CN103357115B - A kind of preparation and application of rodent models - Google Patents
A kind of preparation and application of rodent models Download PDFInfo
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- CN103357115B CN103357115B CN201210102343.XA CN201210102343A CN103357115B CN 103357115 B CN103357115 B CN 103357115B CN 201210102343 A CN201210102343 A CN 201210102343A CN 103357115 B CN103357115 B CN 103357115B
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Abstract
The present invention relates to the preparation method and applications of a kind of rodent models.Specifically, before the present inventor utilizes low frequency electromagnetic field treatability maturation and grow up female rats and sexual maturity before and grow up male rat, obtain oestrusing frequency and adult female rats that Cluth size improves and spermatozoa motility reduces and sperm concentration reduces Adult male rats, thus establish two kinds of animal models.The present invention also relates separately to obtained animal model and is studying premature ovarian failure and related drugs exploitation, the application ahead of time and in the drug development of related drugs exploitation and raising human sperm's vigor of the research Women’s climacteric.
Description
Technical field
The present invention relates to rodent models field, it is more particularly related to male and female Rodents moves
The preparation of object model and their application in the drug development of premature ovarian failure drug development and raising human sperm's vigor.
Background technology
Along with the progress of society, the development of electric utility, the life scope of power frequency high voltage equipment settlement and people is more come
The nearest, it is exposed to for a long time in power frequency electromagnetic field and whether can affect health, cause the common concern of the public.Voltage is had just to have
Electric field, has electric current just to have an electromagnetic field, therefore, all there is electric field, use around socket, switch, the power line that is connected with socket
Electrical appliance and transmission line near all there is power frequency electromagnetic field[1].The power frequency magnetic field that China's (GB exposure draft) is given is sudden and violent
Dew limit value is occupational exposure 78 μ T, and the public exposes as 22 μ T, and the professional exposure limit of European Union and Australia etc. is defined as more
500 μ T, but human body can be damaged by the most great exposure strength and dosage, does not has definite final conclusion[2]。
The earth has earth's magnetic field about (about 3 × 10-7T), tellurian life has been also adapted to earth's magnetic field in evolution process
Existing, some biology make use of earth's magnetic field the most dexterously.Along with the development of science and technology, the application in magnetic field is got more and more by people, number
The Magnetotherapeutic health product that mesh is numerous also emerges in multitude, such as magnetic pad, magnetic pillow, low-frequency pulse Magnetotherapeutic apparatus etc..Magnetic field should in clinic
It is found that many therapeutic effect in, as improved microcirculation in human body, promotes union of fracture, prevent and treat osteoporosis etc..People are having
While effect utilizes magnetic field, more urgently it is to be understood that whether biology is harmful to by magnetic field, and its impact on environment.
Owing to the reproduction of the mankind relates to the quality of population, low frequency electromagnetic field, power frequency electromagnetic field the most therein, to people
The impact of class reproduction is particularly subject to pay close attention to.Many laboratory developments go out magnetic field generating device, observe magnetic exposure to laboratory animal
The impact of physiological function, particularly observes its impact on animals' reproduction function.Multiple laboratory has been had to open both at home and abroad at present
The research of exhibition this respect.Such as CXLY-II type pulsed magnetic field generator, this instrument can produce frequency 0～100Hz, magnetic field intensity
150～800mT continuously adjustable pulsed magnetic fields, can work long hours, and can put in the action of a magnetic field district (85mm × 40mm × 25mm)
Enter the object of the multiple volume profiles such as mice, Tissue Culture Plate, Micro-Organism Culture Dish, can be used for studying magnetic field to animal, cell, thin
The effect of the multiple organism effect such as bacterium[3].But the action of a magnetic field space is less, it is impossible to enough tolerance mices are free in its space
Life.Some magnetic field instrument can have (2006) such as the vivosphere of rat or mice, such as Cao Ya south[4]Institute use
Magnetic field generator, but it have studied magnetic field to pregnant mouse and the impact of Offspring's deyelopment, finds that pregnant Mus is omnidistance in trimester of pregnancy
In, it being exposed to the electromagnetic field 8 hours of 50Hz, 1.2mT every day, result causes fetal development significantly affected.There is presently no
Before sexual maturity, the experimental studies results report of magnetic exposure is proceeded by for rat or mice.In view of people in daily life
From birth, the most do not contact electromagnetic field, then what magnetic exposure has for the female mammal before sexual maturity
Negative effect becomes the problem being badly in need of research the most naturally.
Another the most according to the literature, adult rat be exposed to intensity be 1,4.8,9mT extremely low frequency electromagnetic fields, exposed 2 every 2 hours
Hour, maintain 8 weeks, cause level of serum testosterone to occur being remarkably decreased, and only magnetic field intensity reaches 9mT, just can cause serum
Estrogen level presents and is remarkably decreased, and then causes male During spermatogenesis to be affected[5].Additionally, the document reports such as de Bruyn
Road, from the pregnancy period, makes BALB/c mouse be constantly exposed to the intensity magnetic field intensity Extremely Low Frequency Electromagnetic at 0.5 to 77 μ T range
, find in filial generation, although level of serum testosterone and testis etc.s with adrenal gland not by appreciable impact, but live sperm ratio with
The quality of sperm motility presents significant difference[6], this explanation mice motility of sperm than hormone serum level more susceptible to magnetic
The impact of field.A1-Akhras etc. confirm, are exposed to 25 μ T, the 50Hz magnetic field male rat of 90 days with do not accept magnetic exposure
Female rats post-coitum, the pregnancy rate of female Mus declines and tire absorbance rises[7].Illustrate that extremely low frequency electromagnetic fields exposure can cause hero
Property rat fertility decline, may be reflected in above sperm quality.
