be improved.Myocardium injection in the present invention refers to the ventricle wall muscle that drug or slow virus are injected into heart left ventricle with syringe, mode are as follows: uses micro syringe, throws face length as depth of needle using syringe needle, and carry out three point injections.Accordingly, strict control depth of needle and the effect of drug or slow virus can be reinforced, improves the homogeneity and stability of experiment.
Description
Technical field
The present invention relates to a kind of construction method of the mouse model of myocardium transfection slow virus, this model can guarantee that cardiac muscle is given
The homogeneity and stability of medicine.
Background technique
With social horizontal raising, the aggravation of aging of population, cardiovascular disease illness rate and the death rate rapidly on
It rises.Although preventing and treating cardiovascular disease work in China's has obtained first-stage success, face a severe challenge, simultaneously as cardiovascular and cerebrovascular
Sick hospitalization cost average annual growth rate is much higher than GDP speedup, and the financial burden of cardiovascular disease bring also increasingly aggravates.Closely
Nian Lai studies certain gene pairs heart although gene therapy and stem-cell therapy provide possibility to cure cardiovascular disease
Whether dirty disease works, and optimal method is still injected into cardiac muscular tissue.
Slow virus is widely used in the research of expression RNAi at present, provides strong work to explore gene function
Tool.Slow virus carrier is by the way that foreign gene to be effectively integrated on host chromosome, to reach persistent expression.Feeling
In terms of dye ability, slow virus can effectively infect the multiple types cell such as cardiac muscle cell, tumour cell, endothelial cell, therefore have
Wide application prospect.The slow virus for being currently applied to cardiovascular disease research direction, which is injected, mostly uses tail vein injection, but by
It is big in the viral dose of tail vein injection, easily unsuccessfully cause infection and can not first time confirm transfection efficiency, therefore by
The allogenic materials such as slow virus are efficiently imported in heart tissue and are made it stable expression by myocardial injection technology, will be in cardiovascular disease
Great application value is played in the research of the research of disease.
A kind of model animal of the mouse as classics can be good at the research applied to heart disease, but carry out the heart
When experiment is penetrated in intramuscular injection, since mouse heart is small in size, only 1.0 ~ 1.3mm is thick for ventricle wall, is difficult precisely to be injected, careless slightly
The chambers of the heart will be injected, injection mass is caused to be lost with blood circulation, efficiency can not be played, when serious can also heart bleeding make it is small
Mouse is lethal.
Summary of the invention
The purpose of the present invention is to provide a kind of transfection stabilization, the structure of the mouse model of uniform myocardium transfection slow virus
Construction method.
The present invention focuses on to probe into safety and validity through opening chest intramyocardial injection transfection art, to utilize transgenosis
The research for treating related cardiovascular disease provides a relatively inexpensive feasible channel genes mode.Therefore, the present invention is in body formula
3 points of injection injections of cardiac muscle are carried out using micro syringe under mirror, transfect slow virus to prepare the uniform and stable myocardium of one kind
Animal model.
To achieve the above object, it is as follows to provide technical solution by the present invention.