In sum, in prior art many experimental designs stress have nothing in common with each other, the most contradictory part of conclusion obtained,
Whether the reproductive performance of the mammal of contact electromagnetic field can be affected, how be affected and do not come to a conclusion, the work of various countries
Frequency electromagnetic field exposure limits also has dramatic difference.Therefore, at present how magnetic field is affected to genital conditions and the electricity of mammal
The biological effect in magnetic field had not both had the recognized standard, did not had to obtain the theory of everybody common recognition yet.
Therefore, a kind of animal model of exploitation is badly in need of in this area, and it is raw to mammal that this animal model can be used for studying magnetic field
What negative effect is the situation of growing have, and then for drug development.
Summary of the invention
It is an object of the invention to provide male and female rodent birth pulse.
Therefore, in first aspect, the present invention provides the preparation method of a kind of rodent models, wherein, makes Rodents
Animal is exposed to the electromagnetic field special time of certain strength, thus obtains the male rodent that male fertility reduces, and/
Or the female rodent models that frequency improves or Cluth size improves of oestrusing after growing up,
Wherein, described rodent is selected from lower group:
(a) bull rodent；
Male rodent before (b) sexual maturity；
(c) Adult female rodent；
Female before (d) sexual maturity；
E () (a)～(d) appoint the combination of 2-4 item.
In a preference, intensity 1-2mT of female rodent before described sexual maturity；The most preferably
Intensity described in example is 1mT.
In a preference, before described sexual maturity, the described exposure persistent period of female rodent amounts to 224
Hour；Every day is exposed as 8 hours described in further preference, 4 weeks altogether.
In a preference, for intensity 5mT of described Adult female rodent.
In a preference, amount to 720 hours for the described exposure persistent period of described Adult female rodent；
Every day is exposed as 12 hours described in further preference, 60 days altogether.
In a preference, the frequency of oestrusing of described female rodent models improves more than 30%, more preferably 30-
60%, most preferably 35-50%；Or Cluth size improves more than 20%, more preferably 25-200%, most preferably 50-100%.
In a preference, the intensity of male rodent before described bull rodent or sexual maturity
For 1.5-5mT.
In a preference, amount to 720 hours for the described exposure persistent period of described bull rodent；
Every day is exposed as 12 hours described in further preference, 60 days altogether.
In a preference, the motile sperm ratio reduction by more than 40% of described male rodent models, more preferably
40-90%, most preferably 45-85%；Or sperm concentration reduces by more than 5%, more preferably 5-70%, most preferably 8-65%.
In another preference, when described rodent is exposed to particular magnetic field strength, it is maintained to raise normally
State or animation.
In second aspect, the present invention provides the preparation method of a kind of rodent models, and wherein, make before sexual maturity is female
Property rodent be exposed to the electromagnetic field special time of particular magnetic field strength, thus frequency of oestrusing after being grown up improve female
Property rodent models.
In a preference, described magnetic field intensity 0.5-5mT；Described in further preference, magnetic field intensity is 1-
3mT。
In a preference, described magnetic field is low frequency magnetic field (0-200Hz, preferably 0-100Hz).
In a preference, the described exposure persistent period amounts to 72-480 hour；Every day or every two days expose 2-24 hour,
Continue 2-10 week.In further preference, described in expose as every day 8 hours, 4 weeks altogether.
In a preference, described frequency raising of oestrusing is, compared with normal similar rodent, this animal model
Frequency of oestrusing improves more than 30%, more preferably 30-60%, most preferably 35-50%.
In another preference, when described rodent is exposed to particular magnetic field strength, it is maintained to raise normally
State or animation.
In the third aspect, the present invention provides the preparation method of a kind of rodent models, wherein, makes Adult female grinding tooth
Class animal is exposed to the electromagnetic field special time of certain strength, thus obtains the female rodent that adult rear Cluth size improves
Model.
In a preference, described intensity is 1-10mT, preferably 2-6mT.
In a preference, described magnetic field is low frequency magnetic field (0-200Hz, preferably 0-100Hz).
In a preference, the described exposure persistent period amounts to 240-1440 hour, is more preferably 360-960 hour；?
Further expose as every day 12 hours described in preference, 60 days altogether.
In a preference, the Cluth size raising more than 20%, more preferably 25-200% of described animal model, most preferably
50-100%.
In another preference, when described rodent is exposed to particular magnetic field strength, it is maintained to raise normally
State or animation.
In fourth aspect, the present invention provides the preparation method of a kind of rodent models, wherein, makes male Rodents move
Thing is exposed to the electromagnetic field special time of certain strength, thus obtains the male rodent models that male fertility declines.
In a preference, described magnetic field is low frequency magnetic field (0-200Hz, preferably 0-100Hz).
In another preference, when described rodent is exposed to particular magnetic field strength, it is maintained to raise shape normally
State or animation.
In another preference, described male fertility decline includes that motility of sperm reduces, sperm concentration reduces or it
Combination.
In another preference, described method also includes the step verifying the male rodent models obtained
Suddenly.
In another preference, described checking includes motility of sperm and/or the sperm concentration measuring described animal model.
In a preference, described intensity is 1.5-2.5mT；In further preference, described intensity is 2mT.
In a preference, the described exposure persistent period amounts to 720 hours；Expose as described in further preference
At 12 hours every day, 60 days altogether.
In a preference, described motility of sperm reduction is, compared with normal similar rodent, this animal model
Motile sperm ratio reduces by more than 40%, more preferably 40-90%, most preferably 45-85%.
In a preference, described sperm concentration reduction is, compared with normal similar rodent, this animal model
Sperm concentration reduces by more than 5%, more preferably 5-70%, most preferably 8-65%.
In another preference, described rodent is adult or male rodent before sexual maturity.
At the 5th aspect, the present invention provides the above first female Rodents prepared to described method arbitrary in the third aspect
The purposes of animal model, wherein, described animal model is for testing whether candidate substances affects oestrusing of female rodent
Frequency or Cluth size.
In a preference, described animal model is for testing whether described candidate substances can be used as the medicine of premature ovarian failure
Thing.
In another preference, described animal model is used for testing described candidate substances and whether can be used as Women’s climacteric and carry
Medicine early.
At the 6th aspect, the present invention provide above first or fourth aspect described in the male rodent mould prepared of method
The purposes of type, wherein, described animal model is used for testing whether candidate substances affects motility of sperm or sperm concentration.
In a preference, described animal model be used for testing described candidate substances whether can be used as improving motility of sperm or
The medicine of sperm concentration.
At the 7th aspect, the present invention provides a kind of and tests the method whether candidate substances affects oestrus frequency or Cluth size,
Wherein, comprise the following steps: female rodent prepared by the method described in first or second aspect that test substances is applied to
Model, and by oestrus frequency or the Cluth size of described female rodent models compared with matched group, wherein, frequency of oestrusing has
The change of significance,statistical shows that described candidate substances is to oestrusing frequency or Cluth size has an impact.
In another preference, described method also includes testing whether the candidate substances selected can be used as ovary early further
The medicine declined.
In another preference, described method also includes testing whether the candidate substances selected can be used as women more further
Term medicine ahead of time.
In another preference, described application process be administered orally, buccal, spraying or transdermal administration.
In eighth aspect, the present invention provides whether a kind of test candidate substances affects motility of sperm or density (male fertility
Power) method, wherein, including step: male rodent prepared by the method described in the third aspect that test substances is applied to
Model, and by the motile sperm ratio of described male rodent models or sperm concentration compared with matched group, wherein, described
Motile sperm ratio or sperm concentration have the change of significance,statistical to show, and described candidate substances is close to motility of sperm or sperm
Degree has an impact.
In another preference, described method also includes testing the candidate substances selected further to human spermatogoa vigor or essence
The impact of sub-density.
In another preference, described application process be administered orally, buccal, spraying or transdermal administration.
In another preference, described in any of the above aspect, the animal model of method obtains with following device, described device
Including casing, field generator for magnetic, detector for magnetic field, temperature detection and regulation device and voltage-operated device, wherein:
Described preparation facilities also includes ventilation unit, illuminator and chiller；
Described field generator for magnetic is positioned in described casing and has for placing laboratory animal or experimental animal feeding appearance
The cavity of device；
Having door on the sidewall of described casing, described door is used for putting into or take out laboratory animal or experimental animal feeding holds
Device；
Described chiller contacts with described field generator for magnetic, is used for cooling down described field generator for magnetic；
Described ventilation unit and illuminator are respectively used to provide for described field generator for magnetic ventilate and illumination；And
Described voltage-operated device is connected with described field generator for magnetic, is used for controlling described field generator for magnetic and is produced
Magnetic field intensity.
In a preference, described casing is the casing of armoured magnetic field, and described casing is approximately parallelepiped body, six of casing
Wall is made by iron plate, and four sidewalls are provided with vent, and described door is positioned in described four sidewalls and fills with described magnetic field
Put on the sidewall that axis is vertical.
In another preference, described field generator for magnetic is solenoid, described dynamic for placing laboratory animal or experiment
It is described solenoid inner chamber that thing raises the cavity of container.
In another preference, the magnetic field display device that described detector for magnetic field is positioned on the front side wall of described casing and
It is positioned at the magnet field probe in the cavity placing laboratory animal or experimental animal feeding container of described field generator for magnetic.
In another preference, described temperature detection includes thermindicator and is connected with thermindicator with regulation device
Temperature probe.
In another preference, described ventilation unit is exhaust fan, and described exhaust fan is arranged on the sidewall of described casing.
In another preference, described illuminator includes illuminating lamp and luminous lamp switch controller, and described illuminating lamp is opened
Gateway controller is opened for illuminating lamp described in loop control, it is achieved daytime and the simulation at night.
In another preference, described chiller includes that the cooling tube being wrapped on described field generator for magnetic is with cold
But pipe connects pump installation and refrigerant storage container, described cooling tube, pump installation and refrigerant storage container constitute cooling and follow
Loop back path.
In another preference, the described cavity size for breeding experimental animals of described field generator for magnetic can be held
Receive normal rat rearing-box and/or standard mice rearing-box, described normal rat rearing-box a size of (380 ± 20) × (325 ±
20) × (180 ± 20) mm, described standard mice rearing-box a size of (310 ± 20) × (200 ± 20) × (140 ± 20) mm.
In another preference, described illuminating lamp is LED.
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and having in below (eg embodiment)
Can be combined with each other between each technical characteristic that body describes, thus constitute new or preferred technical scheme.As space is limited, exist
This tires out the most one by one states.
Accompanying drawing explanation
Fig. 1. show the knot of an embodiment of the animal model preparation facilities with field generator for magnetic of the present invention
Structure schematic diagram；
Fig. 2. show the structural representation of an embodiment of animal model preparation facilities shown in Fig. 1；
Fig. 3. show the structural representation of the biopsy cavity marker devices of animal model preparation facilities shown in Fig. 2, for clarity sake, save
Slightly part-structure.
Fig. 4. show that rat is in heat the typical vaginal smear of different phase.Wherein: dioestrus (A) is mainly wrapped
Include leukocyte and mucus；The a large amount of nucleated cell of proestrum (B) and cornified vaginal epithelial cell are also deposited；Oestrus (C) is main
If cornified vaginal epithelial cell；Oestrus the later stage (D) nucleated cell and a large amount of leukocyte deposit.Each vaginal smear is all aobvious
Amplify 100 times of observations under micro mirror to take pictures.
Detailed description of the invention
Inventor through extensively in-depth study, it was unexpectedly found that before utilizing low frequency electromagnetic field treatability maturation with
And grow up female rats and sexual maturity before and grow up male rat, it is possible to respectively obtain oestrus frequency improve and Cluth size
The adult female rats improved and the Adult male rats that spermatozoa motility reduces and sperm concentration reduces, thus establish two
Plant animal model.Complete the present invention on this basis.