A kind of construction method of the mouse model of myocardium transfection slow virus, it is characterised in that the tool of this method of this method
Body step are as follows:
A. modeling is taken to use mouse；
B. animal pattern is anaesthetized, connects ventilator, open chest and exposure heart；
C. cut that the left skin of chest and muscle of mouse are successively laterally cut off 0.8-1cm under stereoscope with microforceps is small using mosquito
Mouthful, microforceps are inserted into the 4th intercostal, expose heart after blunt separation；
D. mouse intercostal being strutted using microforceps and carrying out myocardial injection under stereoscope, face length is thrown as depth of needle using syringe needle
Syringe needle is lunged mouse cardiac muscle to inject, in left ventricle front central location, bottom position, flanking central position in three times into
Row injection, per injection amount are 8 μ l, and three injection points should be uniformly distributed；
E. successively suture disinfection after injecting；
F. ventilator tracheae is taken out, checks the throat muscle and skin for successively suturing mouse after mouse breathing is normal, and to operation
Wound carries out disinfection；
Above-mentioned steps b's method particularly includes:
B-1., ventilator parameter, 120 times/min of frequency, tidal volume 2.0 are set；It is 34 DEG C that hot blanket parameter, which is arranged,；
B-2. 4% chloraldurate is drawn according to every gram of weight standard of 10 μ l after weighing to mouse, in the nearly hip joint position of lower abdomen
Set injection, anesthetized mice；
B-3. after mouse holonarcosis, mouse is faced upward and is fixed under stereoscope, to the throat position and ambition position of mouse
It loses hair or feathers, dips depilatory cream in smearing on hair using cotton swab, wipe hair off using blade after 1 minute and expose skin, and
With 75% alcohol disinfecting exposed section skin；
B-4. it is cut using mosquito and the throat skin and muscle of mouse is successively longitudinally cut off into 0.5- under stereoscope with microforceps
0.8cm osculum starts ventilator after exposing escape pipe, is being inserted into ventilator tracheae, inspection under glottis between two tracheal rings
Whether consistent to ensure that mouse breathing is normal with breathing unit frequency look into the fluctuating of mouse thorax.
Further, the mouse species are as follows: C57BL/6 mouse.It may extend to other experimental rat strains: BALB/c
Mouse, BALB/c Nude mouse, SD rat etc..
Further, muscle layer injection in center of the present invention, which refers to, is injected into heart for drug or slow virus with micro syringe
The ventricle wall muscle of left ventricle, mode are as follows: operated under body formula mirror, using micro syringe, control depth of needle, simultaneously will
Injection system is changed to 3 points of injections, guarantees that injection mass is effectively distributed in the cardiac muscular tissue of mouse.
The myocardial injection of drug is carried out to experimental rat, it is advantageous that: it is injected using micro syringe, for operation
Homogeneity has to be improved well, while using 3 points of injections, 3 points are respectively left ventricle front center position, left ventricle bottom
Portion, left ventricle center side position, greatly enhance the stability of experiment.The micro syringe injection refers to and will infuse
Emitter syringe needle plane gently penetrates cardiac muscle to syringe needle does not throw face excessively upward；The operation homogeneity refers to when carrying out drug injection
Depth of needle is controlled, when carrying out drug injection to more mouse in this way, so that it may guarantee the consistent of the inserting needle depth.Simultaneously
Control depth of needle can also prevent from making syringe needle penetrate the chambers of the heart since inserting needle is too deep, and drug is caused to cannot be introduced into cardiac muscle, or into
Needle is too shallow to will lead to drug injection in heart surface again, leads to the failure of an experiment；The stable experiment, which refers to, changes injection system
After injecting in batches for 3 points, the region of injection is increased, it is therefore prevented that cardiomyocyte cell death caused by largely injecting because of single-point.
The invention proposes a kind of preparation method of the myocardium slow-virus transfection animal model of transfection efficiency stable homogeneous,
Selection for cardiovascular research and cardiovascular gene therapy scheme provides certain help and reference.
Detailed description of the invention
Fig. 1 is injection effect proof diagram, the green fluorescence that mouse heart is sliced after slow virus FUGW myocardial injection.
Specific embodiment
Materials and methods:
Experimental animal: C57BL/6 mouse, 25 ± 3g of weight, age of mouse are greater than 8 weeks.Experiment is in Shanghai University's school of life and health sciences painstaking effort
Pipe research center carries out.The free diet of animal and drinking-water, animal house temperature are 25 ± 1 DEG C, and relative humidity is 40% to 60%.It is all
Mouse starts modeling in normal state.
Experiment reagent and instrument: slow virus FUGW；Surgery Platform is toy Special surgical platform, is breathed containing toy
Machine, Stereo microscope, light source, heating blanket, electronic balance；Surgical instrument is microsurgery instrument, comprising: mosquito is cut, directly
Tweezer, curved tweezer, micro- sharp tweezer, micro- curved tweezer, high-pressure sterilizing pot, 4-0 operation suture thread, 75% cotton ball soaked in alcohol, Iodophor swab stick; 30G
Specification micro-injection syringe needle.