The method of preparation animal pattern
Before the present invention uses the magnetic field treatability maturation of specific induced field intensity and Adult female rodent and
In period certain with bull rodent before sexual maturity, obtain the Adult female that frequency improves and Cluth size improves of oestrusing
The animal model that animal model and bull fertility decline.
The present invention is also by the electromagnetic field special time making rodent be exposed to certain strength, thus obtains male life
Educate power reduce male rodent and/or grow up after oestrus frequency improve or Cluth size improve female rodent mould
Type, wherein, described rodent is selected from bull rodent；Male rodent before sexual maturity；Grow up female
Property rodent；Or the female rodent before sexual maturity.
The concrete magnetic field intensity used for the inventive method and exposure persistent period, those of ordinary skill in the art can root
Make one's options flexibly according to concrete condition.In a preferred embodiment, the magnetic field of female rodent before acting on sexual maturity
Intensity 0.5-5mT；In further preferred embodiment, described magnetic field intensity is 1-3mT.In a particular embodiment,
The described exposure persistent period amounts to 72-480 hour；In a preferred embodiment, every day or every two days expose 2-24 hour, hold
Continuous 2-10 week.In further preferred embodiment, described in expose as every day 8 hours, 4 weeks altogether.
In a particular embodiment, the magnetic field intensity acting on Adult female rodent is 1-10mT, preferably 2-
6mT.In a particular embodiment, described exposure persistent period total 240-1440 hour, more preferably 360-960 hour；?
In further preferred embodiment, described in expose as every day 12 hours, 60 days altogether；
In a particular embodiment, the magnetic field intensity acting on male rodent is 1.5-2.5mT, preferably 2mT.
In a preferred embodiment, described male rodent is adult or male rodent before sexual maturity.
In a particular embodiment, when the exposure of bull rodent and Adult female rodent continues
Between amount to 720 hours；Every day is exposed as 12 hours described in further preferred embodiment, 60 days altogether.
In a particular embodiment, described male fertility decline include motility of sperm reduce, sperm concentration reduce or
A combination thereof.
In another preference, described method also includes the step verifying the male rodent models obtained
Suddenly.
In another preference, described checking includes motility of sperm and/or the sperm concentration measuring described animal model.
In a particular embodiment, when described rodent is exposed to particular magnetic field strength, maintain it normal
Raising state or animation.
In a particular embodiment, described magnetic field is low frequency magnetic field；Described in a preference, magnetic field is 0-200Hz's
Low frequency magnetic field；In another preference, described magnetic field is the low frequency magnetic field of 0-100Hz.
In a particular embodiment, described rodent is selected from rat, mice, Cavia porcellus or rabbit.The most real
Executing in mode, described rodent is rat.
The device of preparation animal pattern
The animal pattern of the present invention can utilize various device known to persons of ordinary skill in the art to prepare.Hereinafter describe
Device be only a kind of representational preferred embodiment.
Fig. 1 is the structural representation of the animal model preparation facilities with field generator for magnetic of the present invention.Such as Fig. 1 institute
Show, animal model preparation facilities 100 include casing 1, field generator for magnetic 2 (i.e. solenoid), detector for magnetic field (14,18),
Temperature detection and regulation device (11,12), voltage-operated device 15, chiller (3,4,5,6), illuminator (9,10), with
And ventilation unit (7,8).
During work, laboratory animal by chiller 3 around solenoid 2 in raise.Voltage-operated device 15 controls spiral shell
The voltage strength of spool 2, and then control the intensity in magnetic field produced by solenoid 2, to obtain the magnetic field needed for experiment.Cooling dress
Put when solenoid works or when with regulation device, temperature detection detects that solenoid temperature exceedes predetermined value, for solenoid 2
Cooling is provided, thus ensures that solenoid 2 can work long hours.Illuminator realizes the mould at daytime and night by on-off control
Intending, the laboratory animal for raising in solenoid 2 provides closer to natural growing environment together with ventilation unit.
Fig. 2 and Fig. 3 illustrates the structural representation of an embodiment of the animal model preparation facilities 100 of Fig. 1.Such as Fig. 1-3
Shown in, in the present embodiment, casing 1 is approximately parallelepiped body, and six walls of casing 1 are all made up of iron plate, wherein, on four sidewalls
All have louver (or hole), be beneficial to air circulation.It is provided with and exhaust fan control on the rear wall (or on other sidewalls) of casing
The exhaust fan 8 that device 7 processed connects, and it is provided with the hole passed through for connecting line and cooling tube 3 (being described in more detail below).Preferably
Ground, is additionally provided with roller (not shown), to be moved easily casing bottom casing 1.
The front side wall of casing 1 is provided with control and display device region 1a and door 1b, door-plate 1c, can be put by door 1b
Entering or take out the rearing-box 22 equipped with laboratory animal, door-plate 1c is also made up of iron plate, with preferably armoured magnetic field.Control and display
Region is provided with on and off switch 16, current sensing means 17, voltage regulator 15, magnetic field display device 18, illumination control apparatus
9 and thermindicator 11.
Wherein, on and off switch 16 is connected with main power, and voltage regulator 15 and current sensing means 17 are connected to solenoid
2, detector for magnetic field includes the magnetic field display device 18 being positioned on the front side wall of described casing and the magnetic field being positioned in solenoid 2
Probe 14, illumination control apparatus 9 is connected to the illuminating lamp 10 being positioned in solenoid 2.It addition, although it is not shown, but exhaust fan 8,
Current sensing means 17, voltage regulator 15, magnetic field display device 18, illumination control apparatus 9 and thermindicator 11 etc. are equal
It is connected with power supply.
In above-mentioned animal model preparation facilities 100, by the size of voltage regulator 15 adjustable voltage, and then regulation
The size of current intensity and the size of magnetic field intensity, meanwhile, the size of current intensity and magnetic field intensity is respectively in real time at electric current
Detection device 17 and magnetic field display device 18 show so that user can observe the change in electric current and magnetic field easily, thus square
Just magnetic field intensity is adjusted to required value.
Illumination control apparatus 9 Automatic Cycle controls the illuminating lamp 10 in solenoid 2 with simulation daytime and night.The present embodiment
In, illuminating lamp is LED, the advantages such as it is long that it has the life-span, lower calorific value.
Above-mentioned field generator for magnetic is solenoid 2, can be according to magnetic field size to be produced and laboratory animal needed for
The cultured space wanted, arranges the diameter of solenoid 2, coil winding density and length thereof.
In the present embodiment, solenoidal diameter (i.e. the diameter of coil) is about 350mm, and solenoid length is about 800mm, energy
The homogeneous area enough providing uniform magnetic field is more thanIndustrial frequency AC magnetic field intensity reaches 0～50.0Gs,
And continuously adjustabe, accuracy reaches 2%.
The big I of serpentine pipe inner chamber accommodates the rat feeding case (size is about 380 × 325 × 180mm) or 4 of two standards
The Mouse feeder case (size is about 310 × 200 × 140mm) of individual standard.It should be understood that can be arranged as required to solenoidal directly
Footpath and length, to accommodate other experimental animal feeding case, such as rabbit rearing-boxes.
Additionally, due to field generator for magnetic, i.e. solenoid 2 caloric value in the course of the work are big, especially for producing height
Intensity magnetic field or provide the solenoid of homogeneous area of big uniform magnetic field, caloric value is the biggest so that it cannot work for a long time
Make.For guaranteeing to work for a long time, being wound with cooling tube 3 on the outer wall of solenoid 2, cooling tube 3 is with solenoid 2 electricity absolutely
Edge.One end of cooling tube 3 is connected to pump installation 5, and the other end is connected to store the container 4 of coolant, and pump installation 5 other end also connects
Receive container 4, thus constitute circulation cooling circuit.It is preferred that cooling tube 3 is made up of copper metal, coolant is water.Should be noted that
, cooling tube 3 contacts with solenoid 2 also by modes such as laminatings, as long as solenoid 2 can be cooled down.
By arranging gang switch, when solenoid 2 is started working, pump installation 5 also begins to work, to enter solenoid 2
Row cooling, thus ensure that solenoid 2 and animal model preparation facilities 100 can work long hours.Or, when temperature detection and tune
Control device, i.e. temperature probe 12 detect when solenoidal temperature exceedes predetermined value, and pump installation starts and lowers the temperature solenoid.
The purposes of animal pattern
The jenny model that the present invention obtains can be used for testing whether candidate substances affects sending out of female rodent
Feelings frequency or Cluth size.
In a preference, described animal model is for testing whether candidate substances can be used as the medicine of premature ovarian failure.
In another preference, described animal model is for testing whether candidate substances can be used as what Women’s climacteric was done sth. in advance
Medicine.
The animal model that the bull fertility that the present invention obtains declines can be used for testing whether candidate substances affects essence
Sub-vigor or sperm concentration.
In a preference, described animal model is used for testing whether candidate substances can be used as improving motility of sperm or sperm
The medicine of density.
Main advantages of the present invention include:
1. the inventive method is easy and simple to handle, reliable, safety；
2. two kinds of animal models that the inventive method obtains are stable, can be used for studying premature ovarian failure and related drugs is opened
Send out, research Women’s climacteric ahead of time and develop and the medicine of raising human sperm's vigor or sperm concentration is opened by related drugs
Send out；With
3. the present invention is sufficiently large and can for a long time, stably produce magnetic field for obtaining the device of animal pattern, thus mould
Type animal can live freedom in the apparatus, cause obtain animal pattern more can simulating reality environment, and relevant experimental data
More accurately, reliable.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.
Universal method and material
1 laboratory animal
Healthy SPF level SD is female and male rat (purchased from Shanghai City western pul-Bi Kai laboratory animal company limited).Raise
Normal condition be that every day, light application time was 6:00 to 18:00, and animal freely drinks water and ingests, indoor temperature control (20 ±
2)℃。
2 magnetic field generators
The biological AC magnetic field experimental box used is as shown in " preparing the device of animal model " part and accompanying drawing 1-3.
3 the action of a magnetic fields are in the experimental technique of rat
Take the female rats of 10 4 week old, be randomly divided into 2 groups.Experimental group 5, raises inside the instrument of magnetic field, maintains magnetic field
Intensity 1mT, stimulates 8 hours from 16:00 to 24:00, persistently stimulates 4 weeks, be used for detecting oestrus and blood steroid hormone every day
Level.During magnetic exposure, animal can freely drink water, ingest, and room temperature controls at 20 ± 2 DEG C.Matched group (5) is raised in
Outer 2 meters of the magnetic field instrument of same room, the noiseless part in experiment magnetic field.
Similar with above experimental design, experimental group and matched group respectively take 8 rats starting to process from 4 week old, maintain 4 weeks
After, mate with 1: 1 ratio with normal adult hero Mus.Whether next day is by having sperm to judge to be in vaginal smear examination reproductive tract
No generation copulation.The successful female rats of copulation, individually raises, the trimester of pregnancy length of record rat, claims when giving a birth latter 28 days
Dams and filial generation body weight.Observing fetal development situation, the conceived natural law of record, filial mice grow and Cluth size, with analysis of magnetic field action
Impact on female Mus fertility.
Take the male rat experimental group 8 of 8 week old at random, maintain magnetic field intensity 2mT, every day stimulation time by 18:00 extremely
06:00, totally 12 hours.Separately take 4 male rats as a control group.After continuing 60 days, put to death rat, take epididymal sperm, analyze
The indexs such as sperm morphology.