Referring to Fig. 1, below in conjunction with specific embodiment, to further illustrate the technical scheme of the present invention.It will be understood that following
The embodiments are used only to illustrate the invention and the invention is not limited in any way.
Embodiment one: the injection of mouse core muscle layer, specific steps are as follows:
(1) surgical instrument uses advance horizontal high voltage steam sterilizing；Operative region is sterilized using 75% ethanol.
(2) ventilator parameter (120 times/min of frequency, tidal volume 2.0) is set；Hot blanket parameter (34 DEG C) are set.
(3) a certain amount of 4% chloraldurate is drawn according to every gram of weight standard of 10 μ l after weighing to mouse, by mouse
It is fixed on the centre of the palm, is injected in the nearly hip joint position of lower abdomen, anesthetized mice.
(4) after mouse holonarcosis, mouse is faced upward and is fixed under stereoscope, to the throat position and ambition portion of mouse
It loses hair or feathers position.Depilatory cream is dipped in smearing on hair using cotton swab, is wiped hair off using blade after 1 minute and is exposed skin,
And with 75% alcohol disinfecting exposed section skin.
(5) it is cut using mosquito and successively longitudinally cuts off the throat skin and muscle of mouse about under stereoscope with microforceps
0.5cm osculum starts ventilator after exposing escape pipe, is being inserted into ventilator tracheae, inspection under glottis between two tracheal rings
Whether consistent to ensure that mouse breathing is normal with breathing unit frequency look into the fluctuating of mouse thorax.
(6) it is cut using mosquito and successively laterally cuts off the left skin of chest and muscle of mouse about under stereoscope with microforceps
0.8cm osculum is inserted into microforceps in the 4th intercostal, exposes heart after blunt separation.
(7) strut mouse intercostal using microforceps and carry out myocardial injection under stereoscope, with syringe needle throw face length be into
Syringe needle is lunged mouse cardiac muscle and injected by needle depth, in left ventricle front central location, bottom position, flanking central position point
It is injected three times, per injection amount is 8 μ l, and three injection points should be uniformly distributed.
(8) front rib cage, muscle and skin are successively sutured after injecting, with Iodophor swab stick to operation wound after suture
Mouth carries out disinfection.
(9) ventilator tracheae is taken out, checks the throat muscle and skin for successively suturing mouse after mouse breathing is normal, and right
Wound carries out disinfection.
(10) mouse is put on 34 DEG C of hot blankets and is restored, if mouse has dehydration sign, sterile life is injected intraperitoneally in time
Manage salt water.Clean mouse cage is put back to after mouse revival, individually raises one week to wound healing.
(11) after note: rat step is consistent with mouse, and anaesthetic concentration is adjusted to 10% chloraldurate, 3 μ l/g weight, breathing
Machine parameter is adjusted to 70-75 beats/min, tidal volume 4.0ml.
Injection effect verifying, frozen section and fluorescence are shot
1. taking heart tissue
Mouse carries out myocardial injection position materials through slow virus FUGW myocardial injection after a week.It weighs to mouse, then presses
A certain amount of 4% chloraldurate is injected intraperitoneally according to 10 μ l/g weight standards.After mouse holonarcosis, have a common boundary in abdomen and thoracic cavity
Transverse incision is done at place, is cut off diaphragm and is exposed heart, clamps the big blood vessel such as aorta along heart uplink using curved tweezer, later carefully
On worry it is dirty, with microscissors below curved tweezer detachment blood vessel take out heart, finally heart is immersed in 1 × PBS buffer solution rapidly,
Heart, which is squeezed, with microforceps removes blood.
2. OCT embedding medium embeds
Heart is cut off the cardiac muscular tissue of slow virus injection site using blade after cleaning, and immerses preprepared OCT
In embedding medium.Then it is put into liquid nitrogen rapidly, is quickly cooled down.It is finally placed in -80 DEG C of refrigerators and saves.