The judgement of 4 female rats Each Stage of Estrus Cycles
Take 4 week old rats, process 4 thoughtful rats through magnetic field and grow up (8 week old), then start vaginal smear examination rat
Estrus cycle.Concrete grammar is, morning every day, 8:30 took female Mus to be checked, conventionally, take greatly with the round end Glass rod of sterilizing
Mus vagina mucus is evenly coated on the microscope slide adding aquesterilisa, and conventional Giemsa staining method dyeing is observed.Reference literature, aobvious
Oestrous Stages residing for micro-lens-belowed identifying rat.General plate coating checking continues more than two cycles.Send out it follows that check
About the 10:30 in the morning on the same day of feelings phase, venous blood sampling 1.2ml under Rat Tongue, separated plasma be used for detecting FSH, estrogen and
The mensuration of progesterone level.
5 hormone test methods
Utilize estradiol and progesterone diagnostic kit (Electrochemiluminescince, Germany's Roche Diagnostics GmbH), according to examination
Agent box operation instructions, measure E2 and P4 concentration on Roche 2010 Electrogenerated chemiluminescent immunoassay instrument.
The analysis method of 6 rat spermatozoas
At rat after death, take its cauda epididymis immediately, be eliminated as much as peripheral adipose, blood and tissue fluid；By tail of epididymis
Move in the PBS (2ml) containing 1%BSA.The side of cauda epididymis is lived, at another side eye scissors with fine forceps sub-folder
A vertical osculum cut by cutter, is extruded by cauda epididymis spermatium.At 37 DEG C, cultivate 1 hour under the conditions of 5%CO2, mixing, draw 6 μ l
Suspension, drips in counting plate for sperm (macro ruby counting chamber, Shanghai micro-figure instrument development in science and technology company limited), counts sperm
Density and activity ratio.Motile sperm in one visual field of counting and spermatium quantity are to estimate sperm motility respectively, every part
Sample counting sum is more than 200 sperms.
Transgreen (Shanghai Shanghai Ganfeng Bio-Tech Development Co., Ltd.) and PI (sigma) is used to redye detection essence
Sub-membrane integrity, concrete grammar is shown in[8].Briefly: Transgreen Yu PI is mixed, add fresh spermatozoa suspension, incubate for 37 DEG C
Educate more than 5min, take 6 μ l suspensions and drip in counting plate for sperm, observe under fluorescence microscope (LEICA, DFC360FX).In indigo plant
Under the exciting of coloured light, the complete sperm of plasma membrane sends green fluorescence, and injury of plasmalemmae sperm sends red fluorescence.Count same regarding respectively
Red and the green sperm quantity in Yezhong, is calculated survival rate, and every part of sample counting sum is more than 200 sperms.
7 statistical analysis methods
Observing and detect each index, recording relevant data, using each index as variable, magnetic field processes and comparison is recorded as
Experiment packet, inserts in Excel form.Then relevant data import SPSS 18.0 statistical software (SPSS Inc.,
Chicago, IL), according to data type and distribution situation, select t inspection or Mann-Whitney U inspection, it is thus achieved that average and mark
Accurate poor, and significant indexes p value.The significance of difference horizontally selected p ＜ 0.05.
Embodiment 1
Inspection magnetic field is on female rats estrus cycle and the impact of steroid hormone blood level
Using the magnetic field intensity of 1mT, start lasting stimulation in rats 4 weeks from 4 week old, action time every day is from 16:00 to 24:
00.Find that rat to put the appearance in 2-3 days that magnetic field space starts into restless, gradually recovers normal performance after 3 days.Rat is existed
After raising 4 thoughtful 8 week old in magnetic field space, each group rat all starts the estrus cycle occur.By conventional vaginal smear method, inspection
Look into the estrus cycle of rat.
Result is as shown in Figure 4 and Table 1.Fig. 4 shows the microscopic findings of the vaginal smear of estrus cycle different phase.
By the vaginal epithelial cell keratinization situation come off therein, and leukocyte and the relative prevalence of mucus, determine week of oestrusing
The process of phase.Table 1 have recorded and starts magnetic field (1mT) from 4 week old and process female rats oestrus and hormonal readiness are affected feelings
Condition.
Table 1. starts magnetic field (1mT) from 4 week old and processes female rats oestrus and the impact of hormonal readiness
Note: * mark represents that the comparison between magnetic field process group and matched group is by non parametric tests Mann-Whitney U side
The p value that formula obtains, the comparison of the blood level of estrogen (E2) and progestogen (P4) obtains p value by t inspection, it is assumed that variance phase
Deng, bilateral truncation.
As can be seen from Table 1, process group frequency of oestrusing in magnetic field significantly improves, and dioestrus is shorter, and the estrogen in oestrus
(estradiol) has risen.But, the blood level change of progestogen is little, without significant difference.
Embodiment 2
Inspection magnetic field is on female rats steroid hormone blood level and the impact of reproduction offspring
The female Mus of 4 week old is similarly processed to 8 week old.From table 2 it can be seen that the blood level of estrogen and progesterone is same
Not having significantly affected, wherein p value is all higher than 0.05.In this case, treated female rats and normal hero of growing up
Property rat copulation, the situation of fertility offspring does not has the difference of statistically significant between process group and experimental group.
Result is as shown in table 2 below, Cluth size, the average weight of every nest filial generation, and the sex ratio of every nest offspring, at magnetic
Field processes the difference (p ＞ 0.05) all not having statistically significant between matched group.Trimester of pregnancy and dams when giving a birth latter 28 days
Body weight processes the difference the most not presenting statistically significant between matched group in magnetic field.
Table 2 starts magnetic field (1mT) from 4 week old and processes female rats hormonal readiness and the impact of reproduction offspring
Annotation: * mark represents and compares between magnetic field process group and matched group by t inspection acquisition p value, it is assumed that variance is equal,
Bilateral truncation.Lycoperdon polymorphum Vitt marks the index of offspring.