3. being sliced
Freezing microtome is opened in advance, is cooled to -20 DEG C in advance.Sample is put on sample carrier, is fixed with embedding medium, it is latter
And it is put into ice and cuts and carry out sharp freezing on heat-exchange device in machine.When the two combine it is firm after be sliced.Take 10 μm of slices
- 80 DEG C of refrigerators preservations are put on glass slide, being inserted into film magazine later.
4. fluorescent collecting
By frozen section after taking out in -80 DEG C of refrigerators, rewarming 20min, then washes off group with 1 × PBS buffer solution at normal temperature
The OCT embedding medium for knitting periphery, be finally placed under fluorescence microscope heart after acquisition injection FUGW virus green fluorescence photo (see
Fig. 1).FUGW is a kind of virus that can translate green fluorescent protein, under the microscope can be with after being overexpressed in heart
It observes green fluorescence, illustrates virus injection success.
Claims (2)
1. a kind of construction method of the mouse model of myocardium transfection slow virus, it is characterised in that this method of this method it is specific
Step are as follows:
A. modeling is taken to use mouse；
B. animal pattern is anaesthetized, connects ventilator, open chest and exposure heart；
C. cut that the left skin of chest and muscle of mouse are successively laterally cut off 0.8-1cm under stereoscope with microforceps is small using mosquito
Mouthful, microforceps are inserted into the 4th intercostal, expose heart after blunt separation；
D. mouse intercostal being strutted using microforceps and carrying out myocardial injection under stereoscope, face length is thrown as depth of needle using syringe needle
Syringe needle is lunged mouse cardiac muscle to inject, in left ventricle front central location, bottom position, flanking central position in three times into
Row injection, per injection amount are 8 μ l, and three injection points should be uniformly distributed；
E. successively suture disinfection after injecting；
F. ventilator tracheae is taken out, checks the throat muscle and skin for successively suturing mouse after mouse breathing is normal, and to operation
Wound carries out disinfection.
2. the construction method of the mouse model of myocardium transfection slow virus according to claim 1, is characterized in that the step
Rapid b's method particularly includes:
B-1., ventilator parameter, 120 times/min of frequency, tidal volume 2.0 are set；It is 34 DEG C that hot blanket parameter, which is arranged,；
B-2. 4% chloraldurate is drawn according to every gram of weight standard of 10 μ l after weighing to mouse, in the nearly hip joint position of lower abdomen
Set injection, anesthetized mice；
B-3. after mouse holonarcosis, mouse is faced upward and is fixed under stereoscope, to the throat position and ambition position of mouse
It loses hair or feathers, dips depilatory cream in smearing on hair using cotton swab, wipe hair off using blade after 1 minute and expose skin, and
With 75% alcohol disinfecting exposed section skin；
B-4. it is cut using mosquito and the throat skin and muscle of mouse is successively longitudinally cut off into 0.5- under stereoscope with microforceps
0.8cm osculum starts ventilator after exposing escape pipe, is being inserted into ventilator tracheae, inspection under glottis between two tracheal rings
Whether consistent to ensure that mouse breathing is normal with breathing unit frequency look into the fluctuating of mouse thorax.
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| CN103509824A (en) * | 2012-06-27 | 2014-01-15 | 上海勃德生物科技有限公司 | Method for establishing heart disease drug screening model |
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2019
- 2019-04-04 CN CN201910268287.9A patent/CN110367188A/en active Pending
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|---|---|---|---|---|
| EP1263757A1 (en) * | 2000-02-11 | 2002-12-11 | Darwin Discovery Limited | Benzthiazoles as tnf and pde-iv inhibitors |
| CN101056539A (en) * | 2004-09-09 | 2007-10-17 | 综合医院公司 | Modulating phosphatase activity in cardiac cells |
| CN101513533A (en) * | 2008-02-20 | 2009-08-26 | 上海美迪西生物医药有限公司 | Method for building models of various diseases in one rat body and application thereof |
| CN103509824A (en) * | 2012-06-27 | 2014-01-15 | 上海勃德生物科技有限公司 | Method for establishing heart disease drug screening model |
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| Title |
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