Embodiment 3
8 week old SD female rats are exposed to 5mT intensity magnetic field to blood steroid hormone level and the impact of reproduction offspring
Taking 8 week old SD rat 9, random packet, experimental group is 8, and matched group is 4.Experimental group is placed in magnetic field, magnetic
Field intensity is 5mT, and every daily exposure time is 18:00 to 06:00, and light application time is 6:00 to 18:00, and rat freely drinks water and enters
Food；Matched group is placed in outside the magnetic field of same room, and magnetic field intensity background value is 0.027 μ T.
Table 3 starts magnetic field (5mT) from 8 week old and processes female rats hormonal readiness and the impact of reproduction offspring
Annotation: * mark represents and compares between magnetic field process group and matched group by t inspection acquisition p value, it is assumed that variance is equal,
Bilateral truncation.Lycoperdon polymorphum Vitt marks the index of offspring.
As shown in table 3.Magnetic exposure continues 60, finds that in blood, estrogen level reduces, and progesterone level raises.Order
People is it was unexpected that with normal with strain male copulatory, and the Cluth size of offspring dramatically increases that (Cluth size meansigma methods rises to from 5.3
9.3), this causes the frequency of oestrusing of female rats to rise with magnetic field and there is certain dependency, and prompting magnetic field is causing female grinding tooth
Animals into heat frequency also results in the increase of number of eggs ovulated while rising, thus causes Cluth size to increase.
Embodiment 4
Magnetic field on male rat fertility about the impact of index
The electromagnetic field using 2mT intensity processes male adult rat (8 week old), every day from 18:00 to 06:00, altogether 12
Hour, continue 60 days.
Result is as shown in table 4 below, and its testis and epididymis weight do not have the most significant difference with matched group；Essence
The membrane integrity of sub-density and sperm is also not significantly different from；But motile sperm proportion significantly drops in the action of a magnetic field group
Low, the meansigma methods of motile sperm ratio drops significantly to 15.25% from 38.5%.
Table 4 starts magnetic field (2mT) from 8 week old and processes the impact on male rat sperm index
Annotation: * mark represents and compares between magnetic field process group and matched group by t inspection acquisition p value, it is assumed that variance is equal,
Bilateral truncation.Lycoperdon polymorphum Vitt marks p ＜ 0.05, processes the index of significant difference between comparison in magnetic field.
Embodiment 5
4 week old SD male rats are exposed to 2mT/5mT intensity magnetic field to fertility about the impact of index
Taking 4 week old SD male rat 15, random packet, 2mT and 5mT magnetic exposure group is respectively 6, and matched group is 3.
Experimental group is placed in magnetic field, and magnetic field intensity is 2mT, and every daily exposure time is 18:00 to 06:00, and light application time is 6:00 to 18:
00, rat freely drinks water and takes food；Matched group is placed in outside the magnetic field of same room, and magnetic field intensity background value is 0.027 μ T.
Result is as shown in table 5.Magnetic exposure continues 60, and result shows, from 4 week old, to be exposed to 2mT or 5mT magnetic field
Test group, its sperm concentration relative to comparison all reduce.Additionally, be exposed to the test group in 2mT or 5mT magnetic field, its movable essence
Ratio shared by son all declines, the most notable with 5mT test group.
Table 5 starts magnetic field from 4 week old, and (2/5mT processes the impact on male rat sperm index
3. discuss
3.1 inventors have made power frequency magnetic field generator, and its space be enough to place common rat breeding cage, it is thus possible to
Raising rat under the effect of certain magnetic field intensity (0-10mT), but rat simultaneously can be the most raw as conventional feeding manner
Live.
3.2 electromagnetic field pollution report [9] and the animal experiment studies [4,10] polluted with reference to relevant power frequency, inventor is by magnetic
The action intensity of field is set to 1-5mT, and the action time of every day is 8-12 hour.
Female rats before the sexual maturity of 4 week old is raised under the magnetic field intensity of 1mT, 8 hours every days, continues 4 weeks；Find
Except the frequency of oestrusing after growing up improves more than 30%, there is not notable change in the situation of sex hormones secretion and fertility offspring
Change.The power frequency electromagnetic field of people's everyday exposure is described, the damage to female fertility will not be produced in normal circumstances.But
It is that the frequency of oestrusing after growing up increases, i.e. dioestrus shortens.In sum, can rationally speculate, if often in contact with up to
The electromagnetic field of 1mT intensity, it is possible to promote frequency increase of ovulating, thus further result in the oocyte stock consumption ahead of time of ovary
Exhaust so that the menopause of female human is ahead of time.Start to stand magnetic stimulation before therefore, it can observe sexual maturity by this rat
The follicle stock whether causing adult rat exhausts in advance, thus ahead of time and related drugs is developed to obtain research Women’s climacteric
Animal model.Such rat model is also the animal model of a kind of premature ovarian failure, and it may be used for studying the physics such as magnetic field
Factor is on the impact of follicular development and the exploitation of associated treatment premature ovarian failure medicine.
Inventor also by 8 week old SD rat feedings under the magnetic field intensity of 5mT, every daily exposure time is 12 hours, continue
60 days.Find that in blood, estrogen level reduces, and progesterone level raises；With normally with strain male copulatory, the nest of offspring is young
Number dramatically increases.Further illustrate often in contact with up to electromagnetic field, it is possible to promote frequency increase of ovulating, and then cause ovary
Oocyte stock exhausts ahead of time so that the menopause of female human is ahead of time.
3.3 about the magnetic field impact on the relevant index of male rat sperm
It is different from the generation of oocyte in view of spermatogenesis, persistently carries out after growing up.Therefore, inventor's first profit
Adult male rats by 8 week old, it is contemplated that the toleration of adult rat may be relatively strong, inventor adds magnetic field intensity with every
The open-assembly time of day, i.e. the magnetic field intensity of 2mT and every day expose 12 hours.As above, shown in table 4, it is exposed to power frequency electromagnetic field 2
Month, testis and epididymis weight on rat do not make a significant impact, and illustrate that the spermatogenesis in testis becomes with the sperm of epididymis
Ripe process is insensitive for power frequency electromagnetic field.But, substantially reducing but occurs in the ratio of motile sperm.This explanation, is exposed to work
Frequency electromagnetic field can affect the motor capacity of sperm.
4 week old SD male rats are also placed under the magnetic field that magnetic field intensity is 2mT and 5mT by inventor, and it is little that every day exposes 12
Time, expose and continue 60.Result shows from 4 week old, is exposed to 2mT magnetic field sperm concentration and notable fall occurs relative to comparison
Low；And it being exposed to 5mT magnetic field, the ratio shared by motile sperm significantly reduces.Thus prove further, it is exposed to power frequency electromagnetic field
Not only affect the motor capacity of sperm, have an effect on sperm concentration.
In sum, in Rat Oocytes and spermatogenesis, before sexual maturity, contact environment magnetic field can cause female
Property oestrus frequency increase, grow up after contact environmental magnetic field steroid hormone blood level can be caused to change；Contact environment after growing up
Magnetic field can cause male motile sperm ratio to decline, and before sexual maturity, contact environment magnetic field can cause male motile sperm ratio
Decline, have an effect on sperm concentration.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document by individually
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the model that the application appended claims is limited equally
Enclose.
List of references
[1] Lv Zijun, Chen Zhongxuan. the power frequency electromagnet interference of transmission line is commonly used in room. journal of Zhejiang university (Edition),
2000,27 (4): 402-8.
[2] Wei Gang, Yang Yi, Zhou Bing. power frequency electromagnetic field is on the impact of human body and relevant criterion analysis. and Shanghai University Of Electric Power is learned
Report, 2009,25 (2): 145-9.
[3] Xi Xiaoli, the development of the CXLY-II type low frequency pulsed magnetic fields generators such as Wei Ming, Wen Jun. biomedical engineering
Magazine, 1999,16 (3): 314-7
[4] Cao Yanan, Zhang Yan, Liu. extremely low frequency electromagnetic fields is on mice female reproduction and the impact of offspring development. in
China's work hygiene occupation disease magazine, 2006, (08): 468-70.
[5] Zhu Shizhong. the experimentation that male adult rat reproductive hormone is affected by extremely low frequency electromagnetic fields. China's radiation is defended
Raw, 2009, (02): 146-7.
[6] de Bruyn L, de Jager L.Effect of long-term exposure to a randomly
varied 50Hz power frequency magnetic field on the fertility of the
Mouse.Electromagn Biol Med, 2010,29 (1-2): 52-61.
[7] Al-Akhras MA, Elbetieha A, Hasan MK, et al.Effects of extremely low
frequency magnetic field on fertility of adult male and female
Rats.Bioelectromagnetics, 2001,22 (5): 340-4.
[8] Zhao Hongxin, the survival rate of the .Transgreen/PI fluorescence staining counter detection sperms such as Yuan Yao, Hua Minmin. China
Andrology magazine, 2008,22 (4): 1-4
[9] Hui Jianfeng, Guan Zhicheng, Liu Yingyan. the limit value of various countries' power frequency electromagnetic field and the foundation determined thereof. high voltage skill
Art, 2006,32 (4).
[10]Al-Akhras MA.Influence of 50Hz magnetic field on sex hormones and
Body, uterine, and ovarian weights of adult female rats.Electromagn Biol Med,
2008,27 (2): 155-63.
Claims (4)
1. the preparation method of a rodent models, it is characterised in that make rodent be exposed to particular magnetic field strength
Electromagnetic field special time, thus oestrus after being grown up frequency improve or Cluth size improve female rodent models,
Wherein, described rodent is selected from:
(c) Adult female rodent；Or
Female rodent before (d) sexual maturity；
Wherein, the magnetic field intensity for described Adult female rodent is 1-10mT；
Amount to 240-1440 hour for the described exposure persistent period of described Adult female rodent；
Before described sexual maturity, the magnetic field intensity of female rodent is 0.5-5mT；
Before described sexual maturity, the described exposure persistent period of female rodent amounts to 72-480 hour.
2. the method for claim 1, it is characterised in that obtain described animal model, described device bag with following device
Including casing, field generator for magnetic, detector for magnetic field, temperature detection and regulation device and voltage-operated device, its feature exists
In,
The device obtaining described animal model also includes ventilation unit, illuminator and chiller；
Described field generator for magnetic is positioned in described casing and has for placing laboratory animal or experimental animal feeding container
Cavity；
Having door on the sidewall of described casing, described door is used for putting into or take out laboratory animal or experimental animal feeding container；
Described chiller contacts with described field generator for magnetic, is used for cooling down described field generator for magnetic；
Described ventilation unit and illuminator are respectively used to provide for described field generator for magnetic ventilate and illumination；And
Described voltage-operated device is connected with described field generator for magnetic, is used for controlling magnetic produced by described field generator for magnetic
Field intensity.
3. the purposes of female rodent models prepared by method described in a claim 1, it is characterised in that described animal
Model is for testing whether candidate substances affects oestrus frequency or the Cluth size of female rodent.
4. testing the method whether candidate substances affects oestrus frequency or the Cluth size of female rodent, its feature exists
In, comprise the following steps: female rodent models prepared by the method described in claim 1 that test substances is applied to, and
By oestrus frequency or the Cluth size of described female rodent models compared with matched group, wherein, frequency of oestrusing has statistics
The change of significance shows that female rodent is oestrused frequency by described candidate substances or Cluth size has an impact.
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